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Biomedical subjects

I Ishii

Publications and source records attributed to I Ishii.

At least 19 recordsLinked to original sources
PubMed2000

Identification of cytochrome P-450 isoform(s) responsible for the metabolism of pimobendan in human liver microsomes.

Pimobendan, 4, 5-dihydro-6-(2-(4-methoxyphenyl)-1H-benzimidazol-5-yl)-5-methyl-3( 2-H )-pyridazinone, is a new inotropic drug that augments Ca(2+) sensitivity and inhibits phosphodiesterase in cardiomyocytes. Pimobendan is well absorbed after oral administration and is metabolized in the liver to the O-demethyl metabolite, which is also active. This study was conducted to identify the cytochrome P-450 (CYP) isoform(s) responsible for the pimobendan O-demethylation in human liver microsomes. Pimobendan O-demethylase activity in human liver microsomes was significantly correlated with phenacetin O-deethylase activity. CYP1A2 antibody and specific inhibitors of CYP1A2 strongly inhibited the metabolism of pimobendan. CYP1A2 was the only one of 10 recombinant human CYP isoforms tested that catalyzed pimobendan O-demethylation at the substrate concentration of 1 microM. At a high substrate concentration (100 microM), recombinant CYP3A4 also catalyzed the reaction, and antibody to CYP3A4 partially inhibited the activity in human liver microsomes. The contribution of CYP1A2 to pimobendan O-demethylation in human liver microsomes varied in the range of 18 to 76%, whereas CYP3A4 accounted for less than 10%, as calculated using the relative activity factor method. We conclude that CYP1A2 is one of the major enzymes responsible for the O-demethylation of pimobendan and CYP3A may make a minor contribution at clinically relevant concentrations of the drug.

Adolescent↗
PubMed1999

Extracellular matrix accumulation on the thickened neointima in a rat double-balloon injury model.

We developed a rat double-balloon injury model and studied the thickened neointima using immunohistochemical and RT-PCR methods. Fourteen days after the first balloon injury of the rat left common carotid artery, a second balloon injury was inflicted in the same place. Histochemical sections taken 14 days after the single- and double-balloon injuries were used for calculating intimal/medial (I/M) area ratios, as an indicator of neointimal formation, and were subjected to immunohistochemical staining. Total RNA was also purified from some arteries and mRNA expression of some extracellular matrices (ECM) and cytokine receptors related to ECM metabolism was estimated using the RT-PCR method. The I/M ratio in the rat double-balloon injury model (II) (1.84+/-0.62 (mean+/-SE, n=5)) was significantly (p<0.05) higher than that in the single-balloon injury model (I) (1.30+/-0.19, n=10). The cell number per neointimal area was less in II than in I. As for the phenotype of smooth muscle cells, alpha-actin staining showed that the neointima of II consisted of more contractile form than synthetic, whereas that of I consisted of more synthetic form than contractile. The neointima of II was strongly stained with laminin and fibronectin, but that of I was stained only weakly. Consistent with these data, laminin and fibronectin mRNAs were markedly expressed in the neointima of II. Neointimas of both I and II were also stained positively with PDGF (alpha and beta) and TGF-beta (types I and II) receptors to the same extent. These results show: that ECM accumulation, particularly of laminin and fibronectin, characterizes the double-balloon injury model; that the marked accumulation of ECM in this model is due to a mechanism other than the PDGF or TGF-beta signalling pathway; and that this model resembles the lesion of post-PTCA re-stenosis, and therefore provides the key for its investigation.

Animals↗
PubMed1999

Expression of LR11, a mosaic LDL receptor family member, is markedly increased in atherosclerotic lesions.

Receptors belonging to the LDL receptor (LDLR) family are thought to play key roles in lipoprotein metabolism in a variety of tissues, including the arterial wall. Here, we report that the expression of a 250-kDa mosaic LDLR family member, which we called LR11 for the presence of 11 ligand-binding repeats, is markedly induced during the process of atherogenesis in 2 animal models. Analysis by reverse transcription-polymerase chain reaction and RNase protection assays revealed that LR11 transcript levels rise in rabbit aortas displaying atheromatous lesions after the rabbits have been fed a high-cholesterol diet. Immunohistochemistry demonstrated that the highest induction of LR11 occurs in intimal smooth muscle cells (SMCs), followed by medial SMCs close to the intimal border of the atheromatous lesions. Experimental intimal hyperplasia by endothelial denudation showed that LR11 mRNA levels were also increased in the arteries after balloon injury, with the transcripts localized primarily in the hyperplastic intimal layer. In agreement with the correlation of LR11 induction during increased cell proliferation, cultured SMCs showed an increase in LR11 expression in the proliferative phase. Furthermore, Northern and Western blot analyses showed that medium conditioned by the monocyte-macrophage cell line THP-1 enhanced LR11 expression in cultured SMCs. These findings suggest that upregulation of LR11 might be contributing to the pathological roles of intimal and medial SMCs during arteriosclerotic lesion development and provide the first insight into the as yet unknown functional significance of this intriguing LDLR family member.

Angioplasty, Balloon↗
PubMed1998

Differential catalytic properties in metabolism of endogenous and exogenous substrates among CYP3A enzymes expressed in COS-7 cells.

The catalytic properties of CYP3A7 in the metabolism of endogenous and exogenous substrates were compared with those of CYP3A4 and CYP3A5 using COS-7 expressing enzymes. The highest activities of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone 3-sulfate (DHEA-S) 16alpha-hydroxylase were observed in COS-7 cells expressing CYP3A7. In contrast, the activity of testosterone 6beta-hydroxylase of CYP3A7 expressed in COS-7 cells was much less than that of CYP3A4 expressed in COS-7 cells. The rate of carbamazepine 10, 11-epoxidation was the greatest in COS-7 cells expressing CYP3A4, followed by CYP3A5 and CYP3A7. On the other hand, the formation of reductive metabolite of zonisamide was the highest in COS-7 cells expressing CYP3A4, followed by CYP3A7 and CYP3A5. Furthermore, the addition of triazolam resulted in a decrease in 6beta-hydroxylation catalyzed by CYP3A7, but not by CYP3A4, whereas the pretreatment of microsomes with triacetyloleandomycin (TAO) resulted in a decrease in the reaction catalyzed by CYP3A4, but not by CYP3A7. Together with these results, it was suggested that CYP3A7 exerts differential catalytic properties not only in metabolism of endogenous substrates but also in drug metabolism compared to CYP3A4 and CYP3A5.

Adult↗
PubMed1998

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. Role of cytoplasmic tail phosphorylation in each process.

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.

Amino Acid Sequence↗
PubMed1998

Molecular evolution of a host-range gene in geminiviruses infecting asexual populations of Eupatorium makinoi.

Asexual plants of Eupatorium makinoi is frequently infected with tobacco leaf curl geminivirus (TLCV). The host range of TLCV is narrow, and ORF C4 is considered to function as a host range determinant. Using this TLCV-Eupatorium system, we tested the expectation that the rate of amino acid replacements will be accelerated in ORF C4 if resistant genes of the host plants drive molecular evolution in ORF C4. ORF C4 is entirely contained within a longer ORF C1 encoding a replication protein. We analyzed 21 sequences containing ORF C4 and a part of ORF C1. While per-site number of synonymous substitutions exceeded that of replacements in ORF C1, per-site number of replacements exceeded that of synonymous substitutions in ORF C4. However, this excess of per-site replacement in ORF C4 was mostly explained by the overlap gene nature, because most synonymous substitutions in ORF C1 change amino acid of ORF C4. In conclusion, not positive but negative selection is a predominant mode characterizing molecular evolution of ORF C4.

Asteraceae↗
PubMed1998

Metabolism of dacarbazine by rat liver microsomes contribution of CYP1A enzymes to dacarbazine N-demethylation.

The N-demethylation of dacarbazine in liver microsomes was significantly increased by treatment of rats with beta-naphthoflavone, dexamethasone, or phenobarbital. However, the extent of increase in the N-demethylation observed in beta-naphthoflavone-treated rats was much greater than that observed in dexamethasone-treated rats. A good correlation between N-demethylation of dacarbazine and O-deethylation of phenacetin was observed when a low concentration of phenacetin was used. Furthermore, the activity of dacarbazine N-demethylase in rat liver microsomes was highly correlated with the amounts of CYP protein immunochemically determined with anti-rat CYP1A2 antibodies. In addition, antibodies to rat CYP1A2, and furafylline and alpha-naphthoflavone, which are known inhibitors of CYP1A enzymes, exhibited inhibitory effects on dacarbazine N-demethylation. These results indicated that CYP1A enzymes may be responsible for N-demethylation of dacarbazine in rat liver microsomes.

Animals↗
PubMed1998

Steroid hydroxylation by human fetal CYP3A7 and human NADPH-cytochrome P450 reductase coexpressed in insect cells using baculovirus.

Human fetal CYP3A7 and human NADPH-cytochrome P450 reductase were coexpressed in insect cells, TN-5, infected with a recombinant baculovirus carrying both cDNAs. The expression of reductase in TN-5 cells was shown to be sufficient for the CYP3A7 dependent 16 alpha-hydroxylation of dehydroepiandrosterone. However, the extra addition of cytochrome b5 and phospholipid was necessary to obtain a maximal activity of CYP3A7 catalyzing the reaction. CYP3A7 expressed in TN-5 cells was capable of metabolizing testosterone, cortisol and dehydroepiandrosterone 3-sulfate as well as dehydroepiandrosterone. The apparent Vmax for 6 beta-hydroxylations of testosterone was similar to that obtained for 6 beta-hydroxylation of cortisol (2.9 versus 2.5 nmol/nmolP450/min). In contrast, the apparent Vmax for 16 alpha-hydroxylation of dehydroepiandrosterone and its 3-sulfate were 20 and 2 times greater than those observed for steroid 6 beta-hydroxylations, respectively (67.5 and 5.8 versus 2.5-2.9 nmol/nmol P450/min). On the other hand, the apparent K(m) for 6 beta-hydroxylations of testosterone and cortisol were greater than those for 16 alpha-hydroxylations (120 and 860 versus 46-58 microM). Thus, CYP3A7 was active for steroid 6 beta-hydroxylations and 16 alpha-hydroxylations, but there were greater differences in Vmax/K(m) ratios between these reactions.

Animals↗
PubMed1998

[A case of choroidal malignant melanoma in which 123I-IMP scintigraphy was useful for diagnosis].

A case of choroidal malignant melanoma in which N-isopropyl-p-[123I]-iodoamphetamine (123I-IMP) scintigraphy was useful for diagnosis is reported. A 62-year-old man first visited our hospital 3 years ago complaining of decreases in left eyesight. CT showed a tumor with an arcuate high attenuation area on the aural side of the optic disk in the left eye. A crescent high attenuation area, evidence of retinal detachment, was also observed on the nasal side of the optic disk. T1-weighted MR images showed low signal intensity in most of the tumor and a high signal intensity area was seen in the center, T2-weighted MR images showed homogeneous and marked low signal intensity area. Differentiation from a hematoma or a vascular tumor associated with bleeding was impossible based on CT and MRI. However, because late-phase images obtained on 123I-IMP scintigraphy showed marked high accumulation in an area corresponding to almost the entire left eye, left eye enucleation was undertaken under a diagnosis of malignant melanoma. Histopathologic examinations showed tumor growth mainly in the subretinal area. The melanin content of the tumor was high in the periphery and relatively low in the center. Infiltration was observed into the sclera and around the vortex vein outside the eyeball, but this change could not be detected by CT or MRI. 123I-IMP scintigraphy was useful not only for qualitative diagnosis of choroidal malignant melanoma, but for the determination of the extent of the lesion as well.

Choroid Neoplasms↗
PubMed1998

Stimulation of cholesterol release from rabbit foam cells by the action of a new inhibitor for acyl CoA:cholesterol acyltransferase (ACAT), HL-004.

A new inhibitor of acyl CoA:cholesterol acyltransferase (ACAT), HL-004 [N-(2, 6-diisopropylphenyl)tetradecylthioacetamide], suppressed the synthesis of cholesterol [14C]oleate at 10(-9) approximately 10(-7) M in a concentration-dependent manner in both THP-1 cell-derived macrophages and foam cells prepared from aortic intima of rabbits fed a high cholesterol diet. Incorporation of [3H]cholesterol oleate-beta very low density lipoproteins was not inhibited by HL-004 at 10(-9) approximately 10(-7) M. Release of radioactivity from the cells loaded with [3H]cholesterol oleate-beta very low density lipoproteins was increased by the inhibition of ACAT activity with HL-004. HL-004 did not affect on acid and neutral cholesterol esterases. As a result, cholesterol ester content in foam cells decreased. These data suggested that HL-004 decreases cholesterol ester in foam cells by increasing the release of cholesterol and therefore might suppress atherosclerotic lesions.

Acetanilides↗
PubMed1997

Malignant transformation by overproduction of translation initiation factor eIF4G.

N4G3, a cell line that overexpresses translation initiation factor eIF4G, one of the components of eIF4F, was made by stable transfection of the human eIF4G cDNA into NIH3T3 cells. The cells expressed 80-100 times greater levels of eIF4G mRNA than did NIH3T3 cells. N4G3 cells formed transformed foci on a monolayer of cells, showed anchorage-independent growth, and formed tumors in nude mice. These results indicate that overexpression of eIF4G caused malignant transformation of NIH3T3 cells. It is also known that overexpression of eIF4E, another component of eIF4F, causes transformation of NIH3T3 cells. However, there was no difference in the amount of eIF4E protein between N4G3 and NIH3T3 cells, indicating that cell transformation does not involve a change in eIF4E levels. The results may be due to an effect of eIF4G on translational control of protein synthesis directed by mRNAs having long 5'-untranslated region.

3T3 Cells↗
PubMed1997

Alanine exchanges of polar amino acids in the transmembrane domains of a platelet-activating factor receptor generate both constitutively active and inactive mutants.

To determine ligand-binding sites of a platelet-activating factor (PAF) receptor, alanine-scanning mutagenesis was carried out. All 23 polar amino acids in the putative 7-transmembrane (TM) domains of a guinea pig PAF receptor were individually replaced with alanine. The ligand-binding properties of mutant receptors were determined after transient expression in COS-7 cells. Mutants in TM II (N58A, D63A), TM III (N100A, T101A, S104A) and TM VII (D289A) displayed higher PAF-binding affinities than seen with the wild-type receptor. In contrast, mutants in TM V (H188A), TM VI (H248A, H249A, Q252A), and TM VII (Q276A, T278A) showed lower affinities. Representative mutants were then stably expressed in Chinese hamster ovary cells to observe PAF-induced cellular signals (arachidonate release, phosphatidylinositol hydrolysis, adenylyl cyclase inhibition). An N100A mutant with the highest affinity was constitutively active and was responsive to lyso-PAF, an inactive derivative of PAF. One nanomolar PAF induced no signals in low affinity mutants, an EC50 value for the wild-type receptor. Three histidines (His-188, His-248, His-249) might form a binding pocket for the phosphate group of PAF, since zinc effectively inhibited ligand binding. Based on these results, a three-dimensional molecular model of PAF and its receptor was generated using bacteriorhodopsin as a reference protein.

Alanine↗
PubMed1997

The differential involvement of von Willebrand factor, fibrinogen and fibronectin in acute experimental thrombosis in rat cerebral and mesenteric microvessels.

Arterial thrombi are primarily composed of platelets. Platelets are bound to injured endothelial cells, sub-endothelial matrices, and other platelets by a range of adhesive proteins. Some of these reactions are governed by shear forces. The role of adhesive proteins in the pathogenesis of arterial thrombosis is not fully understood. The aim of this study was to examine the involvement of von Willebrand factor (vWF), fibrinogen (Fg), and fibronectin (FN) in the formation of microvascular thrombi in vivo using a helium-neon laser-induced thrombosis method. Transmission electron microscopy demonstrated that laser irradiation resulted in platelet-rich thrombosis in arterioles and venules, and revealed that this occurred in the absence of endothelial denudation. The mean wall shear rates in mesenteric arterioles and venules were 641 +/- 40 and 280 +/- 20 s-1, respectively. Shear rates increased approximately fivefold in arterioles and tenfold in venules during the formation of occlusive thrombi. Antibody to vWF inhibited thrombosis in arterioles and venules. Antibodies to Fg and FN inhibited thrombosis in venules but not in arterioles. These results confirm that vWF, Fg and FN were involved in thrombogenesis in vivo and demonstrated that significantly higher shear rates were required for the reactions involving vWF than those involving either Fg or FN.

Adhesiveness↗
PubMed1997

[Multidrug-resistance by induction of inactivation for anti-cancer drugs].

There are several major groups of multidrug resistance mechanisms. 1) The multidrug resistant phenotype. 2) Glutathione S-transferences (GST) and detoxification mechanisms. 3) Topoisomerase I and II. 4) DNA repair. 5) Drug activation by cytochrome P450 (P450). In this article the biochemical functions of GST and P450 are described to show how individual enzymes contribute to resistance to carcinogens and anti-tumor drugs. Cancer cell lines indicated resistant to anti-cancer drugs, such as mitomycin C, doxorubicin, tamoxifen, cyclophosphamide and their derivatives, by a high activity of GST and a low activity of P450 in general. However, the mechanism of change of these enzyme activities is complicated and different in each drug. We show the study on the mechanism of multidrug resistance using cancer cell lines.

Animals↗
PubMed1997

Nucleotide sequence of pi class glutathione S-transferase in human fetal liver.

The GST Fpi and GST Ppi, pi class glutathione S-transferase (GST), were purified from human fetal livers and placentas, respectively. Both GST enzymes were indistinguishable each other in their subunit molecular weights, immunochemical properties and substrate specificities. Three clones (pFGP-1, pFGP-2 and pFGP-3) coding for the pi class GST purified from fetal livers were isolated from a human fetal liver cDNA library. The full-length clone encodes a polypeptide comprising 210 amino acid including the initiator methionine. All of these cDNA clones were nearly identical to a human placental cDNA clone, pGpi 2. The pFGP-1 cDNA had only a single base transition accompanied by an amino acid transition in the coding region, at position 313. The pFGP-2 and pFGP-3 cDNAs were also nearly identical to pGpi 2 cDNA, having only a single silent C-->T transition in the coding region, at position 555.

Amino Acid Sequence↗
PubMed1996

[Influence of multiple-dose administration of cefetamet pivoxil on blood and urinary concentrations of carnitine and effects of simultaneous administration of carnitine with cefetamet pivoxil].

Cefetamet pivoxil (CEMT-PI), a drug of pivaloyloxymethyl group, was investigated for its impact on the carnitine blood homeostasis and renal excretion upon administering CEMT-PI alone, and CEMT-PI simultaneously with carnitine. 500 mg of CEMT-PI (group A) and 500 mg of CEMT-PI and an equimolar amount (200 mg of carnitine) of levocarnitine chloride (group B) were administered twice a day for 7 and 1/2 consecutive days to 5 healthy volunteers (group A) and 3 healthy volunteers (group B). No serious side effects nor abnormal values in physical and laboratory tests were observed throughout the study in both groups. During the treatment period, plasma total carnitine decreased slowly down to 25.5 microM (group A) and 38.8 microM (group B) and plasma free carnitine reached steady state levels at 17.7 microM (group A) and 29.2 microM (group B) on day 5. These concentrations represent 45 and 37% in group A, 66 and 58% in group B of the average pre-treatment baseline levels. Plasma pivaloylcarnitine quickly reached plateau levels of 6.12 microM (group A) and 4.05 microM (group B) on day 4. After treatment stop, plasma total and free carnitine returned to the pretreatment baseline level within 5 days (group A) and 3 days (group B), and plasma pivaloylcarnitine was detectable until day 7 of the treatment-free follow up in both groups. Although carnitine was given concurrently at a dose equimolar to the ingested amount of pivalic acid in group B, the plasma total and free carnitine exhibited a decrease. This was considered attributable to the fact that the bioavailability of carnitine is as low as 16% when administered orally, which is considerably less compared to the 55% bioavailability of cefetamet pivoxil.

Administration, Oral↗
PubMed1995

Cholesterol metabolism in monocyte-derived macrophages from macrophage colony-stimulating factor administered rabbits.

The metabolism of beta-very-low-density lipoproteins (beta-VLDL) in macrophages from the blood monocytes of rabbits, which had been administered macrophage colony-stimulating factor (M-CSF) in vivo, was investigated in order to clarify the mechanism of the suppressive effect of M-CSF on cholesterol accumulation in macrophages. Cholesterol ester content after incubation with beta-VLDL, and [3H]cholesterol oleate-beta-VLDL incorporation remarkably increased in cultured macrophages from blood monocytes in the high cholesterol diet control group compared to those in the normal diet control group. Those in macrophages from M-CSF-treated groups, both normal diet and high cholesterol diet, were the same as in the normal diet control group. The ratio of released [3H]cholesterol to incorporated [3H]cholesterol oleate-beta-VLDL in macrophages from control was smaller than that from the M-CSF group. The acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity was enhanced in the high cholesterol diet groups, and the activity in M-CSF-treated groups decreased. The neutral cholesterol esterase activity was enhanced in the M-CSF-treated groups compared with that in the non-treated control groups. These results suggest that high cholesterol diet induced further cholesterol esterification and that M-CSF treatment enhanced cholesterol clearance in macrophages.

Animals↗