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Biomedical subjects

Yasuo Ohno

Publications and source records attributed to Yasuo Ohno.

At least 37 records · Page 2Linked to original sources
PubMed2005

Interindividual variability in 5-Fluorouracil metabolism and procainamide N-acetylation in human liver cytosol.

We investigated the enzymatic kinetics and interindividual variability of the metabolism of 5-fluorouracil and procainamide by human liver cytosol and/or microsomes. The Km values for the 5-fluorouracil dihydropyrimidine dehydrogenase (DPD) and procainamide N-acetyltransferase activities in pooled liver cytosol, and procainamide hydrolysis in pooled liver microsomes were 3.9, 1670, and 969 microM, respectively, and the intrinsic clearance (Vmax/Km) values for these reactions were 128, 0.192, and 0.0059 microl/min/mg protein, respectively. The cytosolic activities of 5-fluorouracil metabolism and procainamide N-acetylation ranged from 145 to 790 (469+/-156, mean+/-S.D., n=22) and <1 to 152 (52+/-48, n=12) pmol/min/mg protein, respectively, and the DPD activity of 5-fluorouracil was neither gender-related nor age-dependent. Procainamide N-acetylation activities among 12 human cytosol samples were highly correlated with sulfamethazine N-acetylation activities, suggesting that procainamide N-acetylation is catalyzed by N-acetyltransferase-2. These results suggest that the N-acetylation reaction is more important than the hydrolysis in the metabolic pathway of procainamide, and that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of 5-fluorouracil and procainamide in human liver.

Acetylation↗
PubMed2005

Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver.

A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10(8) cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5'-bromo-2'-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.

Bioreactors↗
PubMed2005

[Network for the collection and distribution of Japanese liver tissues resected surgically for drug development and research use].

We focused on the establishment of a trial procedure for the collection and distribution of Japanese liver tissues obtained from waste surgical resections for drug development and research use. The following procedures were prepared for this project: the pretreatment of liver tissues before storage, their storage at 4 degrees C, the transport of liver samples, the setting up of a communication network among the participating hospitals and laboratories and the approval of each ethics committee. Thirteen liver samples (1.6-7.6 g) obtained from patients whose livers were excised due to cirrhosis, hepatocellular carcinoma, cholangiocarcinoma, or metastasis from colorectal carcinoma and were donated for research. Informed consent was obtained from every patient. Freshly isolated human hepatocytes were prepared from nine liver samples (viability 34.3-86.1%). Four samples were unsuitable to prepare hepatocytes. The profile of testosterone metabolism as 6beta-, 2beta-, 16beta-, 16alpha- and 2alpha-hydroxytestosterone and androstenedione in freshly isolated hepatocytes was shown to be specific for human liver. The 6beta-hydroxylation activity catalyzed by CYP3A4/5 indicated a high level of metabolism (139-996 pmol/min/million cells). Levels of 7-ethoxycoumarin O-deethylation and glucronidation activities were sufficient for analysis in freshly isolated human hepatocytes. We conclude that liver tissues from waste surgical resections supplied from a participating hospital can constitute a valuable source of freshly isolated human hepatocytes for drug development and safety evaluation.

Aged↗
PubMed2004

Differential modulation of PI3-kinase/Akt pathway during all-trans retinoic acid- and Am80-induced HL-60 cell differentiation revealed by DNA microarray analysis.

All-trans retinoic acid (ATRA) and Am80 are natural and synthetic derivatives of Vitamin A and have been used in the fields of oncology and dermatology for years. Their action was considered to be achieved mainly through binding to nuclear hormone receptors, retinoic acid receptors (RARs), although they have been observed to have different biological effects. For example, the two compounds have similar effects on differentiation but different effects on proliferation in human promyelocytic leukemia cell line HL-60 cells. To elucidate the genes responsible for this and other differences, we attempted for the first time to determine the genes whose expressions were differentially modulated during the time course of HL-60 cell differentiation by ATRA and Am80 treatment up to 72h utilizing DNA microarray and clustering analyses. As a result, the expressions of 204 genes were found to be modulated differentially by ATRA and Am80. Among them, we focused on two components of the PI3-kinase/Akt signal transduction pathway, phosphoinositide-3-kinase, beta-catalytic subunit and ribosomal protein S6 kinase polypeptide 1, which are related to the regulation of cell proliferation and apoptosis. Their expressions were specifically suppressed by ATRA, which coincided with the suppressive effects of ATRA on the HL-60 cell proliferation. Moreover, PI3-kinase inhibitors suppressed the proliferation of Am80-treated cells to the same extent as ATRA did. These results indicated that these gene products play a role in HL-60 cell growth suppression during the late stage of differentiation. The complete data and a list of the genes are available at .

Antineoplastic Agents↗
PubMed2004

Disposition of low doses of 14C-bisphenol A in male, female, pregnant, fetal, and neonatal rats.

Bisphenol A (BPA) is a weak xenestrogen (ADI = 50 microg kg(-1), US EPA) which is mass-produced, with potential for human exposure. To study absorption, distribution, excretion, and metabolism of BPA, BPA labeled with carbon-14 was administered p.o. to male and female Fischer (F344) rats at relatively low doses (20, 100, and 500 microg kg(-1)), and i.v. injected at 100 and 500 microg kg(-1). 14C-BPA (500 microg kg(-1)) was also administered orally to pregnant and lactating rats to examine the transfer of radioactivity to fetuses, neonatal rats, and milk. Radioluminographic determination using phosphor imaging plates was employed to achieve highly sensitive determination of radioactivity. Absorption ratios of radioactivity after three oral doses were high (35-82%); parent 14C-BPA in the circulating blood was quite low, however, suggesting considerable first-pass effect. After an oral dose of 100 microg kg(-1) 14C-BPA, the radioactivity was distributed and eliminated rapidly, but remained in the intestinal contents, liver, and kidney for 72 h. The major metabolite in the plasma and urine was BPA glucuronide, whereas most of the BPA was excreted with the feces as free BPA. A second peak in the time-course of plasma radioactivity suggested enterohepatic recirculation of BPA glucuronide. There was limited distribution of 14C-BPA to the fetus and neonate after oral administration to the dam. Significant radioactivity was not detected in fetuses on gestation days 12 and 15. On day 18, however, radioactivity was detected in the fetal intestine and urinary bladder 24 h after oral dosing of 14C-BPA to the pregnant rats. Part of radioactivity was transferred to neonatal rats from the milk of the treated lactating dam and remained in the intestine of the neonates after 24-h nursing by an untreated dam.

Administration, Oral↗
PubMed2004

4-Hydroxynonenal modulates the long-term potentiation induced by L-type Ca2+ channel activation in the rat dentate gyrus in vitro.

Increased oxyradical production and membrane lipid peroxidation (MLP) occur under physiological and degenerative conditions in neurons. We investigated whether 4-hydroxynonenal (4HN), one of the membrane lipid peroxidation products, affects long-term potentiation (LTP) in the rat dentate gyrus in vitro. Treatment of hippocampal slices with 4HN (10 microM) enhanced LTP without affecting basal evoked potentials. The enhancement was completely inhibited by 2 microM nifedipine, a blocker of L-type Ca2+ channels. In cultured dentate gyrus neurons, treatment of the cells with 4HN for 24 h resulted in a significant amount of cell death that was detoxified by glutathione, whereas short-term treatment with 4HN (< or = 6 h) had no effect. Nifedipine partially but significantly suppressed the 4HN-induced cell death. These results suggest that 4HN modulates LTP and induces delayed cell death through L-type Ca2+ channel activation in the dentate gyrus. 4HN thereby plays an important role in both physiological and pathophysiological events in the hippocampus.

Aldehydes↗
PubMed2004

Desensitization of P2X2 receptor/channel pore mutants.

Properties of five mutants of P2X2 receptor/channel having amino acid residue-substitution at the pore region were examined by expressing the channels in Xenopus oocytes. When the concentration-response relationship for ATP-evoked current was obtained, the current amplitude was increased along with the concentrations of ATP for the wild type channel whereas the amplitude was rather decreased with highest concentrations for four of the five mutants as if an "inactivation-like" mechanism occurs to these mutants. Upon a long exposure (30 s) to ATP, time-dependent decay in the ATP-evoked current was observed for three of the five mutants, suggesting that desensitization occurs to these mutants. The time course of the desensitization was well fitted with a single exponential time whereas that of the recovery from the desensitization could be better fitted with multiple exponentials than with a single exponential. The relationship between the desensitization and the "inactivation-like" mechanism was discussed.

Adenosine Triphosphate↗
PubMed2004

Characteristics of erythritol and formulation of a novel coating with erythritol termed thin-layer sugarless coating.

The purpose of this study was to clarify the characteristics of erythritol and to develop the optimum basic formulation of a novel coating with erythritol termed thin-layer sugarless coating. Characteristics of erythritol were investigated compared with maltitol, mannitol, sorbitol, xylitol, and sucrose. Furthermore, the optimum basic formulation of thin-layer sugarless coating with erythritol was determined by coating glass beads. We selected a continuous spray mist method for thin-layer sugarless coating due to the formation of a thin sugarless coating layer by a simple method. We demonstrated that erythritol is a suitable coating material for thin-layer sugarless coating compared with maltitol, mannitol, sorbitol, xylitol, and sucrose because of its high water solubility, low hygroscopicity, instant crystallization, and low tackiness. We also demonstrated that thin-layer sugarless coating with erythritol can reduce coating time compared with the coating with maltitol or sucrose due to its characteristics. We developed the optimum basic formulation of thin-layer sugarless coating consists of erythritol, powdered acacia, and talc. We confirmed that a smooth coating layer and high coating efficiency were achieved using the formulation.

Drug Stability↗
PubMed2004

Development and evaluation of the tablets coated with the novel formulation termed thin-layer sugarless coated tablets.

The purpose of this study was to develop and evaluate the thin-layer sugarless coated tablets containing Vitamin C, Vitamin E, Vitamin B2, calcium pantothenate, and L-cysteine. As a result of the formulation study, three coating layers, 2% under coating (UC), 38% build-up coating (BC), and 5% syrup coating (SC) were necessary for sufficient impact toughness, elegant appearance, and improvement of appearance stability after storage at 25 degrees C/75% RH for 6 months under open conditions. We demonstrated that the thin-layer sugarless coated tablets are superior to the sugar-coated tablets in terms of small tablet size and stability of calcium pantothenate. It was due to the coating method, the continuous spray mist method, which can minimize the thicknesses of coating layers and the moisture content in the tablets. We also demonstrated that the thin-layer sugarless coated tablets are superior to the film-coated tablets in terms of masking ability of the unpleasant odor and the appearance, stability of the appearance, and low hygroscopicity. It was due to the dense, opaque, and stable coating layers mainly consist of erythritol. We revealed that thin-layer sugarless coated tablets have both advantages of film-coated tablets and sugar-coated tablets.

Dietary Supplements↗
PubMed2004

Hydrogen peroxide modulates whole cell Ca2+ currents through L-type channels in cultured rat dentate granule cells.

Modification of voltage-gated Ca(2+) channels by hydrogen peroxide, a membrane-permeable form of reactive oxygen species, in cultured dentate granule cells was examined using the whole cell patch clamp technique. Pretreatment with hydrogen peroxide (1 and 10 microM) for 2 h enhanced the Ca(2+) current without affecting its voltage dependence. The enhancement was completely cancelled by 1 mM glutathione, an antioxidant, and 2 microM nifedipine, an L-type Ca(2+) channel blocker. In contrast, the enhancement of the Ca(2+) current was not mimicked by pretreatment with 10 microg/ml tunicamycin, an endoplasmic reticulum stressor. These results suggest that oxidative stress induced by hydrogen peroxide selectively regulates the activity of L-type Ca(2+) channels.

Animals↗
PubMed2004

Amino acid substitutions from an indispensable disulfide bond affect P2X2 receptor activation.

The roles of six amino acid residues downward from an extracellular disulfide bond involving Cys(224) in rat P2X(2) receptor were examined. When Cys(224) or Pro(225) was replaced with alanine, the responsiveness to ATP was lost. When Ile(226) was replaced with other hydrophobic amino acids, the responsiveness to ATP was reduced or abolished. When Phe(227) was replaced with leucine or isoleucine, the responsiveness to ATP was abolished. The responsiveness to ATP was moderately decreased with the alanine-substitution for Arg(228) and it was markedly decreased with the alanine-substitution for Leu(229). As for the alanine-substitution for Gly(230), the sensitivity was changed, but the maximal response to ATP was not reduced. The results suggested that a precise structure is required for amino acid residues close to the disulfide bond and, in general, the amino acid residues at odd number positions and those closer to the disulfide bond are more influential to the ATP responsiveness.

Adenosine Triphosphate↗
PubMed2004

Modulation of voltage-gated Ca2+ current by 4-hydroxynonenal in dentate granule cells.

Although recent studies have suggested that dentate granule cells play a key role in hippocampal functions, electrophysiological properties in these cells have not been sufficiently explored. In the present study, modification of voltage-gated Ca2+ channels by 4-hydroxynonenal (4HN), a major aldehydic product of membrane lipid peroxidation, in cultured dentate granule cells was examined using the whole-cell patch clamp technique. When whole-cell voltage clamp was applied, the cells exhibited a high-voltage-activated Ca2+ current, which was totally sensitive to 30 microM Cd2+ and partially sensitive to 2 microM nifedipine. 4HN enhanced the Ca2+ current in these cells. When L-type Ca2+ channels were blocked by application of nifedipine, the enhancement was completely canceled, whereas application of omega-conotoxin-GVIA or omega-agatoxin-IVA, blockers of N- and P/Q-type Ca2+ channels, respectively, had no effect. These results suggest that 4HN modulates L-type Ca2+ channels in the dentate granule cells, and thereby plays a role in the physiological and pathophysiological responses of these cells to oxidative stress.

Aldehydes↗
PubMed2004

Interindividual variability in 2-hydroxylation, 3-sulfation, and 3-glucuronidation of ethynylestradiol in human liver.

In the current study, we investigated interindividual variability of the 2-hydroxylation, 3-glucuronidation, and 3-sulfation of ethynylestradiol (EE2) using human liver microsomes and cytosol. Km values for the 2-hydroxylation and 3-glucuronidation in pooled liver microsomes and for the 3-sulfation in pooled liver cytosol were 3.34, 23.3, and 2.85 microM, respectively. Vmax/Km (ml/min/g liver) was highest for the 3-sulfation, followed by 2-hydroxylation, suggesting that 3-sulfation is the major metabolic pathway of EE2 in human liver. All further studies were performed at a substrate concentration of 0.1 microM. Microsomal 2-hydroxylation and 3-glucuronidation activities ranged from 0.21 to 5.02 (2.04+/-1.34, mean+/-S.D., n=35) and 0.20 to 4.84 (1.20+/-1.00, n=35) pmol/min/mg protein, respectively. Cytosolic 3-sulfation activity ranged from 4.2 to 24.3 (11.8+/-4.4, n=21) pmol/min/mg protein. All the measured enzyme activities were neither gender-related nor age-dependent, except that 2-hydroxylation was significantly higher in females than in males (p<0.05). The relative contribution of CYP3A to the 2-hydroxylation in liver microsomes was estimated from the degree of inhibition by 1 microM ketoconazole. The degrees of inhibition were between 17.8 and 78.0% (51.6+/-16.0%, n=27). These results indicate that there are large interindividual differences in the enzyme activities towards the respective metabolic pathways of EE2 and the relative contribution of CYP3A to the 2-hydroxylation of EE2 in human liver.

Adolescent↗
PubMed2004

Improvement of impact toughness of sugar-coated tablets manufactured by the dusting method.

The purpose of this study was to improve the impact toughness of sugar-coated tablets manufactured by a dusting method. The effects of sugar-coating formulations, which were the sugar-coating suspension formulations and the dusting powder formulations, on impact toughness of sugar-coated tablets were investigated. Impact toughness of sugar-coated tablets was measured by the friability test. We found that the dusting powder formulation was a control factor in impact toughness of sugar-coated tablets manufactured by the dusting method. The dusting method using dusting powder containing 20% microcrystalline cellulose (MCC, Avicel PH-F20) was a useful method for improvement of the impact toughness of sugar-coated tablets. The mechanism of improvement of impact toughness was that MCC in the subcoating layer resulted in a tight bond between the subcoating layer and the smoothing layer and prevented separation of the two layers on impact, because MCC improved the wettability of the subcoating layer, increased the surface roughness of the subcoating layer, and played the role of a binder between the two layers. We confirmed that the MCC between the subcoating layer and the smoothing layer acts to prevent separation of the two layers by impact. We demonstrated that MCC is a suitable material for sugar-coating in order to improve the impact toughness of sugar-coated tablets.

Carbohydrates↗
PubMed2004

Effect of moisture on impact toughness of sugar-coated tablets manufactured by the dusting method.

The purpose of this study was to clarify the effect of moisture on the impact toughness of sugar-coated tablets manufactured by the dusting method. We demonstrated that moisture plays an important role in the impact toughness of sugar-coated tablets. Moisturizing the sugar-coating layer resulted in enhancement of impact toughness of sugar-coated tablets, while reducing moisture in the sugar-coating layer resulted in weakening of the impact toughness. This was due to the characteristics of sucrose, the main ingredient of the sugar-coating layer, which is a soft and non-fragile material at high moisture levels, but hard and fragile at low moisture levels. We also demonstrated that friability as an indicator of impact toughness changed with time, and friability should be measured at 14 d after manufacture. This is due to moisture movement from outer sugar-coating layer into the inner sugar-coated tablets. Incorporating microcrystalline cellulose (MCC) in the subcoating layer resulted in sugar-coating layers with high resistance against impact even though moisture content of sugar-coated tablets was low. We confirmed the high impact toughness of the sugar-coated tablets with MCC whose moisture content was low from the results of both free fall and friability tests. We suggest that the dusting method using dusting powder containing MCC is a useful method for the production of sugar-coated tablets containing moisture sensitive drugs.

Carbohydrates↗
PubMed2004

Preservative solution for freeze-storage of surgically excised human colon to enable study of smooth muscle function in vitro.

We have compared the reactivity to carbachol and high potassium of circular smooth muscle isolated from segments of human colon which was freeze-stored in different preservative solutions for more than one month following surgical resection. Concentration-dependent contractions in response to carbachol were reduced in terms of both their sensitivity (pEC50) and reactivity (Emax), depending on the preservative solutions used. Similar reduction of reactivity to 100 mM KCl was also observed. The best responsiveness was shown when the tissue was freeze-stored in SFM101. It is concluded that the freeze-storage of surgically excised human colon in SFM101 or phosphate buffer solution for more than one month provided the best preservation of smooth muscle function for in vitro pharmacological examination.

Aged↗
PubMed2004

Improvement in reliability of probabilistic test of significant differences in GeneChip experiments.

A probabilistic test (FUMI theory) for GeneChip experiments has been proposed for selecting the genes which show significant differences in the gene expression levels between a single pair of treatment and control. This paper describes that the reliability of the judgment by the FUMI theory can be enhanced, when the selected genes are referred to biomolecular-functional networks of a commercial database. The genes judged as being differently expressed are grouped into a cluster in the biomolecular networks. It is also demonstrated that false positive genes have a trend in the networks to be isolated from each other, and also away from the clustered genes, since the false positive genes are randomly selected.

Algorithms↗