Biomedical subjects
M Takeuchi
Publications and source records attributed to M Takeuchi.
[The significance of Ulex europaeus agglutinin I lectin binding fibers in various muscular diseases].
In the present study, we have reported that Ulex europaeus agglutinin I (UEA I) lectin labeled muscle fibers in distal myopathy with rimmed vacuole formation (DMRV). UEA I binding to muscle fibers was also observed in a small number of biopsies with inflammatory myopathy, but not in other diseases, including neurogenic muscular atrophies and muscular dystrophies. In order to elucidate the relationship between this UEA I binding, rimmed vacuole formation and active autophagocytosis, we examined the UEA I binding fibers in other myopathies which frequently showed rimmed vacuoles, including adult onset acid maltase deficiency, oculo-pharyngo-distal type myopathy and oculopharyngeal muscular dystrophy. No UEA I lectin labeling fiber was observed in the diseases examined. We then studied UEA I binding behavior on 70 biopsies of inflammatory myopathy to characterize the clinical features of UEA I binding positive patients. UEA I binding fibers were observed in 3 of 28 patients (11%) with other collagen diseases, 11 of 36 (31%) without these disorders, and 2 of 6 (33%) with inclusion body myositis. There were no common clinical histories, complications or laboratory findings among the UEA I binding positive patients. In conclusion, a common process may exist between the muscle fiber degeneration in DMRV and subgroups of inflammatory myopathy patients, but the basic mechanism remains to be elucidated.
Purification and characterization of UDP-N-acetylglucosamine: alpha1,3-D-mannoside beta1,4-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase-IV) from bovine small intestine.
A new beta1,4-N-acetylglucosaminyltransferase (GnT) which involves in branch formation of Asn-linked complex-type sugar chains has been purified 224,000-fold from bovine small intestine. This enzyme requires divalent cations, such as Mn2+, and catalyzes the transfer of GlcNAc from UDP-GlcNAc to biantennary oligosaccharide and produces triantennary oligosaccharide with the beta1-4-linked GlcNAc residue on the Manalpha1-3 arm. The purified enzyme shows a single band of Mr 58,000 and behaves as a monomer. The substrate specificity demonstrated that the beta1-2-linked GlcNAc residue on the Manalpha1-3 arm (GnT-I product) is essential for the enzyme activity. beta1-4-Galactosylaion to this essential beta1-2-linked GlcNAc residue or N-acetylglucosaminylation to the beta-linked Man residue (bisecting GlcNAc, GnT-III product) blocks the enzyme action, while beta1-6-N-acetylglucosaminylation to the Manalpha1-6 arm (GnT-V product) increases the transfer. Based on these findings, we conclude that the purified enzyme is UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV (GnT-IV), that has been a missing link on biosynthesis of complex-type sugar chains.
Binding of neuroligins to PSD-95.
PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins.
Interleukin-18 induces activation and association of p56(lck) and MAPK in a murine TH1 clone.
Interleukin-18 (IL-18) was identified as an inducer of interferon-gamma (IFN-gamma) production by stimulated T cells. In this study, we used an ovalbumin-responsive murine Th1 clone (OVA#4), in which DNA synthesis was reportedly enhanced after IL-18 treatment in the presence of a non-mitogenic TCR/CD3 stimulus, to examine signal transduction pathways. In the presence of the stimulus, IL-18 induced the appearance of tyrosine-phosphorylated proteins and herbimycin A inhibited DNA synthesis. It is suggested that protein tyrosine kinase (PTK) mediated signaling is induced by IL-18. Specifically, IL-18 induced phosphorylation of phosphorylates p56(lck) (LCK) and mitogen-activated protein kinase (MAPK). IL-18 alone induced the kinase activities of both LCK and MAPK, and the activities were increased by the TCR/CD3 stimulus. Simultaneously, IL-18 induced the association of LCK with MAPK and this was also increased by the TCR/CD3 stimulus. The activation of the LCK-MAPK pathway correlated with enhanced DNA synthesis in OVA#4 cells. These results suggest that the LCK-MAPK pathway is involved in IL-18 signaling and that IL-18 may play an important role in modification of TCR/CD3-mediated response.
Treatment with a new synthetic retinoid, Am80, of acute promyelocytic leukemia relapsed from complete remission induced by all-trans retinoic acid.
Differentiation therapy with all-trans retinoic acid (ATRA) has marked a major advance and become the first choice drug in the treatment of acute promyelocytic leukemia (APL). However, patients who relapse from ATRA-induced complete remission (CR) have difficulty in obtaining a second CR with a second course of ATRA therapy alone. We tested the efficacy of a new synthetic retinoid, Am80, in APL that had relapsed from CR induced by ATRA in a prospective multicenter study. Am80 is approximately 10 times more potent than ATRA as an in vitro differentiation inducer, is more stable to light, heat, and oxidation than ATRA, has a low affinity for cellular retinoic acid binding protein, and does not bind to retinoic acid receptor-gamma. Patients received Am80, 6 mg/m2, orally alone daily until CR. Of 24 evaluable patients, 14 (58%) achieved CR. The interval from the last ATRA therapy was not different between CR and failure cases. The clinical response was well correlated with the in vitro response to Am80 in patients examined. Adverse events included 1 retinoic acid syndrome, 1 hyperleukocytosis, 9 xerosis, 8 cheilitis, 16 hypertriglyceridemia, and 15 hypercholesterolemia, but generally milder than those of ATRA, which all patients had received previously. Am80 is effective in APL relapsed from ATRA-induced CR and deserves further trials, especially in combination with chemotherapy.
Discovery of the shortest sequence motif for high level mucin-type O-glycosylation.
The consensus primary amino acid sequence for mucin-type O-glycosylation sites has not been identified. To determine the shortest motif sequence required for high level mucin-type O-glycosylation, we prepared more than 100 synthetic peptides and assayed in vitro O-GalNAc transfer to serine or threonine in these peptides using a bovine colostrum UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyl transferase (O-GalNAcT). We chose the sequence PDAASAAP from human erythropoietin (hEPO) for further systematic substitutions because it accepted GalNAc and was a fairly simple sequence consisting only of four kinds of amino acids. Several substitutions showed that threonine is approximately 40-fold better than serine as the glycosylated amino acid and a proline at position +3 on the C-terminal side is very important. To define the effect of proline residues around the glycosylation site, we analyzed a series of peptides containing one to three proline residues in a parent peptide AAATAAA. The results clearly indicated that prolines at positions +1 and +3 had a positive effect. The O-GalNAc transfer level of AAATPAP was increased approximately 90-fold from AAATAAA. The deletion of amino acids from the N-terminal side of the glycosylation site suggested that five amino acids from position -1 to +3 were especially important for glycosylation. Moreover, the influence of all 20 amino acids at positions -1, +2, and +4 was analyzed. Uncharged amino acids were preferred at position -1, and small or positively charged amino acids were preferred at position +2. No preference was observed at position +4. We propose a mucin-type O-glycosylation motif, XTPXP, which may be suitable as a signal for protein O-glycosylation. The features observed in this study also appear to be very useful for prediction of mucin-type O-glycosylation sites in glycoproteins.
Interleukin-18 activates NF-kappaB in murine T helper type 1 cells.
Interleukin-18 (IL-18) activated T helper type 1 (Th1) cells, OVA#4, and induced production of interleukin-2 (IL-2) in costimulation with anti-CD3 antibody. Upon stimulation with IL-18, IkappaB disappeared from cytoplasm and subsequently nuclear factor-kappaB (NF-kappaB) (p65) accumulated in the nucleus. Corresponding with that, DNA binding activity of NF-kappaB (p65 homodimer or p65/p50 heterodimer) was detected in the nucleus. In the transfection experiments, an IL-2 promoter-driven reporter construct showed the similar responsiveness against IL-18 to that of the intrinsic IL-2 gene, and a construct lacking kappaB site failed to respond to IL-18. These results suggest that IL-18 activates NF-kappaB and it is important for enhancement of IL-2 gene expression by Th1 cells stimulated with IL-18.
Preparation and biological activity of manno- and galacto-validamines, new 5a-carba-glycosylamines as alpha-glycosidase inhibitors.
Manno- and galacto-validamines, which are epimers of validamine, were semi-synthesized by the configurational inversion of validamine, a pseudo-sugar analogue of alpha-D-gluco-pyranose that has inhibitory activity for alpha-glucosidases. The inhibitory activities of these analogues were determined against several mannosidases and galactosidase. Manno-validamine shows potent inhibition for the alpha-mannosidases (competitive. K(i) = 4.6 x 10(-5) M for jack beans, and competitive, Ki = 2.8 x 10(-5) M for almonds), and galacto-validamine shows weak inhibition for the alpha-galactosidases (coffee bean and E. coli). The inhibitory effect of the epimers on the N-linked oligosaccharide-processing mannosidases involved in glycoprotein biosynthesis and lysosomal mannosidase from rat liver were also examined. Mannovalidamine shows potent inhibition on the endoplasmic reticulumal alpha-mannosidase (competitive, K(i) = 1.2 x 10(-6) M), Golgi mannosidases IA, II (competitive, K(i) = 2.8 x 10(-5) M), and lysosomal alpha-mannosidase (competitive, Ki = 1.7 x 10(-5) M).
SAPAPs. A family of PSD-95/SAP90-associated proteins localized at postsynaptic density.
PSD-95/SAP90 is a member of membrane-associated guanylate kinases localized at postsynaptic density (PSD) in neuronal cells. Membrane-associated guanylate kinases are a family of signaling molecules expressed at various submembrane domains which have the PDZ (DHR) domains, the SH3 domain, and the guanylate kinase domain. PSD-95/SAP90 interacts with N-methyl-D-aspartate receptors 2A/B, Shaker-type potassium channels, and brain nitric oxide synthase through the PDZ (DHR) domains and clusters these molecules at synaptic junctions. However, neither the function of the SH3 domain or the guanylate kinase domain of PSD-95/SAP90, nor the protein interacting with these domains has been identified. We have isolated here a novel protein family consisting of at least four members which specifically interact with PSD-95/SAP90 and its related proteins through the guanylate kinase domain, and named these proteins SAPAPs (SAP90/PSD-95-Associated Proteins). SAPAPs are specifically expressed in neuronal cells and enriched in the PSD fraction. SAPAPs induce the enrichment of PSD-95/SAP90 to the plasma membrane in transfected cells. Thus, SAPAPs may have a potential activity to maintain the structure of PSD by concentrating its components to the membrane area.
Histological study of urinary bladder transplantation in rats.
Patients who require cystectomy are usually treated with an ileal conduit or intestinal neobladder for urinary control. In some of them, however, the bowel segment cannot be used because of previous abdominal surgery or radiation treatment. Bladder transplantation from cadavers may be beneficial to these patients, if possible. To obtain basic knowledge about bladder transplantation, we developed an animal model of whole bladder transplantation in rats. Male Lewis rats weighing 270-320 g were used as both donors and recipients. Of the 23 recipients, 12 (52.2%) survived 7 days or longer after surgery. At 1 week after transplantation, the bladder showed loss of transitional epithelium and remarkable cellular infiltration. In the bladder at 5 weeks after transplantation, the transitional epithelium regenerated markedly and submucosal cellular infiltration was much improved. Regeneration of some smooth muscle cells was also noted. At 6 months after transplantation, the nerve fibers were recognized in the bladder and the volume of the transplanted bladder was well preserved (1.0-1.3 ml). This article describes an animal model of whole bladder transplantation in the rat which we produced and the results of our study. Because a large number of pure-bred animals can easily be used, we believe our rat model is very useful for basic studies of bladder transplantation.
Oxygen-saving effect of a new cardiotonic agent, MCI-154, in diseased human hearts.
OBJECTIVES: The aim of this study was to examine the left ventricular mechanoenergetic effects of a novel Ca2+ sensitizing agent, MCI-154, on diseased human hearts compared with dobutamine. BACKGROUND: Unlike conventional cardiotonic agents, a Ca2+ sensitizer that could produce a positive inotropic action by altering the responsiveness of myofilament to Ca2+ could generate force with smaller amounts of Ca2+; thus, it may potentially save energy expenditure. METHODS: The left ventricular pressure-volume relation and myocardial oxygen consumption per beat (Vo2) were measured by a conductance (volume) catheter and a Webster catheter. Left ventricular contractility (Emax), systolic pressure-volume area (PVA [index of left ventricular total mechanical energy]) and Vo2 were assessed before and after infusion of MCI-154 or dobut-amine. The PVA-independent Vo2 (Vo2 mainly for excitation-contraction coupling) was assessed as the Vo2 at zero PVA. RESULTS: Both agents increased Emax comparably (dobutamine: from 3.55 +/- 1.10 [mean +/- SD] to 5.04 +/- 1.16 mm Hg/ml per m2, p < 0.0001; MCI-154: from 3.36 +/- 1.26 to 5.37 +/- 2.14 mm Hg/ml per m2, p < 0.0001); dobutamine increased total Vo2 (from 0.22 +/- 0.08 to 0.27 +/- 0.09 ml O2, p < 0.05) and PVA-independent Vo2 (from 0.019 +/- 0.019 to 0.091 +/- 0.051 ml O2, p < 0.005); but MCI-154 did not change these variables significantly. Consequently, the oxygen cost of contractility (delta PVA-independent Vo2/delta Emax) was less with MCI-154 than with dobutamine (0.14 +/- 0.18 vs. 1.10 +/- 0.80 J/mm Hg per ml per m2, p < 0.05). CONCLUSIONS: These results suggest that the cardiotonic action mediated by MCI-154 could provide an energetic advantage over the conventional cardiotonic action with currently used inotropic agents.
Effect of aortic valve replacement on coronary flow velocity during metabolic stress in a patient with aortic stenosis.
The effect of aortic valve replacement on coronary flow velocity during atrial pacing and papaverine-induced-resistance vessel dilatation was tested in a patient with aortic stenosis. Although systolic flow reversal disappeared early after the valve replacement, rapid atrial pacing caused myocardial ischemia with lactate production. The coronary flow reserve also remained depressed. These results suggest that the alteration in the coronary flow profile early after the aortic valve replacement does not reflect an improvement in the flow increase during metabolic stress in a patient with aortic stenosis.
Comparative effects of dobutamine and amrinone on coronary blood flow in patients with idiopathic dilated cardiomyopathy.
Although amrinone has favorable hemodynamic effects in patients with congestive heart failure, little is known about its effects on coronary blood flow (CBF). We compared the effects of intravenous low-dose dobutamine and amrinone on CBF in 10 patients with dilated cardiomyopathy using a Doppler guidewire. We infused dobutamine at a dose of 5 and 10 microg/kg/min for 5 min. After the end of each stage, coronary flow velocity (CFV) and coronary arterial diameter (CAD) in the proximal left anterior descending coronary artery, and hemodynamic variables were obtained. After the CFV and hemodynamics returned to baseline, we infused 1 mg/kg of amrinone over 5 min, and obtained these variables at 5 and 10 min after the cessation of the infusion. CAD did not increase with dobutamine, but significantly increased after amrinone (% increase: 10 +/- 7%; P < 0.001 vs. baseline). CFV progressively increased with dobutamine (5 microg/kg/min: 21 +/- 26%; P < 0.05 vs. baseline; 10 microg/kg/min: 53 +/- 42%; P < 0.005 vs. baseline and 5 microg/kg/min), but slightly decreased after amrinone (-4 +/- 17%; P = not significant vs. baseline). CBF increased during dobutamine (5 microg/kg/min: 25 +/- 29%; P < 0.05; 10 microg/kg/min: 66 +/- 55%; P < 0.005) and after amrinone (19 +/- 22%; P < 0.05) compared to that at baseline. Although there was a significant correlation between the percent increase in CFV and that in dP/dt during dobutamine infusion (r = 0.82, P < 0.001), this correlation was not observed after amrinone (r = 0.23). In conclusion, although both agents significantly increased CBF in patients with dilated cardiomyopathy, they do so by different mechanisms. Amrinone mainly increases CBF by causing dilatation of epicardial coronary arteries. These results suggest that amrinone has beneficial effects on coronary flow dynamics in dilated cardiomyopathy.
Acute myocardial infarction in a patient during dobutamine stress echocardiography.
We report the case of a patient who developed an acute anteroseptal and inferior myocardial infarction during dobutamine stress echocardiography. The proposed mechanism for this event is discussed based on results obtained during emergency coronary angioplasty and subsequent clinical findings.
Coronary angioplasty of a severe coronary stenosis at the site of a myocardial bridge.
We report the case of a patient who underwent coronary angioplasty for a severe stenosis at the site of a myocardial bridge. Although favorable angiographic results and the disappearance of myocardial ischemia were obtained, a significant pressure gradient across the lesion and abnormal myocardial fractional flow reserve remained immediately following angioplasty. These results suggest that pressure measurements may be of limited value in this subset of patients.
Measurement of myocardial fractional flow reserve during coronary angioplasty in patients with old myocardial infarction.
Although myocardial fractional flow reserve (FFRmyo) has been demonstrated to be a useful index for determining functional significance of coronary stenosis, the data in previous studies was derived from a highly selected group of patients. The aim of this study was to investigate the value of FFRmyo in a more clinically relevant group of patients, especially in patients who also had resistance vessel dysfunction. We measured FFRmyo in 20 consecutive patients who had undergone elective coronary angioplasty. FFRmyo was calculated by the ratio of Pc/Pa during intracoronary adenosine 5'-triphosphate (ATP; 50 micrograms in the left coronary and 20 micrograms in the right coronary artery) induced maximal hyperemia, where Pa represents mean aortic pressure obtained by the guiding catheter and Pc represents mean distal coronary pressure measured by a 2.1 F infusion catheter. In total, 21 vessels were dilated and 14 of them were infarct-related arteries. The percent diameter stenosis significantly decreased from 80 +/- 14% to 27 +/- 17%, and the FFRmyo increased significantly from 0.46 +/- 0.18 to 0.77 +/- 0.15 after angioplasty. There was no significant differences in the FFRmyo between vessels with previous myocardial infarction and those without, after angioplasty (0.78 +/- 0.18 vs. 0.76 +/- 0.08). There was a significant correlation between the percent diameter stenosis and FFRmyo before (r = 0.83, P < 0.001) and after (r = 0.64, P < 0.01) angioplasty. In conclusion, FFRmyo significantly improved immediately after angioplasty in vessels with myocardial infarction as well as those without. These results led us to suggest the usefulness of FFRmyo in patients who had both epicardial stenosis and resistance vessel dysfunction. The significant correlation between FFRmyo and quantitative coronary arterial diameter stenosis would further support the more widespread use of FFRmyo in the clinical setting.
On the mechanisms by which transforming growth factor-beta 2 alters antigen-presenting abilities of macrophages on T cell activation.
Peritoneal exudate cells (PEC) incubated with antigen in the presence of transforming growth factor-(TGF)-beta 2 selectively suppress delayed hypersensitivity and IgG2a antibody production when injected intravenously into naive syngeneic recipients. In this study, we have examined in vitro the effects of TGF-beta 2 on the antigen presenting abilities of PEC to activate DO11.10 T cells that express a transgenic T cell receptor that recognizes ovalbumin peptide fragment 323-339 in the context of I-Ad. PEC were pretreated overnight with TGF-beta 2, washed extensively, then co-cultured with DO11.10 T cells in the presence of native OVA or P323-339. We found that TGF-beta 2-treated PEC induced the production of the T helper type 2 (Th2) cytokine, interleukin-4 (IL-4), but unlike untreated PEC, were unable to stimulate the Th1 cytokines, IL-2 and interferon-gamma (IFN-gamma). Furthermore, TGF-beta 2 was produced in an autocrine fashion by TGF-beta 2-treated PEC and was responsible for this shift to a Th2 response. This conclusion was supported by the following results. First, TGF-beta 2-treated PEC were found to express much more TGF-beta 1 and TGF-beta 2 mRNA than untreated PEC. Second, TGF-beta 2-treated PEC secreted large amounts of TGF-beta including its mature form. Third, addition of neutralizing anti-TGF-beta 2 antibodies, but not neutralizing anti-TGF-beta 1 antibodies, restored the ability of antigen-pulsed, TGF-beta 2-pretreated PEC to stimulate DO11.10 T cells to secrete IL-2 and IFN-gamma. These results indicate that antigen-presenting cells that encounter antigen in a TGF-beta-enriched environment (e.g., in the eye) shift responding native T cells toward Th2 responses by producing TGF-beta during antigen presentation.