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Biomedical subjects

T Nagata

Publications and source records attributed to T Nagata.

At least 253 records · Page 14Linked to original sources
PubMed1997

Inhibition of arachidonate lipoxygenase activities by 2-(3,4-dihydroxyphenyl)ethanol, a phenolic compound from olives.

The effects of olive fruit extract on arachidonic acid lipoxygenase activities were investigated using rat platelets and rat polymorphonuclear leukocytes (PMNL). Olive extract strongly inhibited both 12-lipoxygenase (12-LO) and 5-lipoxygenase (5-LO) activities. One of the compounds responsible for this inhibition was purified and identified as 2-(3,4-dihydroxyphenyl)ethanol (DPE). DPE inhibited platelet 12-LO activity (IC50, 4.2 microM) and PMNL 5-LO activity (IC50, 13 microM) but not cyclooxygenase activity in cell-free conditions. It also inhibited 12-LO activity in intact platelets (IC50, 50 microM) and reduced leukotriene B4 production in intact PMNL stimulated by A23187 (IC50, 26 microM). The inhibition by DPE of both lipoxygenase activities was stronger than that by oleuropein, caffeic acid, or 7 other related phenolic compounds, especially in intact cells. These results suggest that DPE is a potent specific inhibitor of lipoxygenase activities.

Animals↗
PubMed1997

Effect of simultaneous bilateral tibial nerve stimulation on somatosensory evoked potentials (SEP) in dogs.

Since somatosensory evoked potentials (SEP) by hindlimb nerve stimulation are known to ascend bilaterally in the spinal cord, it was investigated whether or not simultaneous bilateral stimulation causes facilitation or inhibition of the stimuli. In an experiment using 36 adult Beagle dogs, the difference between simultaneous bilateral stimulation and unilateral stimulation was studied as to latencies and amplitudes. No significant difference was noted. However, the detection of far-field potentials by bilateral stimulation was not effective in which case far-field potentials did not recorded by unilateral stimulation. It was therefore confirmed that simultaneous bilateral stimulation does not cause facilitation or inhibition in the relay pathways, and does not affect the latency.

Animals↗
PubMed1997

Elevated L-kynurenine level and its normalization by prednisolone in a patient with eosinophilia-myalgia syndrome.

We report a L-tryptophan-induced case of eosinophilia-myalgia syndrome in a Japanese woman and describe the time course of changes in tryptophan metabolism observed during steroid therapy. She had taken 1.0 g of the implicated L-tryptophan daily. When admitted due to painful swelling of her extremities, eosinophil count was 22.3 x 10(9)/L. Before prednisolone treatment, her serum L-kynurenine level was 10.2 mumol/L, a level about three-fold higher than the normal value, while serum tryptophan level was abnormally low (23.1 mumol/L). On the 14th day of prednisolone treatment (40 mg daily), L-kynurenine was declined to 8.1 mumol/L and, concomitantly, L-tryptophan level increased to the normal range (51.0 mumol/L). Subsequently, on the 42nd day of therapy, serum L-kynurenine was normalized. In contrast, serum serotonin level was unchanged throughout the course of this therapy. Prednisolone dramatically reduced the elevated serum L-kynurenine with a reciprocal increase in serum L-tryptophan indicates that abnormal tryptophan metabolism, may play a role in the pathogenesis of eosinophilia myalgia syndrome, and that the observed effect of steroid treatment was due to suppression of elevated activity of indoleamine 2, 3-dioxygenase, a first rate-limiting enzyme of the kynurenine pathway.

Anti-Inflammatory Agents↗
PubMed1997

Development and DNA synthesis in the retina of chick embryo observed by light and electron microscopic radioautography.

The incorporation of 3H-thymidine into DNA was examined in the chick embryo retina in early development during the period of the optic vesicle formation (day 2) before the formation of the basic layers of retina (day 7) by means of light and electron microscopic radioautography. Labeling index with 3H-thymidine reached the maximum on day 2 and declined thereafter. The patterns of localization of labelled cells and average grain counts were compared in the anterior, equatorial and posterior regions of the retina. On day 2, more labelled cells were found in the posterior region than the other two regions of the retina. Most of the labelled cells were found in the outer portion of the retinal layer throughout the retina. On day 3, most labelled cells were found in the anterior region and decreased in the posterior one. On day 4, labelled cells dominated in the anterior and equatorial regions. In these two regions, more labelled cells were present in the outer portion of the retinal layer, while in the posterior region these cells were seen in the inner portion of the retinal layer.

Animals↗
PubMed1997

Field emission SEM, conventional TEM and HVTEM study of submandibular gland in prenatal and postnatal aging mouse.

The development of acinar and ductal cells of the mouse submandibular gland was studied using field emission SEM, conventional TEM and HVTEM methods. The specimens, at 15 and 18 days of gestation and 1, 3, 7, 14, 21, 30, 90 and 180 postnatal days were fixed in 2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.3). At 15 and 18 days of gestation, the structure of mouse submandibular gland contains acinar and ductal cells in proliferation. The cytoplasmic organelles such as mitochondria, granular endoplasmic reticulum and Golgi apparatuses are scattered in the cytoplasm. At 18 prenatal days only several acinar cells present immature secretory granules in the apical portion. In this stage the acinar and ductal cells are enveloped by bundles of fine collagen fibrils disposed in several directions. There are also numerous capillaries located closely to the acinar cell membranes. In the aging stages of 1, 3, 7, 14, 21, and 30 postnatal days, the histo-differentiation of acinar, intercalated and ductal cell components are observed. At newborn day one the cytoplasmic organelles start to place themselves around the nucleus. Several immature secretory granules are observed at day one, however, they increase in the aging days. At postnatal day 30, the cytoplasms of acinar and ductal cells are filled with a large number of secretory granules of different sizes. The stacks of granular endoplasmic reticulum and Golgi apparatus and some vesicles and free ribosomes are noted. The intercellular membranes are attached by desmosomes and cytoplasmic interdigitations. The luminal surface shows several small projections of microvilli. An electron-dense line of basement membranes followed by fine collagen fibrils are recognized. Delicate capillaries are found in the outer surface of acinar cells. At postnatal day 90 and 180 the acinar, intercalated and striated ductal cells reveal numerous secretory granules in the apical portion. The acinar cells showed basal nuclei and the parallel arrangement of granular endoplasmic reticulum. The mitochondria are located at the base of ductal cells showing a typical pattern of cristae. In these stages the intercellular digitations of cytoplasmic protrusions and desmosomes are also noted. The cytoplasm of myoepithelial cells are seen along the cell membranes. The spongy-like structures constituting the basement membrane are followed by bundles of fine collagen fibers.

Animals↗
PubMed1997

Techniques and application of microscopic radioautography.

The techniques for light and electron microscopic radioautography developed in our laboratory are described. The outlines of the applications to various tissues and organs are also briefly summarized. First, the techniques are explained in detail. These techniques can be divided into two categories, i.e., chemical fixation followed by wet-mounting radioautography and cryo-fixation followed by dry-mounting radioautography. The former is limited in application to only insoluble compounds, while the latter is universally applicable to any kind of compounds including soluble compounds. Both procedures, the chemical fixation wet-mounting procedure and the cryo-fixation dry-mounting procedure at light and electron microscopic levels are described. Next, the applications of both the procedures to various compounds are briefly mentioned. Chemical fixation with glutaraldehyde and osmium tetroxide followed by wet-mounting radioautography demonstrates insoluble macromolecular synthesis such as nucleic acids (both DNA and RNA), proteins, glucides and lipids. On the other hand, cyrofixation followed by dry-mounting radioautography demonstrates soluble small molecular compounds such as macromolecular precursors, hormones, neurotransmitters, vitamins, inorganic compounds and drugs or toxins. These procedures are expected to be applied for various inorganic and organic compounds in living organisms to elucidate the sites of their incorporation, synthesis and discharge.

Animals↗
PubMed1996

Induction of recombination activating gene expression in a human lymphoid progenitor cell line: requirement of two separate signals from stromal cells and cytokines.

The activation and expression of recombination activating genes (RAGs) plays critical roles in V(D)J gene recombination machinery and lymphocyte development. We showed that RAG gene expression was induced in freshly isolated human bone marrow cells and a human lymphoid progenitor cell line, FL8.2.4.4, by coculture on a monolayer of a murine bone marrow-derived stromal cell line, PA6, in the presence of a mixture of recombinant cytokines. The RAG transcripts were detected 12 hours after initiation of culture, and the increased level was sustained at 24 hours. Among recombinant cytokines, interleukin-3 (IL-3), IL-6, and IL-7, but not IL-2, IL-4, stem cell factor (SCF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) could induce RAG-1 activation in FL8.2.4.4 cells, and a significant synergistic effect between IL-3, IL-6, and IL-7 was observed. Using a double chamber culture technique, it was shown that a cognate interaction between FL8.2.4.4 cells and PA6 stromal cells was a prerequisite for RAG-1 activation. Furthermore, RAG-1 transcripts were induced in FL8.2.4.4 cells when they were cocultured on paraformaldehyde-fixed PA6 stromal cells in the presence of cytokines. These results suggest that two separate signals are both required for induction of RAG-1 activation in lymphoid progenitors; one from the cell surface molecule(s) on stromal cells, and the other from the recombinant cytokine(s). Finally, we showed that expression of RAG mRNA in FL8.2.4.4 cells was concomitant with induction of recombinase activity. This system may provide useful means for further understanding the mechanisms controlling RAG activation and lymphocyte development in the human system.

Bone Marrow Cells↗
PubMed1996

IFN-gamma induces expression of MHC class I molecules in adult mouse dorsal root ganglion neurones.

The expression of major histocompatibility complex (MHC) class I molecules on adult mouse DRG neurones was analysed using immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). MHC class I molecules were not expressed in untreated dorsal root ganglion (DRG) neurones. The molecules, however, were detected on the surface of neurones following IFN-gamma treatment. In addition, expression of the MHC class I beta 2-microglobulin and genes was induced in neurones following IFN-gamma treatment. Expression of Tap-1 and Lmp-2 genes, which are involved in antigen processing and presentation, was also induced by IFN-gamma treatment. These results indicated that DRG neurones are able to express MHC class I molecules following IFN-gamma treatment and suggest that DRG neurones themselves are susceptible to recognition by cytotoxic T cells.

Animals↗
PubMed1996

Chromatin structure and transcriptional regulation of human RAG-1 gene.

The recombination activating genes (RAGs) play a critical role in V(D)J recombination machinery and lymphocyte development. Their expression is strictly regulated during lymphocyte ontogeny, with expression being rapidly lost as the lymphoid precursors differentiate into their progeny. To elucidate molecular mechanisms of regulation of human RAG-1 gene expression, we examined a chromatin structure of a approximately 24-kb DNA segment adjacent to a human RAG-1 promoter region in various cell lines by analyzing DNase I hypersensitive (DNase I HS) sites. In a RAG-1-expressing human pre-B-cell line, at least four DNase I HS sites (HS1, HS2, HS3, and HS4) were identified. Among these HS sites, one HS site (HS1) was ubiquitously detected in all cell lines examined, but the other three HS sites (HS2, HS3, and HS4) were associated only with RAG-1-expressing lymphoid cell lines. Using transient expression assays, we showed that the 5' upstream region of the major transcription start site showed low but significant promoter activity and that a DNA segment within HS3 located in the promoter region was indispensable to its basal promoter activity. Importantly, this promoter region was shown to be active in both RAG-1-expressing and RAG-1-nonexpressing cell lines. These results suggest that alteration of chromatin structure in the promoter region, in addition to other control elements outside of the promoter region, is one of the mechanisms regulating tissue- and stage-specific expression of human RAG-1 gene.

B-Lymphocytes↗
PubMed1996

Osteotropic factor-stimulated synthesis of thrombospondin in rat dental pulp cells.

The amount of thrombospondin (TSP) mRNA in confluent clonal rat dental pulp cells was increased by transforming growth factor-beta (TGF-beta), 1 alpha-25-dihydroxyvitamin D3 (1,25(OH)2D3), and phorbol 12,13-didecanoate (PDD), with maximal levels 6, 36 and 2 h, respectively, after stimulation. These increases were accompanied by enhanced syntheses of TSP proteins which were found in the different forms in cell layer/matrix fraction (198 and 165 kDa TSP) and the culture medium (180 kDa TSP). These three factors also raised the mRNA level of osteopontin, which is thought to play an important role in mineralization in dentin and bone. The order of effectiveness of these factors was PDD > TGF-beta > 1,25(OH)2D3 for all the stimulations described above. These results suggest that the osteotropic factors enhance TSP synthesis at the pretranslational level and that TSP produced by dental pulp cells participates in formation of reparative dentin.

Animals↗
PubMed1996

Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell.

There is increasing interest in the role of RNA-binding proteins during neural development. Drosophila Musashi is one of the neural RNA-binding proteins essential for neural development and required for asymmetric cell divisions in the Drosophila adult sensory organ development. Here, a novel mammalian neural RNA-binding protein, mouse-Musashi-1, was identified based on the homology to Drosophila Musashi and Xenopus NRP-1. In the developing CNS, mouse-Musashi-1 protein was highly enriched in the CNS stem cell. Single-cell culture experiments indicated that mouse-Musashi-1 expression is associated with neural precursor cells that are capable of generating neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1 expression is lost. This expression pattern of mouse-Musashi-1 is complementary to that of another mammalian neural RNA-binding protein, Hu (a mammalian homologue of a Drosophila neuronal RNA-binding protein Elav), that is expressed in postmitotic neurons within the CNS. In vitro studies indicated that mouse-Musashi-1 possesses binding preferences on poly(G) RNA homopolymer, whereas Hu is known to preferentially bind to short A/U-rich regions in RNA. Based on their differential expression patterns and distinct preferential target RNA sequences, we believe that the mouse-Musashi-1 and Hu proteins may play distinct roles in neurogenesis, either through sequential regulatory mechanisms or differential sorting of mRNA populations during asymmetric division of neural precursor cells.

Amino Acid Sequence↗
PubMed1996

Sensitive determination of bromazepam in human tissues using capillary gas chromatography-mass spectrometry.

A reliable and sensitive gas chromatographic-mass spectrometric method was devised to determine the levels of bromazepam in human tissues. Bromazepam was extracted from body tissues using a three-step solvent extraction procedure. N-Desmethyldiazepam served as the internal standard. Selected ion monitoring with m/z 317 for bromazepam and m/z 270 for internal standard was used for quantitation. Calibration curves in all body tissues were linear over the concentration range from 50-500 ng/g. The lower detection limit in body tissues was 2-5 ng/g and the absolute recovery in body tissues was 27.8-68.0%. This method was used to determine the levels of bromazepam in tissues of an autopsied individual who had been prescribed psychotropic drugs and who was found dead in a car.

Anti-Anxiety Agents↗
PubMed1996

Determination of cyanide and thiocyanate in blood by gas chromatography and gas chromatography-mass spectrometry.

We devised a sensitive and simple method for determining cyanide and its major metabolite, thiocyanate, in blood using an extractive alkylation technique. Pentafluorobenzyl bromide was used as the alkylating agent, and tetradecyldimethylbenzylammonium chloride was used as the phase-transfer catalyst. The derivatives obtained were analyzed qualitatively by gas chromatography-mass spectrometry and quantitatively by gas chromatography with an electron-capture detection. The detection limits of cyanide and thiocyanate were 0.01 and 0.003 mumol/ml, respectively, while the gross recovery of both compounds was 80%. The calibration curve was linear over the concentration range from 0.02 to 1.0 mumol/ml for cyanide and from 0.01 to 1.0 mumol/ml for thiocyanate. The accuracy and precision of the method were evaluated, and the coefficients of variation were found to be within 10%. Using the method, the blood levels of two victims who had died from cyanide poisoning were determined.

Chromatography, Gas↗
PubMed1996

Cell cycle-dependent accumulation of a kinesin-like protein, KatB/C in synchronized tobacco BY-2 cells.

Immunoblot analysis with antibodies prepared against highly purified recombinant truncated kinesin-like proteins, KatB(5-249) and KatC(207-754), encoded by the katB and katC genes of Arabidopsis thaliana revealed the presence of a kinesin-like polypeptide, termed KatB/C, in cultured tobacco BY-2 cells. The KatB/C polypeptide cosedimented with microtubules in the presence of a nonhydrolyzable ATP analogue and was released from microtubules in the presence of ATP, both of which are characteristics of kinesin proteins. The amount of KatB/C polypeptide in synchronous BY-2 cells increased during M phase of the cell cycle. Microtubule-based structures present in cells at M phase, such as the spindle and phragmoplast, may be the site of action of the KatB/C protein.

Arabidopsis↗
PubMed1996

A case of drowning linked to ingested sulfides--a report with animal experiments.

An adult male was found dead beneath a pool of sewage in the pump room of a fish market. Autopsy revealed the cause of death to be suffocation after aspirating sewage into the respiratory tract. Since hydrogen sulfide gas was detected in the atmosphere at the scene of the accident, gas poisoning was suspected and toxicological analysis of sulfides in body tissues was performed. The concentrations of sulfides in the blood, lung and kidney were 0.95 mumol@ml, 0.22 and 0.38 mumol/g, respectively. These values were remarkably higher than those in previously reported cases involving exposure to hydrogen sulfide gas. Therefore, oral intake of sulfides was assumed and the distribution of sulfides in tissues following oral administration of sodium sulfide solution was examined by means of animal experiments using rats. The concentration of sulfides in the blood from rats following oral intake was much higher than that seen following gas exposure. Based on these results, we concluded that the victim had been exposed to hydrogen sulfide gas and had then collapse into a pool of sewage containing sulfides. The sulfides which were distributed throughout the body tissues had mainly issued from the alimentary tract prior to death by drowning.

Adult↗
PubMed1996

Identification of vegetable species in gastric contents using HPLC.

Identification of 16 vegetables, focusing on the influence of digestion in the stomach, was carried out on the basis of the types of flavonoids detected on chromatograms using HPLC. Among the 12 vegetables for which flavonoids were detected, the chromatographic patterns of the flavonoids in digested vegetables were the same as those of the corresponding raw vegetables, making it possible to identify the species of vegetables even after digestion. In our analysis, 5 mg of a freeze-dried sample was found be an adequate quantity to enable the detection of flavonoids. Brief practical cases are also described.

Adult↗
PubMed1996

Triacylated anthocyanins from Ajuga reptans flowers and cell cultures.

Four anthocyanins were isolated from Ajuga reptans flowers and one from the cell cultures. By FAB mass spectrometry measurements, the structures of these pigments were determined as delphinidin and cyanidin glucosides acylated with two cinnamic acids, while three of them were also malonylated. A delphinidin-based pigment in the crude extract from cell cultures was identical to the major flower pigment as shown by HPLC co-chromatography. Moreover, by application of 1H and 13C NMR consisting of DQF-COSY, NOESY, ROESY, 2D-HOHAHA, HSQC and HMBC methods, the structures of two new anthocyanins were identified as delphinidin and cyanidin 3-O-(2-O-(6-O-(E)-p-coumaryl-beta-D-glucopyranosyl)-(6-O-(E)-p- coumaryl)-beta-D-glucopyranosyl)-5-O-(6-O-malonyl-beta-D-glucopyranoside ). The deacylated anthocyanins were confirmed as delphinidin and cyanidin 3-sophoroside-5-glucosides.

Acylation↗