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Biomedical subjects

K Nakashima

Publications and source records attributed to K Nakashima.

At least 73 records · Page 4Linked to original sources
PubMed2001

A new expression cloning strategy for isolation of substrate-specific kinases by using phosphorylation site-specific antibody.

Signal transduction from cell surface receptors to the nucleus is regulated in most part by protein phosphorylation. For the purpose of identification of kinases which play an important role at a particular phosphorylation step in a series of signal transduction pathways, we have developed a new expression-screening method using a phosphorylation site specific antibody and a vector encoding substrate polypeptide. We have applied this method for screening kinases which phosphorylate STAT3 at serine(727). In this screening, antibody (PS727 antibody) specifically recognizing STAT3 in which serine(727) is phosphorylated was first prepared. Escherichia coli, bacteria expressing a serine(727)-containing fragment of STAT3 which was fused to glutathione-S-transferase (GST) (GST-STAT3-WT) were infected by lambda phage cDNA expression libraries. Phosphorylation of GST-STAT3-WT was effectively performed in E. coli as expected, and clones positive for PS727 antibody immunoreactivity were selected. Isolated 53 clones encode four serine/threonine kinases; extracellular signal regulated kinase 1 (ERK1/p44-MAPK), dual specificity Yak1 related kinase (DYRK), dual specificity Yak1 related kinase 2 (DYRK2) and homeodomain interacting protein kinase 2 (HIPK2). These kinases have a potential to phosphorylate serine(727) in STAT3 protein also in mammalian cells. The present method is considered to be applicable in general to isolate kinases.

Amino Acid Sequence↗
PubMed2001

Large-scale, multicenter study of cerebrospinal fluid tau protein phosphorylated at serine 199 for the antemortem diagnosis of Alzheimer's disease.

We surveyed a total of 570 cerebrospinal fluid (CSF) samples from a variety of diseases, including Alzheimer's disease (AD; n = 236), non-AD-demented and nondemented diseases (n = 239), and normal controls (n = 95) to quantitate levels of tau protein phosphorylated at serine 199 (CSF/phospho-tau199) by a recently established sandwich ELISA. The CSF/phospho-tau199 levels in the AD group were significantly elevated compared to those in all the other non-AD groups. Receiver operating characteristics curves showed that the diagnostic sensitivity and specificity for the AD group vs all the other non-AD groups using the CSF/phospho-tau199 were 85.2% and 85.0%, respectively. Furthermore, there was a significant positive correlation between CSF/phospho-tau199 and CSF/total-tau levels in the AD group. Elevated CSF/phospho-tau199 in the AD group was noted irrespective of age, gender, dementia severity, and number of apolipoprotein E4 alleles. Thus, we suggest that CSF/phospho-tau199 may be a novel and logical biomarker in supporting antemortem diagnosis of AD.

Aged↗
PubMed2001

High performance liquid chromatography with UV detection for the simultaneous determination of sympathomimetic amines using 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride as a label.

A high performance liquid chromatographic method has been developed for the simultaneous determination of (+/-) fenfluramine (Fen) and phentermine (Phen) in addition to three other sympathomimetic amines-ephedrine (E), norephedrine (NE) and 2-phenylethylamine (2-PEA), using cyclohexylamine (CX) as an internal standard in plasma. The compounds were derivatized with 4-(4,5-diphenyl-1H-imidazole-2-yl)benzoyl chloride (DIB-Cl) to give the DIB-derivatives. The derivatives were then separated using an isocratic HPLC system with UV detection. The limits of detection for Fen, Phen, E, NE and 2-PEA in plasma ranged from 0.32 to 22.9 pmol on column at a signal-to-noise ratio of 3. The recoveries following alkaline extraction from plasma samples of known concentrations were found to be more than 94% for the studied compounds. This method might be useful for the screening of the studied sympathomimetic amines in human plasma samples in forensic as well as toxicological studies. Furthermore, the developed method was modified for the simultaneous determination of Fen and Phen in human and rat plasma using fluoxetine as an internal standard. The methods are reproducible and precise. Finally, the two drugs were administered intraperitoneally to rats in combination, and their plasma levels over the investigated time course were successfully determined.

Amines↗
PubMed2001

Drugs of abuse in a non-conventional sample; detection of methamphetamine and its main metabolite, amphetamine in abusers' clothes by HPLC with UV and fluorescence detection.

In this paper, we report the detection of methamphetamine and its major metabolite, amphetamine, in garments belong to known-abusers. These compounds were extracted from the textile using a mixture of chloroform:propan-2-ol (3:1, v/v), derivatized with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride and separated using a reversed-phase high-performance liquid chromatography. The derivatives were detected by measuring either fluorescence at 440 nm or absorbance at 330 nm. By using 1-methyl-3-phenyl propylamine as an internal standard, calibration curves of spiked textile samples were linear over a wide range with correlation coefficients of 0.997 or better. Detection limits at a signal-to-noise ratio of 3 were less than or equal to 37.3 and 0.4 pg on column for the high-performance liquid chromatography-ultraviolet and -fluorescence detection methods, respectively. Intra- and inter-day variations at high and low concentrations (n > or = 3) were < or =12.7%. The developed methods were successfully applied to the determination of methamphetamine and amphetamine in clothes samples belong to abusers.

1-Propanol↗
PubMed2001

Structure and tissue distribution of prolactin receptor mRNA in Japanese flounder (Paralichtys olivaceus): conserved and preferential expression in osmoregulatory organs.

In diadromous and euryhaline teleosts, it has been established that prolactin (PRL) is a major hormone regulating the maintenance of water and electrolyte homeostasis by acting on its receptor (PRLR) expressed in the osmoregulatory organs. To investigate the major physiological role of PRL in a marine teleost, cDNA for the Japanese flounder (Paralichtys olivaceus) prolactin receptor (fPRLR) has been cloned and characterized. The predicted fPRLR is composed of 636 amino acids conserving common structural features, such as the WSXWS motif and box 1, that are observed in the members of the cytokine receptor superfamily. By Northern blot analysis, 3.5-kb transcripts for fPRLR were clearly detected in the gill, kidney, and intestine. By RNase protection assay, similarly high levels of mRNA expression were detected in these osmoregulatory organs and lower expression levels were seen in the brain for both males and females. Interestingly, a distinct expression level of fPRLR mRNA was observed in the testis, but not in the ovary. The present results suggest that PRL may play an important role in the control of water and electrolyte balance through PRLR expressed in the osmoregulatory organs in the marine teleost the Japanese flounder as well as in other teleosts. Furthermore, PRL may differentially regulate gonadal functions in males and females of Japanese flounder.

Amino Acid Sequence↗
PubMed2001

Temporospatial relationship between the expressions of superoxide dismutase and nitric oxide synthase in the developing human brain: immunohistochemical and immunoblotting analyses.

To clarify a significant relationship between superoxide dismutase (SOD) and nitric oxide synthase (NOS) in the developing human brain temporospatially, we demonstrate immunohistochemical expression of Cu/Zn-binding SOD1 (SOD1), Mn-containing SOD2 (SOD2), neuronal NOS (nNOS), inducible NOS (iNOS), and nitrotyrosine in human brains from 13 weeks of gestation to 2 years after birth. The immunoreactivities of both SOD1 and SOD2 were detected in fetal neuroblasts at 13 weeks' gestation, as well as mature neurons at the age of 2 years. By contrast, nNOS neurons could be recognized only at 28 and 33 weeks of gestation in the cerebrum, and only at 15, 18, and 23 weeks of gestation in the brain stem. No significant immunoreactivity for iNOS or nitrotyrosine was detected in any type of cell in any region during any stage examined. Immunoblotting analysis using frontal tissue homogenates at 15, 28, 40 weeks of gestation and 18 months of age revealed single band corresponding to SOD1 molecular weight, observed at all stages examined; a single band compatible with the nNOS molecular mass was detected only at the 28th week of gestation. Together with the fact that nitric oxide (NO) plays a potential role in neuronal differentiation, and that large amounts of NO have cytotoxicity from the reaction of NO with superoxide anions, our data suggested that the expressions of both SOD1 and SOD2, as scavengers of superoxide anions, were maintained from an early developmental stage to prepare stage-specific nNOS expression for a potential differentiation role and to elude NO cytotoxicity.

Aging↗
PubMed2001

Long-term change in synaptic transmission in CA3 circuits followed by spontaneous rhythmic activity in rat hippocampal slices.

The relevance of long-term potentiation (LTP) at excitatory synapses in CA3 circuits to generation of spontaneous epileptiform bursts in CA3 was investigated using rat hippocampal slices. CA3 pyramidal cells were antidromically stimulated through Schaffer collaterals. Evoked field potentials were extracellularly recorded from the stratum pyramidale and the stratum radiatum in CA3. Therefore, field potentials reflecting recurrent excitatory post-synaptic potentials (EPSPs) and inhibitory post-synaptic potentials (IPSPs) were positive at the stratum pyramidale and negative at the stratum radiatum. First, we tested how the amplitude of the evoked field potentials depends on a gamma-aminobutyric acid (GABA(A)) antagonist. Both of the positive and negative field potential peaks reduced in the medium containing penicillin (2 mM) or bicuculline (20 microM). This suggests that unmasked EPSPs due to suppression of IPSPs do not result in an increase in the evoked potentials. Second, CA3 pyramidal cells were antidromically stimulated by tetanic stimulation of Schaffer collaterals in order to induce LTP at synapses in CA3 circuits. Both of the positive and negative field potentials increased, suggesting that recurrent EPSPs were enhanced by tetanic stimulation. Induction of LTP at recurrent excitatory synapses was followed by spontaneous epileptiform bursts which persisted throughout experiments (approximately 1.5 h), while LTP of afferent synaptic potential evoked by hilar test stimulation was not induced. These results suggest that LTP at the afferent synapses is not necessary to spontaneous epileptiform bursts in CA3, but LTP at excitatory synapses between CA3 pyramidal cells contribute to spontaneous epileptiform bursts.

Action Potentials↗
PubMed2001

Fate alteration of neuroepithelial cells from neurogenesis to astrocytogenesis by bone morphogenetic proteins.

Bone morphogenetic proteins (BMPs), a class of cytokines belonging to the transforming growth factor-beta superfamily, have been shown to play a wide variety of roles during development including those in the central nervous system. We here report that BMP2, BMP4 and BMP7 have an equivalent potential to inhibit neurogenesis and concomitantly induce astrocytogenesis of mouse fetal neuroepithelial cells. We further show that these BMPs activate a promoter of the gene for negative helix-loop-helix (HLH) factor, Id1, which is known to inhibit the function of such neurogenic transcription factors as Mash1 and neurogenin. These results suggest that BMP2, BMP4 and BMP7 alternate the fate of neuroepithelial cells from neuronal type to astrocytic one via a common mechanism involving negative HLH factor.

Activin Receptors, Type I↗
PubMed2001

Assessment of air pollution in Nagasaki City: determination of polycyclic aromatic hydrocarbons and their nitrated derivatives, and some metals.

Air pollutants such as polycyclic aromatic hydrocarbons (PAHs), their nitrated derivatives (NPAHs), and some metals on airborne particles in Nagasaki city, Japan were determined over a period of 12 months by high-performance liquid chromatography with chemiluminescence, fluorescence and flameless atomic absorption spectrometry. The average concentrations (range) were 18.24 (4.07-41.54) ng/m3 for total PAHs, 0.91 (0.23-4.10) pg/m3 for NPAHs, 7.95 (1.71-16.31) ng/m3 for Pb, 11.56 (3.35-24.96) ng/m3 for Mn and 3.79 (0.97-14.71) ng/m3 for Ni (n = 136). The toxic equivalency factors adjusted concentration of total PAHs determined in Nagasaki city area was 2.33 ng/m3. Concentrations of total PAHs and NPAHs in winter were higher than those in summer. In a weekly variations study, total PAHs and NPAHs concentrations, as well as traffic volume showed a similar tendency with all values higher during weekdays and lower at the weekend. The correlation coefficients between total PAHs or NPAHs and traffic volume were 0.781 and 0.818, respectively. These results suggested that one of the main sources for NPAHs and PAHs in a city area might be motor vehicles.

Air Pollutants↗
PubMed2001

Regulatory mechanisms in the pathways of cartilage and bone formation.

Three transcription factors of the Sox family have essential roles in different steps of the chondrocyte differentiation pathway. Because the transcription factor Cbfa1, which is needed for osteoblast differentiation, also stimulates hypertrophic chondrocyte maturation, it links the chondrocyte and osteoblast differentiation pathways in endochondral bone formation. Signaling molecules, including Indian Hedgehog, PTHrP and FGFs, also establish essential links either between these pathways, between steps in these pathways or between signaling molecules and transcription factors, so that a more comprehensive view of endochondral bone formation is emerging.

Animals↗
PubMed2001

Ligand recognition by the vitamin D receptor.

Three-dimensional structure of the ligand binding domain (LBD) of the vitamin D receptor (VDR) docked with the natural ligand 1 alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] has been mostly solved by the X-ray crystallographic analysis of the deletion mutant (VDR-LBD Delta 165-215). The important focus, from now on, is how the VDR recognizes and interacts with potent synthetic ligands. We now report the docking models of the VDR with three functionally and structurally interesting ligands, 22-oxa-1,25-(OH)(2)D(3) (OCT), 20-epi-1,25-(OH)(2)D(3) and 20-epi-22-oxa-24,26,27-trihomo-1,25-(OH)(2)D(3). In parallel with the computational docking studies, we prepared twelve one-point mutants of amino acid residues lining the ligand binding pocket of the VDR and examined their transactivation potency induced by 1,25-(OH)(2)D(3) and these synthetic ligands. The results indicate that L233, R274, W286, H397 and Y401 are essential for holding the all ligands tested, S278 and Q400 are not important at all, and the importance of S237, V234, S275, C288 and H305 is variable depending on the side-chain structure of the ligands. Based on these studies, we suggested key structural factors to bestow the selective action on OCT and the augmented activities on 20-epi-ligands. Furthermore, the docking models coincided well with our proposed active space-region theory of vitamin D based on the conformational analyses of ligands.

Amino Acids↗
PubMed2001

DNA methylation is a critical cell-intrinsic determinant of astrocyte differentiation in the fetal brain.

Astrocyte differentiation, which occurs late in brain development, is largely dependent on the activation of a transcription factor, STAT3. We show that astrocytes, as judged by glial fibrillary acidic protein (GFAP) expression, never emerge from neuroepithelial cells on embryonic day (E) 11.5 even when STAT3 is activated, in contrast to E14.5 neuroepithelial cells. A CpG dinucleotide within a STAT3 binding element in the GFAP promoter is highly methylated in E11.5 neuroepithelial cells, but is demethylated in cells responsive to the STAT3 activation signal to express GFAP. This CpG methylation leads to inaccessibility of STAT3 to the binding element. We suggest that methylation of a cell type-specific gene promoter is a pivotal event in regulating lineage specification in the developing brain.

Animals↗
PubMed2001

Thymol derivatives from Eupatorium fortunei.

Sixteen new thymol derivatives have been isolated from Eupatorium fortunei and their structures determined based on spectroscopic data. They were classified into three groups (i-iii) depending on the oxidation levels: (i) one oxygen function at the 9-position, (ii) two oxygen functions at the 8- and 9-positions, and (iii) three oxygen functions at the 8-, 9-, and 10-positions. The hydroxyl groups are acylated with tigloyl, angeloyl, acetyl, isobutyryl, 3-methyl-2-butenoyl, or 2-methylbutyryl moieties. The compounds having chiral centers showed no specific rotation and exist as racemic mixtures.

Chromatography, High Pressure Liquid↗
PubMed2001

Alpha-like activity in terminal stage of Creutzfeldt-Jakob disease.

We report a case of Creutzfeldt-Jakob disease showing various changes in electroencephalogram (EEG) throughout the course of the disease. The patient's EEG patterns showed periodic synchronous discharge in the intermediate stage of the disease, delta activity in the advanced stage and alpha-like activity in the terminal stage. The mechanism generating alpha-like activity may resemble, at least in part, that of an alpha coma.

Aged↗
PubMed2001

Inclusion body myositis associated with hepatitis C virus infection.

The case of a 77-year-old woman with hepatitis C virus infection with a 5-year history of muscle weakness and mild disturbance of gait is reported. Steroid therapy did not improve her symptoms. She developed HCV-related liver cirrhosis and hepatocellular carcinoma, and muscle biopsy revealed inclusion body myositis. Immunohistochemistry showed that the nonstructural region of HCV and 8-hydroxy-2'-deoxyguanosine, a marker of DNA damage by reactive oxygen species, were present in striated muscle cells of this patient.

Aged↗
PubMed2001

Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans.

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.

Aggregatibacter actinomycetemcomitans↗
PubMed2001

High performance liquid chromatographic determination of mazindol in human plasma.

A simple and convenient high performance liquid chromatographic method with UV detection is described for the determination of mazindol [5-(p-chlorophenyl)-2,5-dihydro-3H-imidazo[2,1-a]isoindol-5-ol] and its major metabolite, 2-(2-aminoethyl)-3-(p-chlorophenyl)-3-hydroxyphthalimidine (Met), in human plasma. The analytes were extracted with ethyl acetate from plasma samples and separated on a C18 column using acetonitrile-0.067 mol dm(-3) phosphate buffer (pH 3.5) (24 + 76 v/v) as a mobile phase. The eluates were monitored at 220 nm. Following complete validation and stability studies, the proposed method proved to be sensitive and precise. The limits of detection were 0.07 and 0.08 ng ml(-1) of plasma for mazindol and Met, respectively. The accuracy and recovery were in the ranges 94-102% and 91-102%, respectively, for both compounds. The intra- and inter-assay precisions were less than 7.6 and 9.2%, respectively, for both compounds. The stability of mazindol under different storage conditions, i.e., at room temperature (rt) and 4 degrees C and with freeze-thaw cycles, was also examined. Mazindol was unstable in plasma samples left at rt and 4 degrees C. The method was applied to the determination of mazindol and Met in the plasma of a patient treated for obesity with mazindol.

Adult↗