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Biomedical subjects

N Yamada

Publications and source records attributed to N Yamada.

At least 199 records · Page 11Linked to original sources
PubMed1999

Effects of YM90K, a selective AMPA receptor antagonist, on amygdala-kindling and long-term hippocampal potentiation in the rat.

To investigate the role of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) type glutamate receptors in epileptic seizures, we examined the antiepileptogenic and anticonvulsant effects of YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3-(1H,4H)-quinoxalinedione hydrochloride], a potent and selective new AMPA receptor antagonist, in the rat amygdala-kindling model of epilepsy. Pretreatment with YM90K (7.5-30 mg/kg i.p.) markedly retarded the evolution of kindling. Once kindling was established, administration of YM90K (7.5-30 mg/kg i.p.) significantly and dose-dependently suppressed fully kindled seizures. The maximal effects were observed 15-30 min after injection. When the intensity of electrical stimulation was increased to twice the generalized seizure-triggering threshold, the anticonvulsant effects of YM90K were reversed, suggesting that they were due to elevation of the generalized seizure-triggering threshold. Furthermore, an anticonvulsant dose (15 mg/kg) of YM90K affected neither field potentials nor long-term potentiation in the hippocampus in vivo. These results indicate that AMPA receptors play an important role in the seizure expression mechanism and the development of kindling-induced epileptogenesis, and suggest the possible clinical usefulness of AMPA receptor antagonists as antiepileptic drugs.

Amygdala↗
PubMed1999

Expression of recombinant NMDA receptors in hippocampal neurons by adenoviral-mediated gene transfer.

N-methyl-d-aspartate (NMDA) receptors have attracted a great deal of attention because they are intimately involved in brain development, synaptic plasticity and a variety of neurological disorders. The ability to artificially alter the properties of NMDA receptors in central nervous system (CNS) neurons would be useful for elucidating the physiological roles of these receptors. It would also raise the possibility of gene therapy of neurological diseases caused by malfunction of NMDA receptors. In this study, we constructed three recombinant adenoviruses encoding rat NMDA receptor subunit cDNAs, NMDAR1 (NR1), NMDAR2B (NR2B) and mutant NR1(N598R) in which the asparagine (N) site of the wild-type NR1 was replaced with arginine (R) by site-directed mutagenesis. PC12 cells co-infected with recombinant adenoviruses bearing NR1 and NR2B cDNAs expressed conventional NMDA receptors that were permeable to Ca2+ and sensitive to Mg2+, whereas those with viruses bearing NR1(N598R) and NR2B cDNAs expressed Ca2+-impermeable and Mg2+-insensitive receptors. When rat hippocampal neurons in culture were infected with NR1(N598R) and NR2B viruses, both Ca2+ permeability and Mg2+ sensitivity of NMDA receptors were markedly reduced in the infected neurons. Excitatory postsynaptic currents (EPSCs) mediated by NMDA receptors also became much less sensitive to Mg2+. Thus, the NR1(N598R)/NR2B receptors were more dominant than the native NMDA receptors in the infected neurons, and the former receptors introduced by the adenoviral vectors functioned as postsynaptic receptors. These results indicate that the functional properties of postsynaptic NMDA receptors can be manipulated by gene transfer technology using adenoviral vectors.

Adenoviridae↗
PubMed1999

High yield refolding and purification process for recombinant human interleukin-6 expressed in Escherichia coli.

Recombinant human interleukin-6 (hIL-6), a pleiotropic cytokine containing two intramolecular disulfide bonds, was expressed in Escherichia coli as an insoluble inclusion body, before being refolded and purified in high yield providing sufficient qualities for clinical use. Quantitative reconstitution of the native disulfide bonds of hIL-6 from the fully denatured E. coli extracts could be performed by glutathione-assisted oxidation in a completely denaturating condition (6M guanidinium chloride) at protein concentrations higher than 1 mg/mL, preventing aggregation of reduced hIL-6. Oxidation in 6M guanidinium chloride (GdnHCl) required remarkably low concentrations of glutathione (reduced form, 0.01 mM; oxidized form, 0.002 mM) to be added to the solubilized hIL-6 before the incubation at pH 8.5, and 22 degrees C for 16 h. After completion of refolding by rapid transfer of oxidized hIL-6 into acetate buffer by gel filtration chromatography, residual contaminants including endotoxin and E. coli proteins were efficiently removed by successive steps of chromatography. The amount of dimeric hIL-6s, thought to be purification artifacts, was decreased by optimizing the salt concentrations of the loading materials in the ion-exchange chromatography, and gradually removing organic solvents from the collected fractions of the preparative reverse-phase HPLC. These refolding and purification processes, which give an overall yield as high as 17%, seem to be appropriate for the commercial scale production of hIL-6 for therapeutic use.

Amino Acid Sequence↗
PubMed1999

Neuronal acetylcholine receptors with alpha7 subunits are concentrated on somatic spines for synaptic signaling in embryonic chick ciliary ganglia.

Nicotinic acetylcholine receptors containing alpha7 subunits are widely distributed in the vertebrate nervous system. In the chick ciliary ganglion such receptors generate large synaptic currents but appear to be excluded from postsynaptic densities on the cells. We show here that alpha7-containing receptors are concentrated on somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy on thick sections, together with tomographic reconstruction, permitted three-dimensional analysis of finger-like projections emanating from cell bodies. The projections were identified as spines based on their morphology, cytoskeletal content, and proximity to presynaptic elements. Both in situ and after ganglionic dissociation, the spines were grouped on the cell surface and tightly folded into mats. Immunogold labeling of receptors containing alpha7 subunits showed them to be preferentially concentrated on the somatic spines. Postsynaptic densities were present in vivo both on the soma near spines and occasionally on the spines themselves. Synaptic vesicle-filled projections from the presynaptic calyx were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane and sometimes included Omega profiles as if caught in an exocytotic event, even when no postsynaptic densities were juxtaposed on the spine. The results suggest several mechanisms for delivering transmitter to alpha7-containing receptors, and they support new ideas about synaptic signaling via spines. They also indicate that neurons must have specific mechanisms for targeting alpha7-containing receptors to desired locations.

Animals↗
PubMed1999

Screening for glaucoma with frequency-doubling technology and Damato campimetry.

OBJECTIVE: To assess frequency-doubling technology (FDT) perimetry (Humphrey Systems, San Leandro, Calif) and Damato campimetry (Precision Vision, Villa Park, Ill) for detecting glaucoma in a public glaucoma screening. METHODS: A 2-day public glaucoma screening was held at 2 different institutions. Each subject underwent 2 visual field screening tests (Damato campimetry and FDT perimetry in screening mode), an ophthalmologic examination, and Humphrey perimetry (24-2 FASTPAC) for each eye. Eyes were divided into 4 categories: normal, ocular hypertensive, glaucoma suspect, and definite glaucoma. The sensitivity and specificity of FDT perimetry and Damato campimetry for detecting glaucoma were estimated with receiver operating characteristic curves. RESULTS: Among 240 subjects who underwent FDT, the number identified as normal, ocular hypertensive, glaucoma suspect, and definite glaucoma was 151, 28, 35, and 26, respectively; among 175 subjects who underwent Damato campimetry, the numbers for the same groups were 118, 19, 19, and 19, respectively. The areas under the receiver operating characteristic curve for FDT perimetry and Damato campimetry were 0.925 and 0.883, respectively. The optimal sensitivity and specificity for FDT perimetry were 92% and 93%, while those for Damato campimetry were 53% and 90%, respectively. The average test time was 1 minute and 3 minutes per eye for FDT perimetry and Damato campimetry, respectively. CONCLUSION: Frequency-doubling technology perimetry was superior to Damato campimetry in this screening for glaucoma.

California↗
PubMed1999

Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation.

The lone CX3C chemokine, fractalkine (FK), is expressed in a membrane-bound form on activated endothelial cells and mediates attachment and firm adhesion of T cells, monocytes and NK cells. We now show that FK is associated with dendritic cells (DC) in epidermis and lymphoid organs. In normal human skin, dual-color fluorescence microscopy co-localized FK expression with Langerhans cells expressing CD1a. In tonsil, FK-positive DC expressed CD83, a marker for mature DC. Human and murine cultured DC up-regulated FK mRNA expression with maturation. Furthermore, CD40 ligation, but not TNF-alpha or lipopolysaccharide treatment, of activated, migratory DC that had migrated from skin explants resulted in a 2.5-fold increase of surface expression of FK without significant alterations of expression of CD80, CD86, CD54 or MHC class II. Since FK mediates adhesion of T cells to activated endothelial cells, the increased expression of FK during DC maturation (and particularly by CD40 ligation) may play a role in the ability of T cells and mature DC to form conjugates and engage in cell-cell communication.

Animals↗
PubMed1999

Effects of intraocular or systemic administration of neutralizing antibody against vascular endothelial growth factor on the murine experimental model of retinopathy.

Vascular endothelial growth factor (VEGF), the strongest known angiogenic cytokine and also a potent enhancer of vascular permeability, is closely associated with diabetic ocular complications and other intraocular neovascular diseases. The therapeutic effect of VEGF-neutralizing antibody on oxygen-induced retinopathy in an experimental murine model of proliferative retinopathy was investigated. Intraocular and systemic injection of the antibody resulted in 46% and 18% reductions in the number of nuclei of newly formed vessels of this model, respectively. The results demonstrated that a neutralizing antibody against VEGF was highly effective in the treatment of intraocular neovascularization and suggested possible modes of therapy in human intraocular neovascular diseases, including diabetic proliferative retinopathy.

Animals↗
PubMed1999

Role of anchor residues in peptide binding to three HLA-A26 molecules.

To investigate the role of anchor residues in HLA-A26 binding peptides, we analyzed the binding of various peptides to three HLA-A26 molecules using the HLA class I stabilization assay. Of twenty nonamer peptides carrying anchors at P2 and P9, 3, 6 and 3 peptides bound to HLA-A*2601, HLA-A*2602 and HLA-A*2603, respectively The peptide EV-IPMFSAL bound most strongly to these three HLA-A26 molecules. Analysis using mutants of this peptide at P1, P2 or P9 showed that acidic amino acids at P1 and five hydrophobic residues (Val, Thr, Ile, Leu and Phe) at P2 are anchor residues for the three HLA-A26 molecules while with exception of positively charged amino acids, a broad range of amino acids function as P9 anchor residues. These anchors were further evaluated using 38 nonamer peptides carrying anchor residues at P1, P2 and P9. Nineteen of these peptides bound to at least one HLA-A26 molecule. The frequency of HLA-A26 binding peptides was higher for peptides carrying all three anchor residues than for peptides carrying only P2 and P9 anchor residues. These results indicate that in addition to P2 and P9 anchors, the P1 anchor plays an important role in peptide binding to three HLA-A26 molecules.

Amino Acid Sequence↗
PubMed1999

Troglitazone and pioglitazone attenuate agonist-dependent Ca2+ mobilization and cell proliferation in vascular smooth muscle cells.

1. The effects of troglitazone and pioglitazone on agonist-induced Ca2+ mobilization and cell proliferation were studied using fluorescent Ca2+ indicator fura-2 AM and incorporation of [3H]-thymidine in rat aortic smooth muscle cells. The patch clamp techniques were also employed. 2. Vasopressin and platelet-derived growth factor-BB (PDGF) caused a transient elevation in [Ca2+]i by Ca2+ mobilization from intracellular stores, followed by a sustained rise due to Ca2+ entry. Nicardipine partly inhibited the sustained phase, but La3+ completely abolished it. 3. Troglitazone and pioglitazone did not significantly affect the transient rise elicited by these agonists, but preferentially inhibited the sustained phase of [Ca2+]i. 4. Under voltage clamp conditions, troglitazone and pioglitazone inhibited voltage-dependent L-type Ca2+ current (ICa.L). They also inhibited nonselective cation channels (Icat) elicited by vasopressin in a concentration-dependent manner. The half maximal inhibitory concentrations of troglitazone on ICa.L and Icat were 4.6 and 5.7 microM, respectively. On the other hand, nifedipine and nicardipine did not inhibit Icat. 5. Vasopressin and PDGF increased incorporation of [3H]-thymidine, and nifedipine and nicardipine partly suppressed it. However, the inhibitory effects of La3+ and exclusion of extracellular Ca2+ were more potent than the Ca2+ blocking agents. Troglitazone and pioglitazone also inhibited it concentration-dependently. 6. These results suggest that troglitazone and pioglitazone preferentially inhibited agonist (vasopressin and PDGF)-induced Ca2+ entry and proliferation in rat vascular smooth muscle cells, where the inhibitory effects of thiazolidinediones on ICa.L and Icat might be partly involved. Thus, thiazolidinediones may exert hypotensive and antiatherosclerotic effects.

Animals↗
PubMed1999

Presence of telomeric G-strand tails in the telomerase catalytic subunit TERT knockout mice.

BACKGROUND: Telomerase consists of two essential subunits, the template RNA (TR; telomerase RNA) and the catalytic subunit TERT (telomerase reverse transcriptase). Knockout mice with a mTR (mouse TR) deletion have been described and well characterized. However, mice with a mTERT (mouse TERT) deletion have not been reported. RESULTS: mTERT-knockout mice have been constructed. The first generation mTERT -/- mice were fertile, and did not show any noticeable macroscopic or microscopic phenotypic change. All tissue cells derived from mTERT -/- mice that were examined lacked telomerase activity, indicating that mTERT is the only gene encoding the telomerase catalytic subunit. Pulse field gel electrophoresis (PFGE) and nondenaturing in-gel hybridization analyses showed that mouse telomeric DNA has G-strand 5'-overhangs, as demonstrated for human and yeast cells. This telomeric single-stranded G-tail was also observed in MEF (mouse embryonic fibroblast) and liver cells derived from mTERT -/- mice. CONCLUSIONS: mTERT-knockout mice show phenotypes that are apparently normal at least during the early generations. This observation is similar to that obtained with the mTR-knockout mice. The presence of the telomeric G-strand tails in mTERT -/- mice suggests that these telomeric 5'-overhangs are produced by telomerase-independent mechanisms, as has been proposed for yeast and human.

Amino Acid Sequence↗
PubMed1999

Role of the N-terminal region of ribosomal protein S7 in its interaction with 16S rRNA which binds to the concavity formed by the beta-ribbon arm and the alpha-helix.

The ribosomal protein S7, a primary 16S rRNA-binding protein, plays an essential role in stabilizing the 3' major domain of 16S rRNA and also in feedback regulation of the str operon, as a translational repressor. We examined amino acid residues in ribosomal protein S7 from Bacillus stearothermophilus (BstS7) that are essential for 16S rRNA binding. Truncation of the N-terminal 10 residues of BstS7 abolished its binding to 16S rRNA, whereas removal of the C-terminal eight residues had no effect on the binding activity. Subsequently, we used site-directed mutagenesis to identify essential basic residues in the N-terminal region for 16S rRNA binding. Mutation of Arg3 and Lys8 significantly weakened the binding activity, and a smaller decrease in binding activity was observed with Arg2 and Arg9 mutations. These observations indicate that N-terminal basic residues, especially Arg3 and Lys8, play a crucial role as positively charged recognition groups for the negatively charged phosphate backbone of 16S rRNA. In addition, the mutagenesis study showed that Arg75, Arg78, Arg94, and Arg101, which are located in a concavity formed by the beta-ribbon arm and the alpha-helix (alpha4), individually make only a small contribution to 16S rRNA binding, but together probably form a positively charged binding site for 16S rRNA. With regard to aromatic residues, Tyr84 on the tip of the beta-ribbon arm was found to be involved in 16S rRNA binding, whereas the conserved aromatic residues Trp102 and Tyr106 in the concavity had little effect. We then probed the 16S rRNA-binding site(s) for the N-terminal region of S7 with iron tethered to the mutant of BstS7 containing a single cysteine residue at position 4. The N-terminal region of S7 is placed in close proximity to helix 43 in the 16S rRNA. Probing also revealed additional cleavages between nucleotides 1397 and 1438, near the P-site region in 16S rRNA. This finding is consistent with a three-dimensional model of 16S rRNA that shows close proximity of helix 43 to the P-site during three-dimensional folding.

Arginine↗
PubMed1999

A newly developed assay for melatonin using cells expressing human mel-1a receptor.

We have developed a radioreceptor binding assay (RRA) method for melatonin using membranes from Chinese hamster ovary cells that can stably express human mel-1a receptors. We measured melatonin levels in plasma samples collected every 4h for 24h using the RRA and radioimmunoassay (RIA) methods, simultaneously. There was a statistically significant correlation between the melatonin levels measured by the two methods, this newly developed method providing a sensitive bioassay. As it is possible to circumvent the cross-reactivity usually occurring in the RIA method, this method may be an important tool for detecting bioactive substances relative to the mel-1a receptor.

Animals↗
PubMed1999

Effects of sleep deprivation: the phosphorus metabolism in the human brain measured by 31P-magnetic resonance spectroscopy.

Sleep deprivation (SD) has an antidepressant effect in some, but not all, patients with depression, although its biological mechanisms have not yet been characterized. We previously reported altered brain phosphorus metabolism measured by phosphorus-31 magnetic resonance spectroscopy (31P-MRS) in patients with bipolar depression. We preliminarily examined effects of SD on phosphorus metabolism in the frontal lobes of 15 normal subjects using 31P-MRS. No significant differences of membrane phospholipid metabolism, high-energy phosphate metabolism and intracellular pH were found between before and after SD in these subjects. Further studies will be necessary to elucidate the physiological mechanism of SD for depressive patients.

Adolescent↗
PubMed1999

Add-on polytherapy with antidepressants and its significance in inpatients with major depression.

Add-on polytherapy with antidepressant agents was reviewed in 42 inpatients with major depression diagnosed according to the ICD-10 criteria. Twenty-eight (67.7%) patients were treated with two or more antidepressants. The most frequent combination consisted of a tricyclic antidepressant and a non-tricyclic antidepressant. Nineteen (67.8%) patients of the polytherapy group were treated with a dosage equivalent to 150 mg/day of tricyclic antidepressant. Clinically, not every patient with major depression can tolerate the adverse side effects induced by an effective dose of a single tricyclic antidepressant. From this viewpoint, add-on polytherapy with antidepressants could be one of the treatment options, especially for such intolerant patients.

Adult↗
PubMed1999

An evaluative system for the response of antibacterial therapy: based on the morphological change of Helicobacter pylori and mucosal inflammation.

The diagnostic standard is an important factor in the evaluation of the antibacterial effect to Helicobacter pylori (H. pylori). Few studies have evaluated the bacterial morphological change. In the present study, H. pylori was examined by means of electron microscopy (EM), light microscopy (LM) and immunohistochemical staining. Patients were followed up from 6 weeks to more than 1 year after treatment for H. pylori, and the results of the 13C-urea breath test (UBT) were compared. A '4-L' evaluative system was used for histological diagnosis; that is, complete, significant, partial and negative response for H. pylori treatment. Complete response showed no H. pylori in histology, and positive 13C-UBT and negative response showed positive in both diagnoses. A significant response showed the morphology of H. pylori was thick walled by EM, that there was no obvious active inflammation, and was negative for C-UBT. These H. pylori showed a coccoid form and possibly static bacteria, which was resistant to further antibacterial therapy. The '4-L' system could evaluate the antibacterial effect, suggesting the necessity for a second line of therapy for H. pylori. It is suggested that this sensitive evaluative system is suitable for clinical applications for antibacterial therapy.

Anti-Bacterial Agents↗
PubMed1999

An autopsy case of a malignant pericardial mesothelioma in a Japanese young man.

An autopsy case of a malignant pericardial mesothelioma in a 27-year-old man with no history of exposure to asbestos is reported. He was admitted for heart failure due to pericardial effusion of unknown origin and surgically drained, but later died. The diagnosis of a malignant pericardial mesothelioma was made on the basis of histologic, immunohistochemical and ultrastructural findings. The tumor was located on the pericardium, but autopsy revealed that it had spread extensively in the mediastinum and the lungs. Microscopically, the tumor cells were epithelial like and contained histochemically demonstrable glycogen and hyaluronic acid. Immunohistochemical studies of the tumor demonstrated positive immunoreactivity for cytokeratin 19, muscle actin HHF35, epithelial membrane antigen, CA125, p53 and p21WAF1/CIP1 whereas the tumor was negative for cytokeratins 10 and 17, carcinoembryonic antigen, vimentin, epithelial antigen BerEP4, S-100, c-erbB2 and bcl-2. A high MIB-1 labeling index was noted. Under the electron microscope the tumor cells exhibited long, thin villi. The operation and autopsy findings thus revealed this to be a very rare case of malignant pericardial mesothelioma in a young man.

Adult↗