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Biomedical subjects

H Tatsumi

Publications and source records attributed to H Tatsumi.

At least 37 records · Page 2Linked to original sources
PubMed1998

Visualization of calcium channels involved in transmitter release from neuronal growth cones.

Immunocytological localization of omega-agatoxin IVA (omega-aga IVA)-sensitive Ca2+ channels involved in glutamate release from growth cones of cultured rat dorsal root ganglion (DRG) neurons was studied with field emission scanning electron microscopy (FE-SEM) and transmission electron microscopy (TEM). The omega-aga IVA-sensitive Ca2+ channels were visualized by labeling with immuno-gold particles (30 nm). FE-SEM and TEM images showed that immuno-gold particles were present in the area of growth cones as well as somata, and generally absent on neurite stem and fibroblasts. TEM images of vertical ultra-thin sections showed that the immuno-gold particles were present on the surface of the plasma membrane. Since the gold particles indicate the immunological presence of omega-aga IVA-sensitive Ca2+ channels, the Ca2+ channels involved in transmitter release are present on growth cones before making synapse formation.

Animals↗
PubMed1998

Effects of various protease inhibitors on the stability and permeability of [D-Ala2,D-Leu5]enkephalin in the rat intestine: comparison with leucine enkephalin.

The effects of various protease inhibitors on the stability of leucine enkephalin (Leu-Enk) and [D-Ala2,D-Leu5] enkephalin (DADLE) were investigated, and the permeability of these peptides was also examined in an in vitro Ussing chamber. Captopril, thiorphan, bacitracin, bestatin, puromycin, amastatin, and sodium glycocholate (Na-GC) were chosen as protease inhibitors. Regional differences in the stability of Leu-Enk and DADLE were observed, and the rank order of the stability of these peptides was colon > duodenum > ileum > jejunum. Na-GC, amastatin, and puromycin were effective protease inhibitors for improving the stability of these peptides, although captopril and thiorphan did not improve the stability of Leu-Enk. In the transport studies, Leu-Enk did not cross the intestinal membrane in the absence of protease inhibitors, but its transport was improved in the presence of Na-GC. In addition, Na-GC, amastatin, and puromycin improved the permeability of DADLE in both jejunum and colon, while the permeability of DADLE was not improved by the addition of captopril, thiorphan, and bestatin. Furthermore, the permeability of 6-carboxyfluorescein, a poorly absorbable and stable compound, was also improved in the presence of Na-GC and bacitracin at a concentration of 10 mM. These findings indicated that amastatin, puromycin, and Na-GC at a concentration of 0.5 mM might increase the permeability of DADLE due to the improved stability of DADLE in the donor site. However, Na-GC and bacitracin at a concentration of 10 mM had absorption-enhancing activities which might be also related to the enhanced permeability of DADLE across the intestinal membrane.

Animals↗
PubMed1998

Evaluation of highly sensitive thyrotropin assay for detecting thyroid diseases in neonatal screening: preliminary studies.

TSH concentrations in dried blood samples on filter paper were determined by a conventional enzyme-linked immunosorbent assay (ELISA), used for routine neonatal screening for primary hypothyroidism, and a highly sensitive bioluminescence ELISA (BL-ELISA) using firefly luciferase to examine whether central hypothyroidism and hyperthyroidism can be efficiently detected as cases of primary hypothyroidism. Samples were obtained from 3 patients with congenital central hypothyroidism, 5 patients with congenital primary hypothyroidism, 6 patients with hyperthyroidism, 31 neonatal babies with low birth weight (premature babies) and 242 newborn babies with normal birth weight from the general population (normal babies). The TSH values were low in central hypothyroidism and hyperthyroidism. Their deviations from the mean TSH value for normal babies by the BL-ELISA method (-3.12 SD and -4.79 SD in central hypothyroidism and hyperthyroidism respectively) were greater than those by the ELISA method (-2.00 SD and -2.97 SD respectively). The TSH values were high in primary hypothyroidism and normal in premature babies while deviations were the same when BL-ELISA and ELISA were used. These findings indicate that the highly sensitive TSH assay (BL-ELISA) can be used for detecting both primary and central hypothyroidism as well as hyperthyroidism in neonatal screening.

Adult↗
PubMed1998

A high expression of heme oxygenase-1 in the liver of LEC rats at the stage of hepatoma: the possible implication of induction in uninvolved tissue.

We have examined changes in the expression of heme oxygenase-1 (HO-1), an inducible isoform and HO-2, a constitutive isoform, in the liver of Long-Evans with a Cinnamon-like color (LEC) rat, a mutant strain which spontaneously develops acute hepatitis and hepatoma. HO-1 expression was highly enhanced in the LEC rat livers with jaundice, and then decreased slightly, but overall remained at a higher level than in the Long-Evans with Agouti color (LEA) control rats, as judged by Northern blotting analysis of the whole liver extract. The high expression of HO-1 in the LEC rat liver was, however, not due to the actual cancer lesion but, rather, due to the surrounding uninvolved tissues including hepatocytes. Immunohistochemical analysis also supported this conclusion. Among normal tissues, the expression of HO-1 but not HO-2 was high in only the spleen of both LEC and LEA rats. The high expression observed in the stage of acute hepatitis and hepatoma stages in the LEC rat is probably due to the oxidative stress caused by the accumulation of free copper and free iron levels which has been reported earlier by our group (Suzuki et al., Carcinogenesis, 1993, 14, 1881-1884 and Koizumi et al., Free Radical Research, in press) as well as by free heme levels. The inflammatory cytokines produced by the surrounding tissue at the hepatoma stage would also be expected to play a role in the induction mechanism. The physiological relevance of HO-1 induction might be an adaptive response to oxidative stress and vasodilatory effect of carbon monoxide on sinusoidal circulation.

Age Factors↗
PubMed1998

Medical Internet exchange project in Japan.

The Internet has been widely used by medical institutes and hospitals around the world, however; its use for telemedicine is still low. The main reason for this is the availability of bandwidth and poor security through the net. Meanwhile, we have established and have been operating 'Cancer Information Network' among 11 Cancer Centers in Japan, mainly for Multipoint TV Conference using HDTV image. There are also similar projects among 9 cardiovascular centers in Japan. By March, all 240 national hospitals will have been connected by an IP network using an ATM backbone. The above network projects are operated independently, and have an 'Intranet' characteristics within them. There are also many hospitals and clinics connected to the Internet by commercial internet providers. To make a secure and efficient network between these medical networks and medical sites, we started the Medical Internet eXchange project (MDX project) constructing a Medical Network Operation Center to create a link between them. To provide the administrative policy of this project, we established the Medical Internet eXchange Association. We are planning to expand this project to Asian-Pacific countries using the Asian-Pacific Advanced Network (APAN), and also expand it to worldwide connections in the future. For this purpose, we are currently asking other countries to form a structure similar to MDX-Japan. The concept, hardware system, software system, firewall configuration, and routing policy will be also discussed.

Computer Systems↗
PubMed1997

Neurotransmitter release from growth cones of rat dorsal root ganglion neurons in culture.

Growing neurites of rat dorsal root ganglion neurons in culture formed growth cones at the tips. Possible release of glutamate from these growth cones was investigated by using a whole-cell patch-clamp recording from an acutely dissociated hippocampal neuron containing glutamate receptors. The hippocampal neuron was placed in contact to various regions of the dorsal root ganglion neurons. Inward currents were recorded from the hippocampal neuron positioned on the growth cones of the dorsal root ganglion neurons (diameter, 12-16 microm) in response to the dorsal root ganglion cell body stimulation. The inward currents were associated with an increase in membrane conductance, and the reversal potential was estimated at -6.5 mV (n=8). The inward currents were blocked by 6-cyano-7-nitroquinoxaline (10 microM), but not blocked by 2-amino-5-phosphonovaleric acid (50 microM) and bicuculline (10 microM). The inward currents were abolished by tetrodotoxin (1 microM), EGTA-buffered Ca2+-free external solution or omega-agatoxin IVA (300 nM), and were inhibited by omega-conotoxin GVIA (3 microM), but were not affected by nicardipine (10 microM). Intracellular calcium ion concentration ([Ca2+]i) in growth cones of the dorsal root ganglion neurons increased in response to dorsal root ganglion cell body stimulation, whereas the elevation of [Ca2+]i was not observed either in the presence of tetrodotoxin (1 microM) or in a Ca2+-free external solution. These results indicate that the inward currents were evoked by glutamate released from the growth cones via a Ca2+-dependent process, and suggest that the growth cones are already endowed with much of the machinery for neurotransmitter release, even before making a structure for synaptic transmission.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1997

Induction of apoptotic cell death in human endothelial cells treated with snake venom: implication of intracellular reactive oxygen species and protective effects of glutathione and superoxide dismutases.

Human vascular endothelial cells play a pivotal role in atherosclerotic changes but are resistant to apoptotic inducers such as Fas ligand and it has been difficult to induce apoptosis. We developed an experimental model for the apoptosis in the endothelial cells by using snake venom treatment. Snake venom was found to generate intracellular reactive oxygen species (ROS) in the endothelial cells, which leads to apoptosis as judged by electron microscopy as well as by DNA cleavage. Buthionine sulfoximine (BSO) and diethyldithiocarbamate (DDC) accelerated the apoptosis, indicating intracellular glutathione and superoxide levels play a critical role. Pretreatment with tumor necrosis factor (TNF) or phorbol ester (TPA), which increases the Mn-SOD level, prevented the apoptosis. These data suggest that intracellular ROS enhances apoptosis whereas several anti-oxidants are protective in human endothelial cells. The induction of apoptosis by ROS of endothelial cells may be related to initiation of atherosclerotic changes.

Animals↗
PubMed1996

Reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress through the glycation reaction.

Several reducing sugars brought about apoptosis in isolated rat pancreatic islet cells and in the pancreatic beta-cell-derived cell line HIT. This apoptosis was characterized biochemically by inter-nucleosomal DNA cleavage and morphologically by nuclear shrinkage, chromatin condensation and apoptotic body formation. N-Acetyl-L-cysteine, an antioxidant, and aminoguanidine, an inhibitor of the glycation reaction, inhibited this apoptosis. We also showed directly that proteins in beta-cells were actually glycated by using an antibody which can specifically recognize proteins glycated by fructose, but not by glucose. Furthermore, fluorescence-activated cell sorting analysis using dichlorofluorescein diacetate showed that reducing sugars increased intracellular peroxide levels prior to the induction of apoptosis. Levels of carbonyl, an index of oxidative modification, and of malondialdehyde, a lipid peroxidation product, were also increased. Taken together, these results suggest that reducing sugars trigger oxidative modification and apoptosis in pancreatic beta-cells by provoking oxidative stress mainly through the glycation reaction, which may explain the deterioration of beta-cells under conditions of diabetes.

Acetylcysteine↗
PubMed1996

Construction of biotinylated firefly luciferases using biotin acceptor peptides.

A cDNA for a thermostable mutant of Luciola lateralis (a Japanese firefly) luciferase was fused with either a gene for an artificial biotin acceptor peptide No. 84 [P. J. Schatz (1993) Bio/Technology 11, 1138-1143] or a gene for the carboxyl-terminal 87 residues of Escherichia coli biotin carboxyl carrier protein. The fused genes, when introduced into separate E. coli, directed the expression of luciferases that were able to bind with streptavidin. We purified them and showed that their specific activities and thermal stabilities remained unchanged. We also found that more than 95% of each fusion protein was biotinylated, suggesting that the biotin holoenzyme synthetase in the host cells worked efficiently. Using the biotinylated luciferases, we developed a highly sensitive bioluminescent enzyme immunoassay system.

Animals↗
PubMed1996

Evaluation of human skin irritation by carboxylic acids, alcohols, esters and aldehydes, with nitrocellulose-replica method and closed patch testing.

Closed patch testing and the nitrocellulose-replica method are performed as useful clinical methods for the evaluation of human skin irritation by cosmetics and topical medicaments. Comparison of the sensitivity between microscopic scoring by nitrocellulose-replica method and visual scoring by closed patch test in the detection of skin irritation, however, has not been well studied with statistical analysis. Here, we evaluated human skin irritation by carboxylic acids, alcohols, esters and aldehydes, with different chain length (C8-C18), using both methods. The results of closed patch testing showed that, although the score of skin irritation for carboxylic acids (C8, C12), alcohols (C8) and aldehydes (C8), tested at a concentration of 0.5 m-2.0 m, significantly increased with increasing concentration of the test compounds, ester compounds scarcely caused any irritation on the surface of the skin occluded. In addition, an increase of carbon chain length in the test compounds made it impossible to detect skin irritation. In contrast, the nitrocellulose-replica method could evaluate skin reactions against very weak irritants that gave no macroscopic alterations on the skin surface in the closed patch test. However, the scoring system is somewhat subjective and should be improved to make the analysis more objective.

Adult↗
PubMed1995

Synthesis, storage and release of acetylcholine at and from growth cones of rat central cholinergic neurons in culture.

Neurons from the nucleus diagonal band of Broca (DBB) from new born rats protrude neuronal processes and growth cones in culture. Cytochemical observations with the light and electron microscope indicate that growth cones of these neurons take up choline, synthesize acetylcholine (ACh) and store ACh in the vesicles. Electrical stimuli at the soma of DBB neurons evoked inward currents in ACh-sensitive neurons attached to DBB growth cones. These currents were suppressed by TTX, a Ca2+ channel blocker (Cd2+), and an ACh nicotinic antagonist (C6). These results suggest that ACh is synthesized, stored and released from the growth cones of DBB neurons prior to synapse formation.

Acetylcholine↗
PubMed1995

Induction of nitric oxide synthase and concomitant suppression of superoxide dismutases in experimental colitis in rats.

Reactive oxygen species are thought to play an important role in some bowel diseases. In order to evaluate the participation of nitric oxide and superoxide in such diseases, we examined the expression of nitric oxide synthase (NOS) and superoxide dismutase (SOD) as well as their activities in whole excised colons of rats with colitis induced by intralumenal administration of 2,4,6-trinitrobenzenesulfonic acid. A marked increase in the inducible form of NOS mRNA was detected and NOS activity was coincidentally augmented in the group administered unbuffered TNBS (pH 1.0), in which severe inflammation was revealed by microscopic examination and myeloperoxidase activity of invading neutrophils in the tissues. The levels of the Mn- and Cu,Zn-SOD proteins as well as SOD activity were suppressed, although expression of the Mn-SOD mRNA was enhanced in colitis tissues. The elevation of NOS activity and the suppression of SOD activity occurred concomitantly at the stage of severe inflammation. This would increase peroxynitrite formation from superoxide and nitric oxide and enhance the tissue damage in experimental colitis.

Animals↗
PubMed1995

Na+ dependent Ca2+ influx induced by depolarization in neurons dissociated from rat nucleus basalis.

Neurons were acutely dissociated from the rat nucleus basalis, and whole-cell patch clamp recordings were made. Voltage dependent calcium currents (ICa) were recorded and fura-2 microfluorimetric recordings of intracellular free Ca2+ concentration ([Ca2+]i) were made at the same time. In Na(+)-containing solution, a depolarization from -60 to +40 mV evoked the maximal increase in [Ca2+]i, and this decreased to 43% of the maximal with a large depolarization to +120 mV. The [Ca2+]i increase induced by the large depolarization (+20 to +120 mV) was inhibited by perfusion of Na(+)-free external solution, and was less when the recording pipette contained a peptide (PRLLFYKYVYKRYRAGKQRG, named XIP) known to inhibit Na/Ca exchange. These results suggest that the [Ca2+]i increase by the large depolarization is mediated by reverse operation of Na/Ca exchange (Ca2+ inward and Na+ outward).

Animals↗
PubMed1995

Analysis of Ca2+ homeostasis in neurons dissociated from rat nucleus basalis.

Whole-cell patch-clamp recordings of calcium currents (ICa) and fura-2 microfluorimetric measurements of intracellular free Ca2+ concentration ([Ca2+]i) were made simultaneously in neurons acutely dissociated from rat nucleus basalis. Depolarization activated ICa and caused an increase in [Ca2+]i. The relationship between total Ca2+ influx and the increase in [Ca2+]i was studied. After repolarization, [Ca2+]i recovered to control values within a few seconds. A mathematical model was constructed to simulate the mechanisms underlying [Ca2+]i regulation; the parameters were (1) the rate of Ca2+ influx, (2) the rate of the [Ca2+]i increase by the Ca2+ influx, and (3) the rate of Ca2+ clearance from cytosol due to extrusion across the plasma membrane and sequestration into calcium storing organelles. After an appropriate evaluation of parameter values from the experimental results, the model mimicked the processes of [Ca2+]i increase and recovery. The experimental results and simulations suggest that (1) neurons possess a large Ca2+ buffering capacity, (2) systems for Ca2+ clearance are activated by the Ca2+ influx in a saturable manner, (3) the rate of Ca2+ clearance is relatively small compared to the rate of Ca2+ influx evoked by depolarizations, and (4) the shoulder in the [Ca2+]i recovery phase is due to the asymptote of the Ca2+ clearance rate.

Animals↗
PubMed1995

Apoptotic cell death triggered by nitric oxide in pancreatic beta-cells.

Nitric oxide (NO) is believed to be an effector molecule that mediates interleukin (IL)-1 beta-induced destruction and dysfunction of pancreatic beta-cells. We have demonstrated that both exogenous NO and NO generated endogenously by IL-1 beta brought about apoptosis of isolated rat pancreatic islet cells as well as pancreatic beta-cell tumor-derived cell line HIT. This apoptosis was characterized by cleavage of DNA into nucleosomal fragments of 180-200 bp and morphologically by nuclear shrinkage, chromatic condensation, and apoptotic body formation. The IL-1 beta-induced internucleosomal DNA cleavage occurred in a time- and dose-dependent manner. Actinomycin D, cycloheximide, and nitric oxide synthase inhibitors inhibited the DNA cleavage, which was correlated with the amount of NO produced, indicating that NO produced by HIT cells themselves could mediate the apoptosis. Furthermore, in the presence of tumor necrosis factor (TNF)-alpha, large amounts of NO were produced by IL-1 beta and DNA cleavage occurred more noticeably, although TNF-alpha alone did not generate NO. Streptozotocin (STZ), a diabetogenic reagent containing a nitroso moiety, also released NO and induced internucleosomal DNA cleavage in HIT cells. These results suggest that NO-induced internucleosomal DNA cleavage is an important initial step in the destruction and dysfunction of pancreatic beta-cells induced by inflammatory stimulation or treatment with STZ.

Amino Acid Oxidoreductases↗
PubMed1995

Morphological studies on the kidney of the spontaneous nephrotic (ICGN) mice in the late stage.

Spontaneous nephrotic (ICGN) mice develop proteinuria, hypoproteinemia and hypercholesterolemia. These symptoms steadily progress to chronic renal failure. Details of the changes of the kidney, in the late stage (more than 5 months old) were investigated by both light and electron microscopy. The kidney exhibited a slightly whitish, granular surface and the cortex became thinner and contained fibrous lesions, in which clusters of unaffected and occluded renal tubules were randomly scattered. In the juxtamedullary and outer medullary zone, there were highly dilated renal tubules, which sometimes contained urinary casts. The glomerulus exhibited basement membrane thickening in the capillary loops and the capillary lumen was narrowed in size and sometimes occluded. No detachment of the podocyte from the basement membrane was observed and the podocyte foot-processes were extensively fused, causing their characteristic slits to be lost. The thickened basement membranes were found both in the glomerulus and around the occluded renal tubules, while the basement membrane in the dilated renal tubule appeared normal. Therefore, the basement membranes of the glomerulus and renal tubules appear to react differently in the pathogenesis of the condition. In conclusion, ICGN mice are a good model for not only the nephrotic syndrome but also for chronic renal failure.

Animals↗