Biomedical subjects

T Inoue

Publications and source records attributed to T Inoue.

At least 703 records · Page 39Linked to original sources

PubMed1999

Vasopressin regulates apical targeting of aquaporin-2 but not of UT1 urea transporter in renal collecting duct.

In the renal inner medullary collecting duct (IMCD), vasopressin regulates two key transporters, namely aquaporin-2 (AQP2) and the vasopressin-regulated urea transporter (VRUT). Both are present in intracellular vesicles as well as the apical plasma membrane. Short-term regulation of AQP2 has been demonstrated to occur by vasopressin-induced trafficking of AQP2-containing vesicles to the apical plasma membrane. Here, we have carried out studies to determine whether short-term regulation of VRUT occurs by a similar process. Cell surface labeling with NHS-LC-biotin in rat IMCD suspensions revealed that vasopressin causes a dose-dependent increase in the amount of AQP2 labeled at the cell surface, whereas VRUT labeled at the cell surface did not increase in response to vasopressin. Immunoperoxidase labeling of inner medullary thin sections from Brattleboro rats treated with 1-desamino-8-D-arginine vasopressin (DDAVP) for 20 min revealed dramatic translocation of AQP2 to the apical region of the cell, with no change in the cellular distribution of VRUT. In addition, differential centrifugation of inner medullary homogenates from Brattleboro rats treated with DDAVP for 60 min revealed a marked depletion of AQP2 from the low-density membrane fraction (enriched in intracellular vesicles) but did not alter the quantity of VRUT in this fraction. Finally, AQP2-containing vesicles immunoisolated from a low-density membrane fraction from renal inner medulla did not contain immunoreactive VRUT. Thus vasopressin-mediated regulation of AQP2, but not of VRUT, depends on regulated vesicular trafficking to the plasma membrane.

Animals↗

PubMed1999

Estradiol increases spine density and NMDA-dependent Ca2+ transients in spines of CA1 pyramidal neurons from hippocampal slices.

To investigate the physiological consequences of the increase in spine density induced by estradiol in pyramidal neurons of the hippocampus, we performed simultaneous whole cell recordings and Ca2+ imaging in CA1 neuron spines and dendrites in hippocampal slices. Four- to eight-days in vitro slice cultures were exposed to 17beta-estradiol (EST) for an additional 4- to 8-day period, and spine density was assessed by confocal microscopy of DiI-labeled CA1 pyramidal neurons. Spine density was doubled in both apical and basal dendrites of the CA1 region in EST-treated slices; consistently, a reduction in cell input resistance was observed in EST-treated CA1 neurons. Double immunofluorescence staining of presynaptic (synaptophysin) and postsynaptic (alpha-subunit of CaMKII) proteins showed an increase in synaptic density after EST treatment. The slopes of the input/output curves of both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) postsynaptic currents were steeper in EST-treated CA1 neurons, consistent with the observed increase in synapse density. To characterize NMDA-dependent synaptic currents and dendritic Ca2+ transients during Schaffer collaterals stimulation, neurons were maintained at +40 mV in the presence of nimodipine, picrotoxin, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). No differences in resting spine or dendritic Ca2+ levels were observed between control and EST-treated CA1 neurons. Intracellular Ca2+ transients during afferent stimulation exhibited a faster slope and reached higher levels in spines than in adjacent dendrites. Peak Ca2+ levels were larger in both spines and dendrites of EST-treated CA1 neurons. Ca2+ gradients between spine heads and dendrites during afferent stimulation were also larger in EST-treated neurons. Both spine and dendritic Ca2+ transients during afferent stimulation were reversibly blocked by D, L-2-amino-5-phosphonovaleric acid (D,L-APV). The increase in spine density and the enhanced NMDA-dependent Ca2+ signals in spines and dendrites induced by EST may underlie a threshold reduction for induction of NMDA-dependent synaptic plasticity in the hippocampus.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1999

Serotonergic modulation of the hyperpolarizing spike afterpotential in rat jaw-closing motoneurons by PKA and PKC.

Intracellular recordings were obtained from rat jaw-closing motoneurons (JCMNs) in slice preparations to investigate the effects of serotonin (5-HT) on the postspike medium-duration afterhyperpolarization (mAHP) and an involvement of protein kinases in the effects. Application of 50 microM 5-HT caused membrane depolarization and increased input resistance in the most cells without affecting the mAHP, whereas not only membrane depolarization and an increase in input resistance, but also the suppression of the mAHP amplitude was induced by higher dose of 5-HT (100 or 200 microM). On the other hand, when the mAHP amplitude was increased by raising [Ca(2+)](o) from 2 to 6 mM, 5-HT-induced attenuation of the mAHP amplitude was enhanced, and even 50 microM 5-HT reduced the mAHP amplitude. This 5-HT-induced suppression of the mAHP could be mimicked by application of membrane-permeable cAMP analogue 8-Bromo-cAMP, potentiated by the cAMP-specific phosphodiesterase inhibitor Ro 20-1724 and antagonized by protein kinase A (PKA) inhibitor H89. The enhancement of the mAHP attenuation induced by 50 microM 5-HT under raised [Ca(2+)](o) was blocked by a protein kinase C (PKC) inhibitor chelerythrine, suggesting an involvement of PKC in this enhancement. On the other hand, the attenuation of the mAHP induced by PKC activator phorbol 12-myristate 13-acetate was blocked almost completely by H89, suggesting that the PKC action on the mAHP requires PKA activation. Neither 5-HT(1A) antagonist NAN-190 or 5-HT(4) antagonist SB 203186 blocked 5-HT-induced attenuation of the mAHP. We conclude that 5-HT induces dose-dependent attenuation of the mAHP amplitude through cAMP-dependent activation of PKA and that PKC-dependent PKA activation is also likely to be involved in the enhancement of 5-HT-induced attenuation of the mAHP under raised [Ca(2+)](o). Because the slope of the linear relationship between firing frequency and injected current was increased only when the mAHP amplitude was decreased by 5-HT, it is suggested that the relation between incoming synaptic inputs and firing output in JCMNs varies according to serotonergic effects on JCMNs and calcium-dependent modulation of its effects.

Animals↗

PubMed1999

Behavior of jaw muscle spindle afferents during cortically induced rhythmic jaw movements in the anesthetized rabbit.

The regulation by muscle spindles of jaw-closing muscle activity during mastication was evaluated in anesthetized rabbits. Simultaneous records were made of the discharges of muscle spindle units in the mesencephalic trigeminal nucleus, masseter and digastric muscle activity (electromyogram [EMG]), and jaw-movement parameters during cortically induced rhythmic jaw movements. One of three test strips of polyurethane foam, each of a different hardness, was inserted between the opposing molars during the jaw movements. The induced rhythmic jaw movements were crescent shaped and were divided into three phases: jaw-opening, jaw-closing, and power. The firing rate of muscle spindle units during each phase increased after strip application, with a tendency for the spindle discharge to be continuous throughout the entire chewing cycle. However, although the firing rate did not change during the jaw-opening and jaw-closing phases when the strip hardness was altered, the firing rate during the power phase increased in a hardness-dependent manner. In addition, the integrated EMG activity, the duration of the masseteric bursts, and the minimum gape increased with strip hardness. Spindle discharge during the power phase correlated with jaw-closing muscle activity, implying that the change in jaw-closing muscle activity associated with strip hardness was caused by increased spindle discharge produced through insertion of a test strip. The increased firing rate during the other two phases may be involved in a long-latency spindle feedback. This could contribute to matching the spatiotemporal pattern of the central pattern generator to that of the moving jaw.

Afferent Pathways↗

PubMed1999

Sevoflurane induced suppression of inhibitory synaptic transmission between soma-soma paired Lymnaea neurons.

The cellular and synaptic mechanisms by which general anesthetics affect cell-cell communications in the nervous system remain poorly defined. In this study, we sought to determine how clinically relevant concentrations of sevoflurane affected inhibitory synaptic transmission between identified Lymnaea neurons in vitro. Inhibitory synapses were reconstructed in cell culture, between the somata of two functionally well-characterized neurons, right pedal dorsal 1 (RPeD1, the giant dopaminergic neuron) and visceral dorsal 4 (VD4). Clinically relevant concentrations of sevoflurane (1-4%) were tested for their effects on synaptic transmission and the intrinsic membrane properties of soma-soma paired cells. RPeD1- induced inhibitory postsynaptic potentials (IPSPs) in VD4 were completely and reversibly blocked by sevoflurane (4%). Sevoflurane also suppressed action potentials in both RPeD1 and VD4 cells. To determine whether the anesthetic-induced synaptic depression involved postsynaptic transmitter receptors, dopamine was pressure applied to VD4, either in the presence or absence of sevoflurane. Dopamine (10(-]5) M) activated a voltage-insensitive K(+) current in VD4. The same K(+) current was also altered by sevoflurane; however, the effects of two compounds were nonadditive. Because transmitter release from RPeD1 requires Ca(2+) influx through voltage-gated Ca(2+) channels, we next tested whether the anesthetic-induced synaptic depression involved these channels. Individually isolated RPeD1 somata were whole cell voltage clamped, and Ca(2+) currents were analyzed in control and various anesthetic conditions. Clinically relevant concentrations of sevoflurane did not significantly affect voltage-activated Ca(2+) channels in RPeD1. Taken together, this study provides the first direct evidence that sevoflurane-induced synaptic depression involves both pre- and postsynaptic ion channels.

Action Potentials↗

PubMed1999

Serum levels of circulating adhesion molecules after coronary angioplasty.

The activation of platelets, leukocytes, and vascular endothelial cells mediated by cell adhesion molecules may play a role in the mechanism of restenosis, which is still a significant complication after coronary angioplasty. We observed serial changes in the circulating soluble forms of adhesion molecules in 25 patients with coronary artery disease who underwent coronary angioplasty for a single lesion of the left anterior descending artery. Serum levels of sICAM-1 (p < 0.05) and sP-selectin (p < 0.05) were significantly increased immediately after angioplasty in the coronary sinus blood samples. These increases continued during the 48-hour observation period, and the maximum increase was seen 48 h after angioplasty for sICAM-1 (p < 0.01) and 24 h after angioplasty for sP-selectin (p < 0. 01). The level of sL-selectin increased 24 h (p < 0.01) and 48 h (p < 0.001) after angioplasty. These changes were not observed in the peripheral blood samples. The sE-selectin level did not change after angioplasty. A multiple regression analysis showed that the late loss index obtained from quantitative angiographic (QCA) analysis was correlated with the changes in sICAM-1 (r = 0.31, p < 0.05), sL-selectin (r = 0.28, p < 0.05), and sP-selectin (r = 0.26, p < 0. 05) 48 h after angioplasty in the coronary sinus blood samples, but was not correlated with procedural variables, other QCA variables, or the change in the sL-selectin level. The measurements of these adhesion molecule levels may help to evaluate traumatic vessel wall injury and inflammation at the intervention site after coronary angioplasty.

Analysis of Variance↗

PubMed1999

Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome.

Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Ultrastructural localization of S100A3, a cysteine-rich, calcium binding protein, in human scalp hair shafts revealed by rapid-freezing immunocytochemistry.

We have characterized the subcellular distribution of S100A3, a cysteine-rich calcium binding protein, in human scalp hair shaft. This was accomplished using rapid-freezing immunocytochemistry, a technique that combines rapid-freezing, freeze-substitution fixation without chemical fixatives, and subsequent electron microscopic detection of immunocytochemical labeling. This technique preserves both the antigenicity and the ultrastructural integrity of fully keratinized tissues, which are highly unmanageable when prepared for immunoelectron microscopy. In the hair shaft, S100A3 was primarily identified in the endocuticle and was also present in the intermacrofibrillar matrix surrounding macrofibril bundles of intermediate filament keratins in cortex cells. Double immunolabeling of S100A3 and hair keratins revealed the in situ spatial relationship between them. In the endocuticle, S100A3 was present on the inner portion of the endocuticle adjacent to the cell membrane complex, whereas hair keratins were present on the outer portion. These results provide the first ultrastructural evidence that an S100 protein is localized in specific subcompartments in human hair cells. (J Histochem Cytochem 47:525-532, 1999)

Adolescent↗

PubMed1999

Clinical and immunoregulatory effects of roxithromycin therapy for chronic respiratory tract infection.

The clinical and immunoregulatory effects of long-term macrolide antibiotic therapy for patients with chronic lower respiratory tract infections (CLRTI) were investigated. Clinical parameters and neutrophil chemotactic mediators in the epithelial lining fluid (ELF) of CLRTI patients (n = 10) were examined before and after 3 months oral administration of roxithromycin (RXM). The in vitro effects of RXM were also examined on the release of these mediators from alveolar macrophages (AM) and neutrophils. Arterial oxygen tension (p<0.05), vital capacity (VC) (p<0.001), %VC (p<0.05) and forced expiratory volume in one second (p<0.01) were improved after RXM treatment, but airway bacteria were not eradicated. Among the mediators, the levels of interleukin (IL)-8, neutrophil elastase (NE) and leukotriene B4 (LTB4) were higher in ELF than in plasma of CLRTI patients and they decreased after RXM treatment (n = 7, p<0.05 for each). RXM concentrations were significantly increased in the bronchoalveolar lavage cells of the treated patients. In in vitro experiments, RXM showed inhibitory effects on IL-8 release from AM and neutrophils. In conclusion, interleukin-8, neutrophil elastase and leukotriene B4 contribute to the neutrophilic inflammation in the airways of chronic lower respiratory tract infection patients and the clinical effects of roxithromycin may, in part, be attributable to the suppression of excess release of the chemotactic mediators from inflammatory cells.

Anti-Bacterial Agents↗

PubMed1999

Profile of urinary bile acids in infants and children: developmental pattern of excretion of unsaturated ketonic bile acids and 7beta-hydroxylated bile acids.

Unusual bile acids, such as unsaturated ketonic and 7beta-hydroxylated bile acids, have been detected in urine early in life. To elucidate the normal profiles of usual and unusual urinary bile acids in the neonatal and pediatric periods, we measured the concentrations of 28 kinds in urine from normal newborns, infants, and children by gas chromatography-mass spectrometry. The mean total bile acid/Cr ratio in 7-d-old infants was significantly higher than in subjects of other age groups (birth, 2-4 mo, 5-7 mo, 11-12 mo, 2-3 y, 9-14 y, and adult) (p < 0.05). Relatively large amounts of unusual bile acids were detected during infancy, especially during the period up to 1 mo of age. At that time, 1beta,3alpha,7alpha,12alpha-tetrahydroxy-5bet a-cholan-24-oic, 7alpha, 12alpha-dihydroxy-3-oxo-5beta-chol-1-en-24-oic, and 7alpha,12alpha-dihydroxy-3-oxo-4-cholen-24-oic acids were predominant among the unusual urinary bile acids present. Moreover, the levels of 3alpha,7beta,12alpha-trihydroxy-5beta-cholan+ ++-24-oic acid increased significantly after 2-4 mo of age. These results indicate that bile acid synthesis and metabolism in the liver of developing infants are significantly different from that occurring in the liver of adults. Significant amounts of urinary isomerized 7beta-hydroxylated bile acids were detected after late infancy, probably because of changes in the intestinal bacterial flora response to a change in nutrition. We describe, for the first time, evidence of the epimerization of the 7alpha-hydroxyl group of cholic acid, which may be unique to human development.

Adolescent↗

PubMed1999

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.

Animals↗

PubMed1999

Synthesis of benzimidazole derivatives as antiallergic agents with 5-lipoxygenase inhibiting action.

Syntheses were conducted of novel benzimidazole derivatives that suppress histamine release from mast cells, inhibit 5-lipoxygenase, and possess antioxidative action. Among the compounds synthesized, 1-[2-[2-(4-hydroxy- 2,3,5-trimethylphenoxy)ethoxy]ethyl]-2-(4-methyl-1- homopiperazino)benzimidazole (22) potently suppressed histamine release from rat peritoneal mast cells triggered by the antigen-antibody reaction, inhibited 5-lipoxygenase in rat basophilic leukemia-1 (RBL-1) cells, and prevented the NADPH-dependent lipid peroxidation induced by Fe(3+)-ADP in rat liver microsomes, in addition to an antagonizing the contraction of guinea pig ileum caused by histamine.

Animals↗

PubMed1999

Dynamic effects of intravenous procainamide infusion on the electrophysiological properties during atrial fibrillation.

Although the mechanism of atrial fibrillation (AF) is still controversial, multiple wandering reentry is considered the primary mechanism in most AF. It has been suggested that prolongation of the wavelength would make it impossible for the reentry to continue and would lead to the termination of the AF. In the present study a dynamic fluctuation in the electrophysiological properties was observed with procainamide infusion during AF. In 12 patients, both the local electrogram and monophasic action potentials (MAP) during AF were recorded from the right atrium before, during and after infusion of procainamide (10 mg/kg). The minimum AF cyclelength (CLmin), MAP duration at 90% repolarization (MAPD90) and widths of the intraatrial potentials (WAP) were measured with custom-made computer software. The conduction velocity index (CVI) was determined from the WAP. The wavelength index (WLI=CVIxCLmin) and postrepolarization refractoriness (PRR= CLmin-MAPD90) were calculated. In 6 patients, AF was terminated by procainamide infusion (group A), but not in the other 6 patients (group B). Group A patients showed a biphasic change in the parameters following procainamide infusion. In phase I, the CLmin, MAPD90 and PRR increased, while the CVI decreased, and the WLI remained unchanged. In phase II, the PRR, CVI and WLI increased and the AF was terminated. No restoration of the CVI nor increase in the WLI were observed in group B. The biphasic fluctuation in the CVI and the remarkable increase of the PRR and WLI were observed before termination of AF by procainamide infusion.

Adult↗

PubMed1999

Meiotic contraction of CAG repeats in Saccharomyces cerevisiae.

Several human neurodegenerative disorders are caused by expansion of CAG repeats that occurs during meiosis or gametogenesis. We anticipated that the CAG repeats cloned in a plasmid of Saccharomyces cerevisiae might undergo a change in the number of repeats during meiosis and sporulation. To test this possibility, we devised a new method to change in vitro the number of CAG repeats and constructed plasmids carrying (CAG)39, (CAG)65 or (CAG)123 from a plasmid carrying (CAG)18. We monitored the number of colonies showing an altered length of the repeat tracts during mitosis and meiotic growth. Contraction of long CAG repeat was found to occur frequently, whereas a few cases of expansion were observed. The contraction was equally enhanced in both orientations when the host cells grew through meiosis. Thus, our results suggest that long CAG repeats are destabilized during meiosis or gametogenesis in S. cerevisiae.

Base Sequence↗

PubMed1999

Expression of bovine cytokines in Escherichia coli.

Glutathione S-transferase fusion proteins of bovine interleukin-2 (IL-2), IL-4, IL-6 and interferon-gamma (IFN-gamma) were expressed in Escherichia coli. Complementary DNA (cDNA) for open reading frame of each cytokine without signal peptide encoding region was amplified by reverse transcriptional polymerase chain reaction method and was subcloned into pGEX-5X-1. In result, IL-6 and IFN-gamma fusion proteins in bacteria were soluble, but IL-2 and IL-4 fusion proteins were insoluble. The insoluble IL-2 fusion protein successfully refolded by urea became soluble. The recombinant IL-2, IL-6 and IFN-gamma could be obtained by the batch method using Glutathione Sepharose 4B and Factor Xa digestion, which may be useful for preparation of antisera as antigens and functional studies.

Animals↗

PubMed1999

Expression of Recombinant bovine L-selectin in Escherichia coli and insect cells.

Bovine L-selectin was expressed in bacteria using pGEX vector and in insect cells infected with recombinant baculovirus in order to obtain recombinant protein for preparation of specific antiserum and its functional studies. In bacterial expression, L-selectin fusion protein with glutathione S-transferase was detected in the insoluble fraction with the expected molecular weight of 60 kDa by SDS-PAGE and reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. In insect cells infected with recombinant baculovirus, a band corresponding to L-selectin was not observed in SDS-PAGE with protein staining, but they apparently reacted with anti-bovine CD62L monoclonal antibody in immunoblot analysis. Furthermore, the indirect immunofluorescence test revealed that bovine L-selectin was efficiently expressed on the surface of Sf9 cells infected with recombinant baculovirus, and flow cytometric analysis showed that the percentage of CD62L positive cells in bovine PBMC was about 66% and that most Sf9 cells infected with recombinant baculovirus had specific immunofluorescence.

Animals↗

PubMed1999

Some characteristics of bacteria found in a bioreactor to treat trichloroethylene-contaminated groundwater.

A mixture of bacteria, having a methane-utilizing ability, was separated from a bioreactor supplied with air and methane gas. The bioreactor was operated to treat trichloroethylene (TCE)-contaminated groundwater. The mixture was composed of an obligate methane-utilizing bacterium and a heterotroph, identified as Methylomonas methanica and Pseudomonas sp., respectively. The mixed culture of these two strains removed TCE. In addition, it appeared that a cooperative metabolic interaction of these strains enabled Meth.methanica to maintain the TCE degradation ability.

Bioreactors↗

PubMed1999

Expression of bovine interleukin-2 in insect cells by recombinant baculovirus.

Bovine interleukin-2 (bIL-2) was expressed in insect cells infected with recombinant baculovirus. The cDNA for bIL-2 was amplified by reverse transcriptional polymerase chain reaction and recombinant baculovirus was constructed by homologous recombination. The recombinant baculovirus was plaque-purified, amplified, and then infected to Sf9 cells for expression of recombinant bIL-2 (rbIL-2). In result, the protein band corresponding to rbIL-2 could be detected on SDS-PAGE with coomassie brilliant blue staining and on immunoblot analysis reacted with mouse antiserum against bIL-2. In addition, cell proliferation assay for bIL-2 activity demonstrated that the culture supernatant of Sf9 cells infected with recombinant baculovirus enhanced the proliferation of bovine peripheral blood mononuclear cells in a dose-dependent manner.

Animals↗