Biomedical subjects
Publications and source records attributed to T Okada.
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The clinical usefulness of a new myocardial perfusion imaging agent, technetium-99m tetrofosmin, was assessed in 58 patients for a total of 76 times (mean age 9.7 years, 1-15 years) including 20 with and 26 without significant coronary stenotic lesions in Kawasaki disease. Pharmacological (dobutamine or adenosine triphosphate disodium) or exercise stress technetium-99m tetrofosmin single photon emission computed tomography was performed under stress and at rest on the same day. The sensitivity for detection of stress-induced perfusion defects by this method was 90% (18/ 20) and the specificity was 85% (22/26). The dose was 9.3 +/- 2.5 MBq/kg under stress and 18.7 +/- 5.6 MBq/kg at rest. No significant correlation was recognized between the dose and the image quality. Adequate image quality was provided by projection time 20-40 sec per frame. Increased liver accumulation was seen in 24% (18/76), especially in younger and pharmacological stress cases. The acquisition starting time after tetrofosmin injection was 58.4 +/- 18.7 min in the negative increased liver accumulation group and 43.7 +/- 18.3 min in the positive group (p < 0.01). We recommend that the following protocol is used for pediatric imaging. 1) The standard tetrofosmin dose is rougly 10 MBq/kg (upper limit 370 MBq) at stress and the double dose for the rest imaging. 2) SPECT projection time of 20-40 sec per frame. 3) In practice, the patient should be fasting prior to stress injection, and the imaging should be done 1 hour after eating. The rest injection should be done immediately thereafter, and the rest image should be obtained 1 hour later.
To investigate the mechanism of intraocular neovascularization, we studied how vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) are expressed in the ocular tissues under hypoxic conditions. Prior to proliferation of vascular endothelial cells resulting in neovascularization, the retinal tissues such as pericytes, retinal glial cells, ganglion cells, and ciliary epithelium react directly to hypoxia expressing VEGF and/or IL-8 and stimulate endothelial cell proliferation in a paracrine manner. We demonstrated that transcription factor activator protein-1 (AP-1) is activated for expression of VEGF messenger ribonuculeic acid (mRNA) and in a similar way nuclear factor kappa B (NF-kappa B) is activated for expression of IL-8 mRNA. However, hypoxia-induced expression of VEGF and/ or IL-8 is only one aspect of the complicated processes in intraocular neovascularization. We hope that further detailed analysis of the mechanism will make it possible to inhibit and treat clinically intraocular neovascularization in the near future.
To investigate the usefulness of an intravascular ultrasound (IVUS)-bearing balloon catheter, we analyzed results in 17 patients with 19 lesions who underwent percutaneous transluminal coronary angioplasty (PTCA) with IVUS-bearing balloon catheters (group A) and 25 patients with 28 lesions who underwent PTCA without IVUS (group B). The patients had angina pectoris with or without old myocardial infarction. The success rate (less than 50% residual stenosis on coronary angiography) in group A was 17/17 patients (100%) and 19/19 lesions (100%), and that in group B was 21/25 patients (84%) and 24/28 lesions (86%) (NS). The percent stenosis, by coronary angiography, in groups A and B was 71.3% and 76.8% before PTCA (NS) and 4.8% and 22.3% in groups A and B after PTCA (P = 0.005). Complication rates in the two groups were not significantly different, but non-Q myocardial infarction was observed in one patient with a type C lesion (according to the American Heart Association/American College of Cardiology [AHA/ACC] classification) in group A. Postdilation of Wiktor stents was determined by IVUS in four patients. These results suggest that the IVUS-bearing balloon catheter is useful to achieve larger lumen size for optimal PTCA.
We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyltransferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans. The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.
Phospholipase A2 is an enzyme which hydrolyzes the sn-2 position of certain cellular phospholipids. The liberated lysophospholipid and arachidonic acid are precursors in the biosynthesis of various biologically active products. As human nonpancreatic sPLA2 is present in high levels in the blood of patients in several pathological conditions, the potent sPLA2 inhibitors have been suggested to be useful drugs. Here we describe the synthesis, structure-activity relationship, and inhibitory activities of indolizine and indene derivatives. 1-(Carbamoylmethyl)indolizine derivatives and 1-oxamoylindolizine derivatives exhibited very potent inhibitory activity. The former was unstable to air oxidation, but the latter exhibited an improvement both in stability and in potency. Some compounds approached the stoichiometric limit of the chromogenic assay.
In order to develop novel antiasthmatic agents based on a new mechanism of action, a series of 3-substituted 5-amino-1-[(methylamino)(thiocarbonyl)]-1H-1,2,4-triazole derivatives were synthesized and evaluated in a model in which eosinophilia was induced in the airway through intravenous (iv) injection of Sephadex particles on days 0, 2, and 5. After screening of several hundred derivatives, we finally identified the highly potent eosinophilia inhibitor 5-amino-3-(4-chlorophenyl)-1-[(methylamino)(thiocarbonyl)]-1H-tria zole (23c, GCC-AP0341), which had ID50 values of 0.3 and 0.07 mg/kg when administered orally (os) and intraperitoneally (ip), respectively. This compound showed complete inhibition of the hypersensitivity induced by ascaris inhalation at an ip dose of 1 mg/kg as well as low toxicity, with an LD50 value of > 2.0 g/kg in mice. Extensive study of its mechanism of action revealed that 23c inhibited eosinophil survival induced by interleukin-5 (IL-5), but had little or no effect on leukotriene D4 (LTD4) or platelet-activating factor (PAF)-induced responses. Taken together, these results suggest 23c as a novel candidate for the treatment of chronic asthma. Further studies are now underway.
Stimulation of differentiated THP-1 cells by insulin led to rapid accumulation of PtdIns(3,4,5)P3, a product of PtdIns 3-kinase. Stimulation of the GTP-binding-protein-linked receptor by N-formylmethionyl-leucyl-phenylalanine (fMLP) also induced the accumulation of PtdIns(3,4,5)P3 in the cells. The effect of insulin was, while that of fMLP was not, accompanied by increased PtdIns 3-kinase activity in the anti-phosphotyrosine immuno-precipitate. The combination of insulin and fMLP induced more PtdIns(3,4,5)P3 production than the sum of the individual effects. The insulin-induced recruitment of PtdIns 3-kinase activity in the anti-phosphotyrosine immunoprecipitate was unaffected by the combined treatment with fMLP. To investigate the mechanism underlying the synergistic accumulation of PtdIns(3,4,5)P3, we separated the cytosolic proteins of THP-1 cells on a Mono Q column. PtdIns 3-kinase activities were eluted in two peaks, and one of the peaks markedly increased on the addition of beta gamma-subunits of GTP-binding proteins (G beta gamma). The other peak was affected only slightly by G beta gamma, but was synergistically increased by G beta gamma and a tyrosine-phosphorylated peptide which was synthesized accordingly to the amino acid sequence of insulin receptor substrate-1. The activity in the latter fraction was completely immunoprecipitated by an antibody against the regulatory subunit of PtdIns 3-kinase (p85). These results suggest that the conventional PtdIns 3-kinase (p85/p110), which has been implicated in insulin-induced cellular events, or a closely related isoenzyme is controlled by a combination of a tyrosine-phosphorylated protein and a GTP-binding protein in intact cells.
To study the existence of the erythropoietin receptor (Epo-R) mRNA in brain capillary endothelial cells, the reverse transcription (RT) PCR was performed using total RNAs from rat brain capillary endothelial cells (RBECs) and MBEC4, which is one of the established mouse brain capillary endothelial cell lines. Southern analysis of the RT-PCR products indicated that both RBECs and MBEC4 expressed an authentic form of Epo-R mRNA as a minor form and an intron-5-inserted form of Epo-R mRNA, thus a soluble form of Epo-R mRNA, as a major form. Furthermore, the effect of recombinant human erythropoietin (rHuEpo) on the DNA synthesis in RBECs was analyzed. rHuEpo showed a dose-dependent mitogenic action on RBECs as a competence factor. Radioiodinated rHuEpo was bound specifically to RBECs with time, cell number and dose dependencies. Binding studies with 125I-rHuEpo showed that RBECs had a single class of receptors with low-affinity (Kd = 860 pM) and that the number of sites/cell (10300) was abundant. These results suggest that brain capillary endothelial cells express not only an authentic form of Epo-R but also a soluble form of Epo-R and that erythropoietin acts directly on brain capillary endothelial cells as a competence factor.
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Congenital aganglionosis rat is a mutant with an autosomal recessive gene (sl). Recent studies have revealed that the endothelin ETB receptor gene of sl/sl rat has a deletion of 301-bp region spanning exon 1 and intron 1 corresponding to the first and the second transmembrane domains of the receptor. In the present experiments, we examined the functions of ETB receptors in the sl/sl rats. In the membranes of cerebellum, heart, and lung of control (+/+ and sl/+) rats, ET-1 induced a monophasic, competitive displacement of [125I]ET-1 binding, whereas ET-3, IRL 1620, and BQ-123 showed biphasic displacement. In the membranes of sl/sl rats, in contrast, ET-1, BQ-123, ET-3, and IRL 1620 showed only monophasic displacement. Scatchard analysis revealed a single [125I]ET-3 binding site in the membrane of control heart but not in the sl/sl rat heart, and the specific binding sites for [125I]ET-1 in both control and sl/sl rat hearts. In the control rat aorta but not in the sl/sl rat aorta, ET-3 induced endothelium-dependent relaxation. These results suggest that sl/sl rats do not have functional ETB receptors.
We detected a novel nuclear protein, MRF, that binds to multiple sites on the modulator which is located upstream of the human cytomegalovirus major immediate early gene enhancer. The expression of MRF is differentiation specific; the DNA binding activity is present in nuclear extracts from undifferentiated Tera-2 and THP-1 cells, but significantly reduced after these cells are induced to differentiate. In undifferentiated cells the enhancer activity is repressed by the modulator and upon differentiation the enhancer becomes active. Competitive binding assays demonstrate that MRF requires the presence of multiple A+T stretches for binding to DNA, rather than binding to a specific DNA sequence. Mutations of these stretches in the modulator reduce the binding activity of MRF, as well as the repressing activity on the enhancer. These results suggest that MRF may act as a repressor of enhancer function. We propose that MRF binds over the entire modulator and exerts repressor activity.
B-Raf is regulated by Ras protein and acts as a mitogen-activated protein (MAP) kinase kinase kinase in PC12 cells and brain. Ras protein undergoes a series of post-translational modifications on its C-terminal CAAX motif, and the modifications are critical for its function. To elucidate the role of the post-translational modifications in interaction with, and activation of, B-Raf, we have analyzed a direct association between H-Ras and B-Raf, and constructed an in vitro system for B-Raf activation by H-Ras. By using methods based on inhibition of yeast adenylyl cyclase or RasGAP activity and by in vitro binding assays, we have shown that the segment of B-Raf corresponding to amino acid 1-326 binds directly to H-Ras with a dissociation constant (Kd) comparable to that of Raf-1 and that the binding is not significantly affected by the post-translational modifications. However, when the activity of B-Raf to stimulate MAP kinase was measured by using a cell-free system derived from rat brain cytosol, we observed that the unmodified form of H-Ras possesses an almost negligible activity to activate B-Raf in vitro compared to the fully modified form. H-RasSer-181,184 mutant, which was farnesylated but not palmitoylated, was equally active as the fully modified form. These results indicate that the post-translational modifications, especially farnesylation, are required for H-Ras to activate B-Raf even though they have no apparent effect on the binding properties of H-Ras to B-Raf.
The 190 kDa outer membrane protein of Rickettsia rickettsii is a major immunodominant protective antigen which contains 13 tandem nearly identical repeating sequences. We have identified a deletion in the 190 kDa antigen gene of R. rickettsii strain Smith maintained in the laboratory. The deletion occurred within the repeat region of this gene and the mutated repeat region corresponded to one repeating unit in size.
BACKGROUND: Vogt-Koyanagi-Harada disease (VKH) consists of uveitis with associated non-ocular symptoms, such as vitiligo or central nervous system disorders. A cell-mediated immune disorder is believed to play an important role in VKH; however, the skin lesion has not been well studied. Therefore, for this study, we examined the skin of a 56-year-old Japanese man with Vogt-Koyanagi-Harada disease (VKH). METHODS: Skin biopsy specimens from the patient's vitiligo were obtained 1 month after the onset of ocular symptoms. Immunohistochemical analysis of the specimens study was performed using the following monoclonal antibodies: anti-T cell, anti-B cell, anti-major histocompatibility complex class II (HLA-DR), anti-T helper/inducer (CD4), and anti-T suppressor/ cytotoxic lymphocytes (CD8). RESULTS: Histopathologic analysis revealed mononuclear cell infiltration of the slightly edematous dermis, especially surrounding the hair follicles and sweat glands. Melaninladen cells in the epidermis were partially lost. The infiltrating mononuclear cells consisted primarily of T lymphocytes with a smaller number of B lymphocytes. Most showed expression of HLA-DR. CD4-positive lymphocytes were dominant over CD8-positive cells (3:1). CONCLUSIONS: The results indicate that vitiligo of VKH is infiltrated by mostly activated helper/inducer lymphocytes and that cell-mediated immunity plays an important role in the pathogenesis of the dermal lesions of VKH as well.
The effects of ultraviolet A (UVA) radiation and reactive oxygen species (ROS), generated with a xanthine and xanthine oxidase (XOD) system, on collagen enzymatic degradation involving the matrix metalloproteinase (MMP) and its tissue inhibitor of metalloproteinase (TIMP) were investigated using cultured human dermal fibroblasts. Total RNA was isolated and subjected to Northern blot analysis using cDNA clones for human interstitial collagenase (MMP-1), 72-kDa type IV collagenase (MMP-2) and TIMP-2. UVA irradiation resulted in an increase in MMP-1 mRNA up to 2.3-fold, but did not stimulate MMP-2 or TIMP-2 mRNA expression. In contrast, ROS induced by the xanthine and XOD system resulted in a dose-related increase in the level of MMP-2 mRNA up to 2.1-fold and a decrease in the level of TIMP-2 mRNA by 49% in the same fibroblasts. Catalase, used as scavenger, essentially prevented the ROS-induced alterations in MMP-2 and TIMP-2 mRNA levels. These results suggest that ROS produced in the dermis may contribute to biological changes in the connective tissue matrix observed in photoaging skin by accelerating the MMP-2-related matrix degradation system.
OBJECTIVE: In vascular strips, hydrogen peroxide (H2O2) relaxes alpha 1-adrenergic agonist-induced but not high-K(+)-induced contractions. The aim of this study was to explore H2O2-induced changes in [Ca2+]i of vascular smooth muscle and to elucidate the mechanisms of action of H2O2. METHODS: Isolated rabbit aortic strips were isometrically contracted with high-K+ (64.7 mM) or phenylephrine (PE, 0.3 microM). The effects of 300 microM H2O2 on [Ca2+]i of endothelium-denuded vascular smooth muscle and tension were determined simultaneously by the fura-2 method. Changes in [Ca2+]i were expressed as percentages of high-K(+)-induced values measured at the beginning of the experiments. In another series of experiments, the relaxant effect of 300 microM H2O2 was examined in high-K+ (20 mM)-induced contraction in the presence of the protein kinase C activator, phorbol 12,13-dibutyrate (PDBu). RESULTS: Hydrogen peroxide caused a reversible rise in [Ca2+]i of vascular smooth muscle under both resting conditions and in the precontracted state. During high-K(+)-induced contraction, H2O2 further increased [Ca2+]i by 26.6(s.e.m. 1.7)% accompanied by a small increase in tension of 6.5(1.9)% of high-K(+)-induced tension. By contrast, during PE-induced contraction, although H2O2 caused a comparable additional increase in [Ca2+]i (26.4(4.7)%), muscle tension fell by 28.9(2.2)% of the steady-state PE-induced tension. Hydrogen peroxide had a relaxant effect on augmented high-K(+)-induced contraction in which Ca2+ sensitivity of the contractile apparatus was elevated by PDBu. CONCLUSIONS: In spite of its effect of increasing [Ca2+]i of vascular smooth muscle, hydrogen peroxide causes relaxation of endothelium-denuded, PE-precontracted rabbit aorta. The mechanism is probably through suppression of agonist-induced augmentation of Ca2+ sensitivity of the contractile apparatus.