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T Kita

Publications and source records attributed to T Kita.

At least 73 records · Page 4Linked to original sources
PubMed2001

Identification of N-acetyltransferase 2 and CYP2C19 genotypes for hair, buccal cell swabs, or fingernails compared with blood.

Genotyping of polymorphic drug metabolizing enzymes may be useful to estimate the blood concentration, efficacy, and toxicity of drugs before administration. Blood samples are most generally used for genotyping; however, sampling is invasive and complicated by handling and transport. Therefore, the authors developed genotyping methods using nonblood specimens, and then each genotype was compared with that from blood. Healthy Japanese volunteers provided hairs (n = 50), buccal cell swabs (n = 50), and fingernails (n = 30) for N-acetyltransferase 2 and CYP2C19 genotyping. Recovery of genomic DNA from each nonblood specimen was lower than that from 0.5 mL blood. Using a modification of the DNA extraction and polymerase chain reaction amplification method, genotypes were diagnosed without failure, even for those with very low levels of DNA. Both genotypes from these specimens completely matched the genotypes from the blood of the same subject. These nonblood specimens can be convenient, accessible, and economical alternatives to blood as a source of DNA for genotyping.

Aryl Hydrocarbon Hydroxylases↗
PubMed2001

Roles of lectin-like oxidized LDL receptor-1 and its soluble forms in atherogenesis.

Lectin-like oxidized LDL receptor (LOX)-1 is a type II membrane protein that belongs to the C-type lectin family of molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. LOX-1 can support binding, internalization and proteolytic degradation of oxidized LDL, but not of significant amounts of acetylated LDL, which is a well-known high-affinity ligand for class A scavenger receptors and scavenger receptor expressed by endothelial cells (SR-EC). LOX-1 is initially synthesized as a 40-kDa precursor protein with N-linked high mannose-type carbohydrate, which is further glycosylated and processed into a 50-kDa mature form. LOX-1 expression is not constitutive, but can be induced by proinflammatory stimuli, such as tumour necrosis factor-alpha, transforming growth factor-beta and bacterial endotoxin, as well as angiotensin II, oxidized LDL itself and fluid shear stress. In addition, LOX-1 expression is detectable in cultured macrophages and activated vascular smooth muscle cells. In vivo, endothelial cells that cover early atherosclerotic lesions, and intimal macrophages and smooth muscle cells in advanced atherosclerotic plaques can express LOX-1. Cell-surface LOX-1 can be cleaved through some protease activities that are associated with the plasma membrane, and released into the culture media. Purification of soluble LOX-1 and the N-terminal amino-acid sequencing identified the two cleavage sites (Arg86-Ser87 and Lys89-Ser90), both of which are located in the membrane proximal extracellular domain of LOX-1. Measurement of soluble LOX-1 in vivo may provide a novel diagnostic tool for the evaluation and prediction of atherosclerosis and vascular disease.

Animals↗
PubMed2001

Role of oxidized LDL in atherosclerosis.

A critical event in the early stages of atherosclerosis is the focal accumulation of lipid-laden foam cells derived from macrophages. In various cholesterol-fed animal models of atherosclerosis, localized attachment of circulating monocytes to arterial endothelial cells appeared to precede the formation of foam cells. It is suggested that monocyte recruitment into early lesions depends on the endothelial adhesiveness for monocytes and lymphocytes. In vivo and in vitro experiments have identified molecules, such as ICAM-1, VCAM-1, and P-selectin, that can support the adhesion of monocytes and lymphocytes. Moreover, oxidized LDL, lysophosphatidyl-choline, and oxidized fatty acids induce the expression not only of these adhesion molecules but also of scavenger receptors, such as CD-36, SR-A, and LOX-1. Recently, we isolated and characterized the novel receptors for oxidized LDL, namely, LOX-1 and SR-PSOX. Expression of LOX-1 is found on endothelial cells, smooth muscle cells, and macrophages, whereas SR-PSOX is expressed on macrophages. In this paper the significance of oxidized LDL and its receptors, LOX-1 and SR-PSOX, in terms of atherogenesis is discussed.

Animals↗
PubMed2001

Study on PDGF receptor beta pathway in glomerular formation in neonate mice.

The assembly of vascular endothelial cells (ECs) and smooth muscle cells is a critical event in the development of the cardiovascular system. Although the role of ECs in this event has been studied intensively, the cross-talk between the two cell components remains poorly understood. In this study, we blocked platelet-derived growth factor receptor (PDGFR) pathways in mice by antagonistic rat monoclonal antibody APB5 against murine PDGFR-beta and examined glomerular capillary formation.

Aging↗
PubMed2001

Peroxisome proliferator-activated receptor a ligands increase lectin-like oxidized low density lipoprotein receptor-1 expression in vascular endothelial cells.

Lectin-like oxidized low density lipoprotein receptor-1 (LOX-1) is a receptor for atherogenic oxidized LDL expressed in vascular endothelial cells, smooth muscle cells, and macrophages. Peroxiosme proliferator-activated receptors (PPARs) include subfamilies PPARalpha and PPARgamma, which are expressed in endothelial cells and involved in transcriptional regulation of a large number of genes that affect lipoprotein and fatty acid metabolism as well as inflammation. We therefore explored the effects of PPARalpha and PPARgamma ligands on expression of LOX-1. Treatment of cultured bovine aortic endothelial cells (BAECs) with PPARalpha ligands (fenofibrate and WY14643) remarkably upregulated LOX-1 expression, which was detected by western and northern blot analyses. LOX-1 promoter-reporter gene assay showed that PPARalpha ligands activate transcription of the LOX-1 gene. PPARgamma ligands, in contrast, did not significantly affect LOX-1 expression in BAECs. PPARalpha appears to be a key regulator in promoting LOX-1 expression in BAECs. Upregulated expression of LOX-1 may modulate the catabolism of oxidized LDL and atherosclerotic progression.

Animals↗
PubMed2001

Expression of scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX) in human atheroma.

Recently, we identified a novel macrophage cell-surface receptor for oxidized low-density lipoprotein (Ox-LDL), designated SR-PSOX (scavenger receptor for phosphatidylserine and oxidized lipoprotein). Here we examine SR-PSOX expression in human atherosclerotic lesions using carotid endarterectomy specimens from 21 patients, directional coronary atherectomy specimens from 11 patients, and normal aortas from 2 patients. RT-PCR analysis demonstrated that SR-PSOX expression was upregulated in atherosclerotic lesions, but undetectable in normal aortas. Immunohistochemistry showed that SR-PSOX was abundantly expressed by macrophages in the intima of atherosclerotic lesions. Taken together, SR-PSOX may be involved in Ox-LDL uptake and subsequent foam cell transformation in macrophages in vivo and therefore may play important roles in human atherosclerotic lesion formation.

Arteriosclerosis↗
PubMed2001

Differential signaling for MCP-1-dependent integrin activation and chemotaxis.

Transmigration of monocytes to the subendothelial space is the initial step in atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we investigated the roles of MAPK in THP-1 cells, a monocytic cell line. MCP-1 stimulated beta1 integrin-dependent, but not beta2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor, but not by a p38-MAPK inhibitor. By contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor, but not by the MEK inhibitor. These data indicate that ERK is responsible for integrin activation and that p38-MAPK is responsible for chemotaxis mediated by MCP-1. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades, leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells.

Cell Line↗
PubMed2001

Molecular dissection of alpha- and dense-core granule secretion of platelets.

Upon activation, platelets release many active substances stored in alpha- and dense-core granules. However, the molecular mechanisms governing the regulated exocytosis are not yet fully understood. We have established an assay system using streptolysin-O-permeabilized platelets to analyze the Ca2+-induced secretions of von Willebrand factor stored in alpha-granules and [3H]5-hydroxytryptamine (5-HT) in dense-core granules. Using the assay, we found that small GTPase Rab4 regulates alpha-, but not dense-core, granule secretion in platelets. Furthermore, we purified a cytosolic essential protein and currently are analyzing its function.

Blood Platelets↗
PubMed2001

Effects of Choto-san on microcirculation, serum nitric oxide and lipid peroxides in patients with asymptomatic cerebral infarction.

The effects of Choto-san on microcirculation, serum nitric oxide and lipid peroxides were investigated. Fifteen patients with asymptomatic cerebral infarction were investigated before and after a four-week administration of Choto-san. The variables of microcirculation of the bulbar conjunctiva, which were the internal diameter of vessels, flow velocity and flow volume rate, were increased. Serum NO2(-)/NO3(-) tended to increase and lipid peroxides were decreased. Total serum cholesterol was also decreased. These results suggest that Choto-san may improve microcirculation affected by endothelial function and fat metabolism.

Blood Flow Velocity↗
PubMed2001

Lysophosphatidylcholine induces early growth response factor-1 expression and activates the core promoter of PDGF-A chain in vascular endothelial cells.

Lysophosphatidylcholine (lyso-PC), a polar phospholipid that is increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to transcriptionally induce the expression of endothelial genes relevant to atherogenesis. In cultured bovine aortic endothelial cells (BAECs), we show that lyso-PC induces the expression of early growth response factor (Egr)-1 and thereby activates the proximal promoter of the platelet-derived growth factor (PDGF)-A chain located 55 to 71 bp upstream from the transcription start site, which has been shown to be crucial for PDGF-A chain expression induced by fluid shear stress and fibroblast growth factor-1. Northern blot analyses showed that lyso-PC (10 to 20 micromol/L) transiently (30 minutes to 1 hour) induced expression of Egr-1 mRNA. Induced expression of Egr-1 mRNA, which was associated with increased amounts of Egr-1 protein in nuclei, preceded PDGF-A chain mRNA induction in lyso-PC-activated BAECS: Nuclear runoff assay revealed that lyso-PC stimulates transcription of the Egr-1 gene. Transient transfection of the oligonucleotide corresponding to the proximal promoter of the PDGF-A chain (oligo A) linked to the luciferase reporter gene revealed that lyso-PC can activate the core promoter of the PDGF-A chain by 5-fold. Insertion of a guanine at 3 sites in the oligo A abolished the lyso-PC-induced increases in luciferase activities. Electrophoretic mobility shift assay with use of radiolabeled oligo A showed a lyso-PC-inducible shift band, which was suppressed by excess amounts of unlabeled oligo A or an anti-Egr-1 antibody. In addition, lyso-PC-induced Egr-1 expression was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase-1 (MEK1), suggesting that lyso-PC-induced expression of Egr-1 depends on the MEK1/extracellular signal-regulated kinase pathway. Taken together, transcriptional activation of Egr-1-dependent genes by this atherogenic lipid may be a key regulator of atherogenesis.

Animals↗
PubMed2001

Oxidized LDL modulates Bax/Bcl-2 through the lectinlike Ox-LDL receptor-1 in vascular smooth muscle cells.

Oxidized low density lipoprotein (Ox-LDL) induces apoptosis in vascular smooth muscle cells (VSMCs), which may increase atherosclerotic plaque instability. In this study, we examined the molecular mechanisms causing the Ox-LDL-induced apoptosis in VSMCs, especially focusing on the involvement of Bax/Bcl-2 and the lectinlike Ox-LDL receptor-1 (LOX-1). In cultured bovine aortic smooth muscle cells (BASMCs), Ox-LDL at high concentrations (>60 microg/mL) induced cell death as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. DNA fragmentation was increased in BASMCs treated with high concentrations of Ox-LDL, indicating that the Ox-LDL-induced cell death in VSMCs was apoptosis. Ox-LDL upregulated LOX-1 expression through phosphorylation of extracellular signal-regulated kinase in BASMCs, and a neutralizing anti-LOX-1 monoclonal antibody, which can block LOX-1-mediated cellular uptake of Ox-LDL, prevented the Ox-LDL-induced apoptosis in BASMCs. This antibody also suppressed the increase in the Bax to Bcl-2 ratio induced by Ox-LDL in BASMCs. Furthermore, LOX-1 expression was well colocalized with Bax expression in the rupture-prone shoulder areas of human atherosclerotic plaques in vivo. LOX-1 may play an important role in Ox-LDL-induced apoptosis in VSMCs by modulating the Bax to Bcl-2 ratio. These molecular mechanisms may be involved in destabilization and rupture of atherosclerotic plaques.

Animals↗
PubMed2001

Expression of SR-PSOX, a novel cell-surface scavenger receptor for phosphatidylserine and oxidized LDL in human atherosclerotic lesions.

Receptor-mediated endocytosis of oxidized low density lipoprotein (Ox-LDL) by macrophages and the subsequent foam cell transformation in the arterial intima are key events in early atherogenesis. Recently, we have identified a novel macrophage cell-surface receptor for Ox-LDL by expression cloning from a cDNA library of phorbol 12-myristate 13-acetate-stimulated THP-1 cells, designated as the scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX). Here, we examined SR-PSOX expression in human atherosclerotic lesions. Total cellular RNA and fresh frozen sections were prepared from human carotid endarterectomy specimens (from 21 patients) and directional coronary atherectomy specimens (from 11 patients). Fragments of human aortas of 2 patients without visible atherosclerotic lesions served as negative controls. Quantitative reverse transcription-polymerase chain reaction demonstrated that SR-PSOX mRNA expression was prominent in atherosclerotic lesions but undetectable in normal aortas. Immunohistochemistry showed that SR-PSOX was predominantly expressed by lipid-laden macrophages in the intima of atherosclerotic plaques in carotid endarterectomy and directional coronary atherectomy specimens, although its expression was not detectable in normal arterial wall. Double-labeled immunohistochemistry confirmed that SR-PSOX is expressed by intimal macrophages. Taken together, SR-PSOX may be involved in Ox-LDL uptake and subsequent foam cell transformation in macrophages in vivo and thus may play important roles in human atherosclerotic lesion formation.

Animals↗
PubMed2001

N-Acetyltransferase 2 genotype correlates with sulfasalazine pharmacokinetics after multiple dosing in healthy Japanese subjects.

Sulfapyridine (SP) is metabolized by polymorphic N-acetyltransferase 2 (NAT2) [EC 2.3.1.5]. In this study, the correlation between the NAT2 genotype and the pharmacokinetics of SP after multiple oral dosing of sulfasalazine (SASP) was examined to elucidate the effect of multiple dosing on the predictability of the phenotype by NAT2 genotyping. Seven healthy subjects were classified into two groups; the homozygotes for the wild-type allele, NAT2*4/*4 (Group I) and the compound heterozygotes for the mutant allele (NAT2*4/*6A or NAT2*4/*7B) (Group II). All received once-daily 1 g of SASP (Salazopyrin) orally for 8 d. Plasma concentrations and urinary recoveries of SASP, SP and N-acetylsulfapyridine (AcSP) were monitored for 8 d. At 24 h on Day 1, the plasma concentration of SASP was lower and those of SP and AcSP were higher in Group II compared with Group I, but there was no significant difference. The plasma concentration ratio of AcSP to SP (AcSP/SP) tended to be lower in Group II. Urinary recoveries of SP and AcSP were increased in Group II, and their ratio was slightly reduced in Group II. Multiple dosing for 8 d resulted in an increase in the plasma concentrations of SASP, SP and AcSP. The difference between Group I and II was marked compared with single dosing, resulting in a significant difference in the plasma concentration of SP and the ratio of AcSP/SP. The simple input-output pharmacokinetic model applied for the analysis of plasma concentrations and urinary recoveries of SP and AcSP suggested the acetylation of SP into AcSP was 2.7-fold reduced in Group II (p=0.064).

Acetylation↗
PubMed2001

N-Acetyltransferase2 genotype correlated with isoniazid acetylation in Japanese tuberculous patients.

Isoniazid (INH) is metabolized by polymorphic N-acetyltransferase2 (NAT2). In the present study, the relationship between the NAT2 genotype and the INH acetylator phenotype was examined in Japanese tuberculous patients and compared with healthy subjects. Subjects were classified according to the genotyping into NAT2*5B (allele4), NAT2*6A (allele3) and NAT2*7B (allele2), using the PCR-RFLP method. Twelve healthy subjects and 7 tuberculous patients participated in the INH acetylator phenotyping study, in which each subject was administered an oral dose of INH, followed by urine sampling for 24 h. Urinary concentrations of INH and N-acetylisoniazid (AcINH) were measured by the HPLC method. The urinary recoveries of INH (% of dose) in healthy subjects in relation to NAT2 genotyping were as follows: 6.4+/-2.2 in the homozygotes for the wild-type allele, 10.7+/-2.2 in the compound heterozygotes for the mutant allele, and 38.6+/-6.4 in the homozygotes for the mutant allele. In the patients study, the findings in the corresponding three groups were 4.0+/-1.7, 8.8 and 18.3+/-9.3. Although no significant difference was found because of the lower systemic exposure of INH in patients compared with healthy subjects, there were differences in the disposition kinetics of INH between subjects with and without mutations in the NAT2 gene, and these findings were observed not only in healthy subjects but also in patients who had comedicated drugs and hepatic dysfunctions. The findings indicated that the metabolism of INH by NAT2 is clearly impaired in subjects with mutations in the NAT2 gene, and thus genotyping for three NAT2 point mutations was adequate to predict the metabolism of INH in Japanese tuberculous patients as well as healthy subjects. This NAT2 genotyping could become a useful alternative to TDM for INH.

Acetylation↗
PubMed2001

Thymidine phosphorylase inhibitors with a homophthalimide skeleton.

Several N-phenylhomophthalimide derivatives were prepared and their inhibitory activity on thymidine phosphorylase/ platelet-derived endothelial cell growth factor (TP/PD-ECGF) was assessed. Among them, 2-(2,6-diethylphenyl)-7-nitro-1,2,3,4-tetrahydroisoquinoline-1,3-dione (9) was found to be a more potent inhibitor than the classical inhibitor, 5-nitrouracil (1). Lineweaver-Burk plot analysis indicated that 9 shows mixed-type competitive inhibition of TP/PD-ECGF, while 1 is a competitive inhibitor.

Endothelial Growth Factors↗
PubMed2001

Magnitude of sustained multiple risk factors for ischemic heart disease in Japanese employees: a case-control study.

A case-control study was performed to clarify the cause of ischemic heart disease (IHD), such as acute myocardial infarction and angina pectoris, in Japanese employees. Among 122,051 workers from 31 industries, 94 cases of IHD were the subjects of the study, and a total of 191 age-matched subjects from the same department, but who did not develop IHD, served as the controls. Compared with the control group, body mass index, blood pressure, fasting plasma glucose, serum total cholesterol and serum triglyceride were significantly higher, and cigarette consumption and serum uric acid also tended to be higher, in the patient group from at least 10 years prior to onset. The frequency of moderate-drinkers tended to be lower in the case group. Electrocardiograms showed that, compared with the control group, the frequency of myocardial ischemia was higher in the case group from 9 years prior to onset and further rapidly increased from 3 years prior. The frequency of subjects with arrhythmia was the same as the control group until 3 years before onset and increased rapidly from 2 years prior. The frequency of subjects with multiple risk factors, particularly obesity, hypertension, hyperlipidemia and hyperglycemia, was consistently higher in the case group compared with the control group from 10 years prior to onset. Conditional logistic regression analysis demonstrated that having more than one risk factor greatly increased the risk; in particular, the combination of 3 or more factors increased the relative risk to 10.56 (95% confidence interval: 3.30-33.78). These findings suggest that a long duration of multiple risks is involved in the onset of IHD in Japanese employees, and that annual ECG monitoring as part of the medical examination was important in the prognosis.

Adult↗
PubMed2001

Expression of basic helix-loop-helix proteins in the glomeruli.

BACKGROUND: Basic helix loop helix (bHLH) proteins play a critical role in the differentiation of not only striated muscle cells but also adipocytes, neuron cells and smooth muscle cells. Previous studies have established in vitro mouse mesangial cells (MCs) to maintain the differentiated smooth muscle phenotype. MATERIALS AND METHODS: The purpose of the present study was to clone bHLH proteins from these MCs using the primers designed from a homologous sequence specific to bHLH, and to analyze the presence of bHLH proteins in normal kidney in vivo. From the cloning of MCs in vitro, we identified myf5 and herculin mRNA but not myoD. The expression of bHLH proteins in vivo was examined by immunohistochemistry with each specific antibody. RESULTS: The MCs in newborn mice possessed Id but did not express either protein herculin or myoD. On the other hand, mature MCs expressed both myf5 and herculin. The Id protein disappeared in mature glomeruli. CONCLUSION: These results suggest that bHLH proteins are an important factor for mature MCs in vivo.

Aging↗