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Biomedical subjects

S Nakamura

Publications and source records attributed to S Nakamura.

At least 361 records · Page 20Linked to original sources
PubMed2000

Enthalpic destabilization of glycosylated lysozymes constructed by genetic modification.

To understand the role of polyglycosylation in protein stability, the thermodynamic changes in the denaturation of various polymannosyl lysozyme mutants (R21T, G49N, R21T/G49N) constructed by genetic modification were analyzed using differential scanning calorimetry (DSC). The denaturation temperature and the enthalpy change for unfolding of the lysozymes were reduced with an increase in the length of the polymannose chain and the number of binding sites to a protein, although the polymannosyl lysozymes revealed apparent heat stability in that no aggregation was observed and the enzymatic activity was conserved under conditions in which the wild-type lysozyme coagulated [S. Nakamura et al., J. Biol. Chem. 268 (1993) 12706-12712]. The reversibility of the denaturation of polymannosyl lysozymes was observed in the DSC curves obtained by reheating after heat denaturation, while it was not observed for the wild-type lysozyme. Based on these results, the polymannosyl lysozyme seems to easily refold due to the excellent reversibility of denaturation, despite the decreases in the enthalpic stabilization due to the strain in the protein molecule by the introduction of a polysaccharide chain.

Binding Sites↗
PubMed2000

Bovine lactoperoxidase and its recombinant: comparison of structure and some biochemical properties.

Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined.

Animals↗
PubMed2000

Neocortical origin and tangential migration of guidepost neurons in the lateral olfactory tract.

The early-generated neurons designated as lot cells specifically mark the future site of the lateral olfactory tract (LOT) and guide LOT axons. We investigated the mechanism of how lot cells develop and get localized in the LOT position. Lot cells differentiated from neuroepithelial cells in all regions of the neocortex but not from those in the ganglionic eminence in culture. Cell tracing analyses demonstrated that lot cells generated from the neocortex subsequently followed a tangential migration stream ventrally toward the LOT position. Mutant mouse embryos lacking the function of transcription factor Gli3 showed disturbances of the migration stream and translocation of lot cells in the dorsal telencephalon. These results reveal a new type of neuronal migration in the telencephalon and introduce an unexpected dramatic feature of the earliest regionalization of the telencephalon.

Animals↗
PubMed2000

In vivo electrical activity of brainstem neurons in fetal rats during asphyxia.

To see changes in the activity and the sensitivity to glutamate of fetal brain neurons during asphyxia, the electrical activity of brainstem neurons was recorded extracellularly in fetal rats which were still connected with the dams by the intact umbilical cord. In urethan-anesthetized pregnant rats, fetal asphyxia (2-10 min) was induced by occluding the umbilical cord with a surgical clip, while reperfusion of the umbilical blood flow was performed by local application of a relaxant of blood vessels to the occlusion site. The spontaneous discharge of fetal brainstem neurons was suppressed for a long period of time by umbilical cord occlusion. The suppression of the firing occurred 48-150 (78+/-7) s after the start of umbilical cord occlusion, and lasted even after fetal cortical PO(2) recovered to control level after reperfusion. The changes occurred with a marked reduction in spike amplitude. A similar suppression was observed for the spikes induced by iontophoretic application of glutamate, although fetal brainstem neurons were extremely sensitive to glutamate before asphyxia. The suppression of the spontaneous spikes became more notable and longer when asphyxia was repeated. These findings suggest that the long-lasting suppression of fetal neurons during asphyxia may contribute to a reduction of cellular energy requirements in the fetal brain, thereby playing a role in the resistance of fetal neurons to brain damage caused by asphyxia. Furthermore, the reduced sensitivity of fetal neurons to glutamate during asphyxia may also contribute to prevent brain damage due to excitotoxity of glutamate.

Action Potentials↗
PubMed2000

Analysis of genes under the downstream control of the t(8;21) fusion protein AML1-MTG8: overexpression of the TIS11b (ERF-1, cMG1) gene induces myeloid cell proliferation in response to G-CSF.

The AML1-MTG8 fusion transcription factor generated by t(8;21) translocation is thought to dysregulate genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors to cause acute myelogenous leukemia (AML). Although AML1-MTG8 has been shown to repress the transcription of AML1 targets, none of the known targets of AML1 are probably responsible for AML1-MTG8-mediated leukemogenesis. In this study, 24 genes under the downstream control of AML1-MTG8 were isolated by using a differential display technique. Analysis with deletion mutants of AML1-MTG8 demonstrated that the regulation of the majority of these genes requires the region of 51 residues (488-538) containing the Nervy homology region 2 (NHR2), through which AML1-MTG8 interacts with MTGR1. Among the 24 genes identified, 10 were considered to be genes under the control of AML1, because their expression was altered by AML1b or AML1a or both. However, the other 14 genes were not affected by either AML1b or AML1a, suggesting the possibility that AML1-MTG8 regulates a number of specific target genes that are not normally regulated by AML1. Furthermore, an up-regulated gene, TIS11b (ERF-1, cMG1), was highly expressed in t(8;21) leukemic cells, and the overexpression of TIS11b induced myeloid cell proliferation in response to granulocyte colony-stimulating factor. These results suggest that the high-level expression of TIS11b contributes to AML1-MTG8-mediated leukemogenesis. (Blood. 2000;96:655-663)

Animals↗
PubMed2000

Morphological and functional changes of rabbit mesenteric artery cultured with fetal bovine serum.

The aim of this study was to examine the morphological and functional changes in rabbit mesenteric arterial tissue cultured with fetal bovine serum. In the endothelium-denuded arteries cultured under a serum-free condition for one week (serum-free arteries), morphology of the smooth muscle layer was intact. In the serum-free arteries, high K+ -induced contraction did not change but norepinephrine-induced contraction slightly decreased compared with that in the freshly isolated arteries, whereas the sensitivity to these stimulants was significantly augmented. In the medial layer of the arteries cultured with 10% fetal bovine serum for one week (serum-treated arteries), proliferation, disorientation and death of smooth muscle cells were observed. In the serum-treated arteries, both the amplitude of contractions induced by high K+ and norepinephrine and the sensitivity to these stimulants were significantly reduced compared with those of the serum-free arteries. The reduced norepinephrine-induced contraction in the serum-treated arteries was partially recovered by adding NG-monomethyl-L-arginine (L-NMMA), a nitric oxide (NO) synthase inhibitor, to the assay medium. In alpha-toxin permeabilized arteries, the amplitude of Ca2+ -induced contraction and the sensitivity of the contractile apparatus to Ca2+ were significantly reduced after serum-treatment. These results suggest that chronic serum-treatment of rabbit mesenteric arteries impairs muscle contractility by the morphological and phenotypic changes in smooth muscle cells. NO production in smooth muscle cells is also responsible for the decreased contractility after the serum-treatment.

Animals↗
PubMed2000

Protein-tyrosine kinase Pyk2 is involved in interleukin-2 production by Jurkat T cells via its tyrosine 402.

We established Jurkat transfectants that overexpress Pyk2 or its mutants, K457A (lysine 457 was mutated to alanine), Pyk2-Y402F (tyrosine 402 to phenylalanine), and Pyk2-Y881F to investigate the role of Pyk2 in T cell activation. Pyk2 as well as kinase-inactive Pyk2-K457A, was phosphorylated at tyrosine residues 402, 580, and 881 upon T cell antigen receptor cross-linking, indicating that these residues are phosphorylated by other tyrosine kinase(s). However, no tyrosine phosphorylation of Pyk2-Y402F was detected while more than 60% of the tyrosine phosphorylation was observed in Pyk2-Y881F. Pyk2-Y402F inhibited the activation of endogenous Pyk2. The degree of activation of both c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinase but not extracellular signal-regulated protein kinase after concurrent ligation of T cell antigen receptor and CD28 was reduced by more than 50% in the clones expressing Pyk2-Y402F. Consistent with this inhibition, IL-2 production was significantly diminished in the Pyk2-Y402F-expressing clones. Furthermore, we found that Pyk2, when overexpressed, associates with Zap70 and Vav. Taken together, these findings suggest that Pyk2 is involved in the activation of T cells through its tyrosine 402.

CD28 Antigens↗
PubMed2000

Down syndrome cell adhesion molecule DSCAM mediates homophilic intercellular adhesion.

Down Syndrome (DS) caused by trisomy 21 is the most common birth defect associated with mental retardation. Recently, a novel gene named, DSCAM, has been identified in the DS critical region. DSCAM is predicted to be a transmembrane protein with a very high structural and sequence homology to Ig superfamily of cell adhesion molecules and is expressed in the developing nervous system with the highest level in fetal brain. Diverse glycoproteins of cell surfaces and extracellular matrices operationally termed as 'adhesion molecule' are important in the specification of cell interactions during development, maintenance and regeneration of the nervous system. To understand the cellular function of DSCAM protein, we transfected human DSCAM cDNA into mouse fibroblast L cells and analysed its expression. On Western blot analysis, antibodies raised against recombinant DSCAM-Ig3 recognized a 198 kDa protein band in the membrane fraction of DSCAM transfected L cells. Stable transformants expressing DSCAM showed uniform surface expression. DSCAM-expressing transfectants exhibited enhanced adhesive properties, aggregating with faster kinetics and forming aggregates in a homophilic manner. Divalent cations are not required for this cell aggregation. These results demonstrate that DSCAM is a cell adhesion molecule that can mediate cation-independent homophilic binding activity between DSCAM expressing cells.

Animals↗
PubMed2000

The roles of platelet-activating factor and endothelin-1 in renal damage after total hepatic ischemia and reperfusion.

BACKGROUND: This study was designed to verify the involvement of platelet-activating factor (PAF) in renal damage associated with hepatic ischemia and reperfusion (HIR) injury through the release of endothelin (ET)-1 and to determine the modulating effect of a specific PAF receptor antagonist on these insults in rats. METHODS: Male rats pretreated with either normal saline as a vehicle (NS group) or intravenous TCV-309, a PAF receptor antagonist (TCV group), were subjected to 120 min of total hepatic ischemia under an extracorporeal portosystemic shunt. Plasma aspartate transaminase, creatinine, blood urea nitrogen, and ET-1 levels and the relative renal wet weight were determined under nonischemic conditions and at 1, 3, and 6 hr of reperfusion after hepatic ischemia. Changes in mean arterial blood pressure and renal tissue blood flow measurements in the kidney were determined throughout the experiment. RESULTS: Increased plasma aspartate transaminase, creatinine, blood urea nitrogen, and ET-1 levels and the relative renal wet weight after HIR in the NS group were significantly suppressed by TCV-309 pretreatment. Mean arterial blood pressure and renal tissue blood flow after HIR in the TCV group were significantly improved when compared with those in the NS group. These effects resulted in attenuation of structural hepatic and renal damage with the improvement of 7-day survival (62%). CONCLUSIONS: The present study demonstrates that renal damage as well as critical liver injury is produced after reperfusion following 120 min of total hepatic ischemia. A PAF receptor antagonist may be therapeutically useful to protect against these types of damage via indirect modulation of plasma ET-1 levels.

Animals↗
PubMed2000

Local directional cues control growth polarity of dopaminergic axons along the rostrocaudal axis.

The vertebrate CNS is composed of a variety of longitudinal axonal tracts extending rostrally and caudally. Although recent studies have demonstrated that chemoattraction and chemorepulsion play key roles in axon guidance along the circumferential axis in the neural tube of the vertebrate, mechanisms of axonal elongation along the longitudinal axis, and most importantly, what determines rostrocaudal polarity of axonal growth, remains unknown. Here, we examined the mechanism that guides midbrain dopaminergic axons rostrally, using flat whole-mount preparations of embryonic rat brain both in vivo and in vitro. At embryonic day 11 (E11) and early stage E12, dopaminergic neurons in the ventral midbrain extended short axons dorsally. By middle stage E12, these axons had increased in number, some deflecting rostrally and others caudally. At E13, almost all axons showed rostrally oriented growth heading toward the forebrain targets. In in vitro whole-mount preparations prepared from an E12 embryo and cultured for 24 hr, these axons showed rostrally oriented growth, but when they were forced to grow on substratum of reversed rostrocaudal polarity, they turned abruptly and grew following the polarity of the reversed midbrain substratum. These results suggest that local directional cues in the midbrain guide these axons rostrally and support the idea that substratum-associated polarized cues play an important role in axon guidance along the longitudinal axis.

Animals↗
PubMed2000

Inhibition of cytokinesis by a lipid metabolite, psychosine.

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.

Actins↗
PubMed2000

Cloning of rabbit alpha(1b)-adrenoceptor and pharmacological comparison of alpha(1a)-, alpha(1b)- and alpha(1d)-adrenoceptors in rabbit.

We have isolated a cDNA clone of the rabbit alpha(1b)-adrenoceptor which has an open reading frame of 1557 nucleotides encoding a protein of 518 amino acids. The sequence shows higher identity to those of hamster, human, and rat alpha(1b)-adrenoceptors than to those of rabbit alpha(1a)- and alpha(1d)-adrenoceptors. The pharmacological binding properties of this clone expressed in Cos-7 cells showed a characteristic profile as alpha(1b)-adrenoceptor; high affinity for prazosin (pK(i)=10.3), relatively high affinity for tamsulosin (9.5) and low affinity for (-)-(R)-1-(3-hydroxypropyl)-5-[2-[[2-[2-(2,2, 2-trifluoroethoxy)phenoxy]ethyl]amino]propyl]indoline-7-carboxamid e (KMD3213) (8.5), 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1, 4-benzodioxane hydrochloride (WB4101) (8.7), and 8-[2-[4-(2-methoxy-phenyl)-L-piperazinyl]-8-azaspiro[4,5]decane-7, 9-dione dihydrochloride (BMY7378) (7.3). We have compared the levels of mRNA expression of three alpha(1)-adrenoceptor subtypes in rabbit tissues using the competitive reverse transcription/polymerase chain reaction (RT/PCR) assay. In most rabbit tissues except heart, alpha(1a)-adrenoceptor mRNA was expressed 10 folds more than the other two subtypes. However, binding experiments with [3H]prazosin and [3H]KMD3213 in rabbit tissues revealed a poor relationship between binding density and mRNA level. Especially, alpha(1b) binding sites were exclusively predominant in spleen, whereas the alpha(1b) subtype was minor at the mRNA level. These results indicate a high identity of structural and pharmacological profiles of three distinct alpha(1)-adrenoceptor subtypes between rabbit and other species, but there are species differences in their distribution.

Adrenergic alpha-Antagonists↗
PubMed2000

Appropriate method of administration of propofol, fentanyl, and ketamine for patient-controlled sedation and analgesia during extracorporeal shock-wave lithotripsy.

PURPOSE: The aim of this study was to identify the appropriate method for administering propofol, fentanyl, and ketamine (PFK) for patient-controlled sedation and analgesia (PCSA) during extracorporeal shock-wave lithotripsy (ESWL). METHODS: Twenty-one unpremedicated patients were randomly assigned to three groups that received different drug administration regimens. (group 1: low loading dose and high demand bolus, group 2: high loading dose and demand bolus, group 3: high loading dose and low demand bolus). RESULTS: The patients in all groups were hemodynamically stable during ESWL. Oxygen desaturation was recognized in all groups, but was avoided by 2 l.min(-1) of oxygen supply via a nasal prong. The total administration dose of the drugs was significantly higher ( P < 0.05) in group 2 than in groups 1 and 3. The median level of sedation was the same, but the episodes of oversedation were not recognized in group 3 ( P < 0.05). A significant difference in the frequency of episodes of oversedation was found between groups 2 and 3 ( P < 0.05). The results were good or excellent for almost all patients, and were assessed as fair by only one patient in group 2. CONCLUSION: We concluded that the method used for group 3 is the most appropriate for administering PFK for PCSA during ESWL.

Clinical Trial↗
PubMed2000

Inhibitory effects of PGD2, PGJ2 and 15-deoxy-delta12,14-PGJ2 on iNOS induction in rat mesenteric artery.

PGD2 and its metabolites PGJ2 and 15-deoxy-delta12,14-PGJ2 have been reported to inhibit iNOS induction in cultured vascular smooth muscle cells. The present study was undertaken to determine whether these prostanoids inhibit iNOS induction in the isolated rat mesenteric artery. The artery without endothelium was incubated with and without lipopolysaccharide (LPS) at 37 degrees C for 6 hrs, then washed and mounted in an organ bath to measure isometric changes in tension. L-arginine but not D-arginine (10(-6) - 10(-3) M) induced concentration-dependent relaxations only in the artery preincubated with LPS, the relaxations of which were attenuated by L-N(G)-nitroarginine methyl ester (LNAME, 10(-4) M), a non-selective iNOS inhibitor, and 1400W (10(-5) and 10(-4) M), a selective iNOS inhibitor. Co-treatment of cycloheximide (10(-5) M), a protein synthesis inhibitor, or actinomycin D (10(-7) M), an RNA synthesis inhibitor with LPS inhibited the development of relaxing ability in response to L-arginine, indicating iNOS induction by LPS. PGD2, PGJ2 and 15-deoxy-delta12,14-PGJ2 but not PGE2, PGI2 or PGF2alpha also inhibited the development of relaxing ability in response to L-arginine when added during incubation with LPS. Incubation of the artery with LPS at 37 degrees C for 6 hrs markedly increased production of nitric oxide (NO), which was abolished by 15-deoxy-delta12,14-PGJ2 (10(-5) M). An imunohistochemical study using antibody against murine iNOS showed that 15-deoxy-delta12,14-PGJ2 (10(-5) M) inhibited the expression of iNOS protein in isolated rat mesenteric arteries. These results demonstrated that PGD2 and its metabolites inhibit iNOS induction by LPS in isolated rat mesenteric arteries, resulting in reduced relaxing ability in response to L-arginine.

Animals↗
PubMed2000

Geranylgeranyl-pyrophosphate, an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by 3-hydroxy-3-methylglutaryl-CoA reductase inhibition.

We investigated the role of the intrinsic mevalonate cascade in the neuronal cell death (NCD) induced by the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in rat primary cortical neurons cultured from the brains of 17-d-old fetal SD rats. HMG-CoA reductase inhibitors induced NCD [HMG-CoA reductase inhibitor-induced NCD (H-NCD)] in time- and dose-dependent manners. The apoptotic characteristics were revealed by the formation of the DNA ladder and by the electron microscopical observation. During the progression of H-NCD, p53 was induced followed by the expression of Bax. Although the mevalonate completely inhibited H-NCD, the cholesterol did not. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, prohibited H-NCD with inhibition of the induction of p53 and Bax. The inhibition of HMG-CoA reductase decreased the amount of membrane-associated Rho small GTPase families, but not Ras small GTPase, and GGPP restored the blockage by HMG-CoA reductase inhibitor in the translocation or redistribution of Rho small GTPase families to membrane. These data indicated that (1) the inhibition of the intrinsic mevalonate cascade induces the apoptotic NCD with the induction of p53 followed by that of Bax, (2) the inhibition of HMG-CoA reductase concomitantly causes blockage of the translocation or redistribution of Rho small GTPase families, not Ras small GTPase, to membrane, and (3) GGPP, not FPP, is one of the essential metabolites in the mevalonate cascade for protecting neurons from H-NCD.

Animals↗
PubMed2000

Effect of rabeprazole on histamine synthesis in enterochromaffin-like cells of mast cell-deficient (Ws/Ws) rats.

The effect of rabeprazole, the latest proton pump inhibitor, on the serum gastrin concentration, histidine decarboxylase activity and histamine content of the oxyntic mucosa in Wistar rats, mast cell-deficient (Ws/Ws) rats, and their normal type, +/+, rats was investigated. In Wistar rats, 2 weeks of treatment with rabeprazole (30 mg/kg/day, s.c.) induced a 1.8-fold increase in serum gastrin concentration and a 3.9-fold increase in histidine decarboxylase activity of the oxyntic mucosa over the control levels, whereas neither 2- nor 4-week treatment affected the histamine content of the oxyntic mucosa. In Ws/Ws and +/+ rats, the serum gastrin concentration, histidine decarboxylase activity and even histamine content of the oxyntic mucosa were increased significantly as compared with control levels after the 4-week treatment with rabeprazole. Immunohistochemistry using a histamine antibody confirmed the increase in the histamine content of the oxyntic mucosa after the 4-week treatment with rabeprazole. The finding that there were no differences in serum gastrin concentration and histidine decarboxylase activity between Ws/Ws and +/+ rats, both with and without the 4-week treatment, indicates that mast cells do not respond to endogenous hypergastrinemia elicited by acid-inhibitory treatment. Moreover, the present study clarified for the first time that enterochromaffin-like (ECL) cells in Ws/Ws rats synthesize and store histamine in response to gastrin.

2-Pyridinylmethylsulfinylbenzimidazoles↗