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Biomedical subjects

M Tohyama

Publications and source records attributed to M Tohyama.

At least 163 records · Page 9Linked to original sources
PubMed1996

Reg gene expression is increased in rat gastric enterochromaffin-like cells following water immersion stress.

BACKGROUND & AIMS: Reg gene has been isolated from regenerating rat pancreatic islets, and subsequent studies have shown a trophic effect of Reg protein on islet cells. However, little is known about the role of Reg protein in the stomach. The aim of this study was to clarify the localization of Reg messenger RNA (mRNA) and its product in the stomach and to examine changes in the level of their expression during regeneration of gastric mucosal cells. METHODS: Gastric lesions were experimentally induced in Sprague-Dawley rats by water immersion stress. Northern blot analysis and in situ hybridization studies were performed to examine changes in mRNA levels. Immunohistochemical studies were performed to identify the cellular localization and to investigate the change in Reg protein level. RESULTS: Reg mRNA and its product were distributed in the basal part of the oxyntic mucosa and were expressed mainly in enterochromaffin-like cells. Levels of both Reg mRNA and its product were markedly increased in the water immersion-induced gastric lesions. CONCLUSIONS: Reg mRNA and its product are expressed in gastric enterochromaffin-like cells, and their levels are increased during the healing process of water immersion-induced gastric lesions.

Animals↗
PubMed1996

Induction of Na+/myo-inositol cotransporter mRNA after focal cerebral ischemia: evidence for extensive osmotic stress in remote areas.

Myo-inositol is one of the major organic osmolytes in the brain. It is accumulated into cells through an Na+/ myo-inositol cotransporter (SMIT) that is regulated by extracellular tonicity. To investigate the role of SMIT in the brain after cerebral ischemia, we examined expression of SMIT mRNA in the rat brain after middle cerebral artery occlusion, which would reflect alteration of extracellular tonicity. The expression of SMIT mRNA was markedly increased 12 h after surgery in the cortex of the affected side and lasted until the second day. Increased expression was also found in the contralateral cingulate cortex. Up-regulated expression was found predominantly in the neurons in remote areas, although nonneuronal cells adjacent to the ischemic core also expressed this mRNA. These results suggest that cerebral ischemia causes extensive osmotic stress in brain and that the neuronal cells respond to this stress by increasing SMIT expression.

Animals↗
PubMed1996

Enhancing effect of oxygen radical scavengers on murine macrophage anticryptococcal activity through production of nitric oxide.

We examined the roles of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) in interferon-gamma (IFN-gamma)-induced cryptococcostatic activity of murine peritoneal macrophages using N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of RNI synthesis, and superoxide dismutase (SOD) and catalase, oxygen radical scavengers. IFN-gamma-activated macrophages produced nitric oxide (NO) in a dose-dependent manner, as measured by increased nitrite concentration in the culture supernatant. IFN-gamma also enhanced the suppressive effect on cryptococcal growth in a similar dose-dependent manner. The induction of killing activity and NO production by an optimal dose of IFN-gamma (100 U/ml) was virtually suppressed by 500 microM L-NMMA. These results confirmed the importance of the RNI-mediated effector mechanism in anticryptococcal activity of macrophages. SOD and catalase significantly enhanced the cryptococcostatic activity of macrophages induced by a suboptimal dose of IFN-gamma (20 U/ml). The augmenting effect of these reagents was mediated by NO, since they potentiated the production of NO by macrophages and their effects were totally blocked by L-NMMA. Our results indicate that the IFN-gamma-induced anticryptococcal activity of macrophages is dependent mostly on RNI, and suggest that the ROI system down-regulates the effector mechanism for cryptococcostasis by suppressing the RNI system.

Animals↗
PubMed1996

Anticryptococcal effect of amphotericin B is mediated through macrophage production of nitric oxide.

Amphotericin B (AmB) is a classical antifungal drug and one of the most effective antifungal drugs for the treatment of systemic fungal infection. It is also known to have various immunomodulating activities other than its direct antifungal effect. In the present study, we demonstrated that AmB augmented gamma interferon (IFN-gamma)-induced killing potentials of murine peritoneal macrophages against Cryptococcus neoformans in a dose-dependent manner. This effect was strongly blocked by NG-monomethyl-L-arginine, a competitive inhibitor of nitric oxide (NO) synthesis. In addition, AmB markedly augmented macrophage NO production induced by IFN-gamma with a dose-response curve similar to that seen with its effect on the anticryptococcal activity. These effects were partially mediated by either tumor necrosis factor alpha or interleukin-1, because AmB enhanced IFN-gamma-induced production of these cytokines by macrophages and their specific antibodies partially inhibited the AmB-induced enhancement of NO generation when they were used separately. Our results indicate that AmB induces the production of tumor necrosis factor alpha and IL-1 by macrophages and augments their anticryptococcal activity through triggering the NO-dependent pathway.

Amphotericin B↗
PubMed1996

[CFTR as cAMP-dependent chloride channels and as cAMP-dependent regulator of sodium channels].

Cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in cystic fibrosis (CF) patients. It has been established that CFTR functions as a cAMP-dependent chloride channel. Therefore mutation of CFTR causes the abnormality of the chloride ion secretion. Furthermore, it has been shown recently that CFTR functions as cAMP-dependent negative regulator of sodium channels. The evidence accounts an abnormally high rate of sodium ion in the airway epithelia of the CF patients.

Chloride Channels↗
PubMed1996

Visualization of ischemic insult in caudate putamen with beta-CIT.

UNLABELLED: Dopamine (DA) has been considered to play an important role in the development of ischemic neuronal injury in the caudate putamen (CPu). The goal of this study was to examine the change in the dopamine transporter (DAT) after ischemic insult in CPu. METHODS: Male Mongolian gerbils (n = 10) were exposed to 10-min forebrain ischemia. Animals were decapitated 24 hr (n = 5) and 96 hr (n = 5) after ischemia. The change in the amount of DAT binding sites in CPu was evaluated by in vitro autoradiography with [125I]-beta-CIT (3 beta-(4-iodophenyl)tropan-2 beta-carboxylic acid methyl ester). In addition, the expression of DAT mRNA in CPu and the substantia nigra pars compacta (SNC) was examined. RESULTS: Iodine-125-beta-CIT specific binding was significantly increased in dorsolateral CPu with ischemic damage both 24 hr and 96 hr after ischemia, with greater increase at 96 hr. DAT mRNA in SNC was also significantly increased 96 hr after ischemia, which corresponded with the increase of [125I] beta-CIT binding. However, DAT mRNA in SNC was decreased 24 hr after ischemia. In the ischemic lesion in CPu, no expression of DAT mRNA could be detected both 24 hr and 96 hr after ischemia. CONCLUSION: The change in DAT after ischemic insult is clarified with [125I] beta-CIT. This increase of [125I] beta-CIT binding does not come from de novo expression of DAT in glial cells in the damaged area in CPu. This increase of beta-CIT binding reflects increase of DAT synthesis in DA neurons in SNC (96 hr) or other factors such as the impairment of the degradation of DAT in the damaged area in CPu.

Animals↗
PubMed1995

Localization of novel receptor tyrosine kinase genes of the eph family, MDK1 and its splicing variant, in the developing mouse nervous system.

In the course of studies to identify new members of the eph family of receptor tyrosine kinases, MDK1 and one of its splicing variants lacking a kinase domain, MDK1-T1, were identified. To gain insight into the functions of these subtypes, expression patterns of their mRNAs in the developing mouse nervous system were examined by Northern blotting and in situ hybridization histochemistry. Colocalization of their mRNAs was observed, but the levels of expression of each mRNA were developmentally regulated. These findings suggest functional differences between full-length and truncated forms of MDK1 receptor tyrosine kinase.

Animals↗
PubMed1995

Cloning of a novel RNA binding polypeptide (RA301) induced by hypoxia/reoxygenation.

Astrocytes have a critical role in the neuronal response to ischemia, as their production of neurotrophic mediators can favorably impact on the extreme sensitivity of nervous tissue to oxygen deprivation. Using a differential display method, a novel putative RNA binding protein, RA301, was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed two ribonucleoprotein domains and serine/arginine-rich domains, suggestive of their function as RNA splicing factor. Northern analysis displayed striking induction only in cultured astrocytes within 15 min of reoxygenation and reached a maximum by 60 min after hypoxia/reoxygenation. Immunoblotting demonstrated expression of an immunoreactive polypeptide of the expected molecular mass, 36 kDa, in lysates of hypoxia/reoxygenated astrocytes. Induction of RA301 mRNA was mediated, in large part, by endogenously generated reactive oxygen species, as shown by diphenyl iodonium, an inhibitor of neutrophil-type nicotinamide adenine dinucleotide phosphate oxidase which blocks oxygen-free radical formation by astrocytes. Similarly, increased expression of RA301 in supporting a neurotrophic function of astrocytes was suggested by inhibition of interleukin-6 elaboration, a neuroprotective cytokine, in the presence of antisense oligonucleotide for RA301. These studies provide a first step in characterizing a novel putative RNA binding protein, whose expression is induced by oxygen-free radicals generated during hypoxia/reoxygenation, and which may have an important role in redirection of biosynthetic events observed in the ischemic tissues.

Aerobiosis↗
PubMed1995

A relationship between substance P receptor and retinal fibers in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the location of substance P receptor (SPR)-containing neurons in the rat SCN, using a specific antibody against SPR, which corresponds to the NK-1 subtype of tachykinin receptors, and also examined the synaptic relationship between SPR-containing neurons and retinal fibers at the ultrastructural level. An SPR-immunoreactive meshwork of labeled somata and dendrites was identified in the SCN. The strongest SPR-immunoreactivity was observed in the dorsal and lateral parts of the SCN. Many labeled somata were identified there and their dendrites protruded ventrally from their somata. A few SPR-immunoreactive somata were observed also in the ventral part of the SCN and within the optic tract. In the SCN of eye-enucleated animals, degenerating retinal fibers were shown to terminate on SPR-immunoreactive dendrites forming asymmetrical axo-dendritic contacts.

Animals↗
PubMed1995

Distribution of the basic fibroblast growth factor and its receptor gene expression in normal and degenerated rat intervertebral discs.

STUDY DESIGN: Using a rat spondylosis model, the distributions of cells expressing the basic fibroblast growth factor and its receptor were investigated in normal and degenerated intervertebral discs. Cell-proliferating activity in degenerated discs was also assessed. OBJECTIVES: This study was conducted to determine whether basic fibroblast growth factor is related to intervertebral disc degeneration. SUMMARY OF BACKGROUND DATA: Basic fibroblast growth factor stimulates proliferation and matrix synthesis of cultured intervertebral disc cells. METHODS: Immunohistochemistry and in situ hybridization histochemistry were conducted to detect cells with basic fibroblast growth factor-like immunoreactivity and fibroblast growth factor receptor messenger RNA, respectively. Cell-proliferating activity was evaluated by AgNOR staining. RESULTS: In degenerated discs, round chondrocytes with basic fibroblast growth factor-like immunoreactivity and fibroblast growth factor receptor messenger RNA are scattered instead of spindle-shaped cells in the normal anulus (normal anular cells), which are devoid of basic fibroblast growth factor-like immunoreactivity and fibroblast growth factor receptor messenger RNA. The proliferating activity of these chondrocytes is suggested to exceed that of normal anular cells. CONCLUSION: Basic fibroblast growth factor is suggested to promote proliferation of chondrocytes in degenerated discs in an autocrine or paracrine manner. Basic fibroblast growth factor may be related to intervertebral disc degeneration as a proliferation-stimulating factor of chondrocytes that replace normal anular cells during disc degeneration.

Animals↗
PubMed1995

Expression of glutamate (AMPA type) and gamma-aminobutyric acid (GABA)A receptors in the rat caudal trigeminal spinal nucleus.

The localization of GABAA receptor gamma 1 and gamma 2 subunits and the AMPA-type glutamate receptor subunits GluR1 and GluR2/3 were identified in the caudal trigeminal spinal tract nucleus (TNC) by immunohistochemistry using specific antibodies. The receptor species on the projecting neurons to the thalamus in TNC were also examined. A retrograde tracer, Fluoro-gold (FG), was injected into the thalamus, and the sections were simultaneously labeled with the antibodies. Injection of FG into the ventral posteromedial nucleus of the thalamus resulted in labeling of scattered neurons contralaterally in the TNC. Most of the neurons labeled by retrograde tracing also showed gamma 1- and gamma 2-like immunoreactivity, while many of the neurons containing FG lacked GluR1- and GluR2/3-like immunoreactivity. These findings show that neurons projecting to the thalamus from the TNC receive GABAergic input via GABAA receptors containing gamma 1 and gamma 2 subunits, while many neurons expressing the AMPA-type receptor did not project to the thalamus.

Animals↗
PubMed1995

Postnatal development of the substance P-, neuropeptide Y- and serotonin-containing fibers in the rat suprachiasmatic nucleus in relation to development of the retino-hypothalamic projection.

The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the development of substance P (SP)-, neuropeptide Y (NPY)- and serotonin (5-hydroxytryptamine, 5-HT)-immunoreactive fibers in the rat SCN and the development of the retino-hypothalamic tract using cholera toxin beta subunits (CTB), in order to understand which parts of the SCN participate in diurnal rhythm regulation and entrainment. In newborn rats, SP-, NPY- and 5-HT-immunoreactive fibers were scarcely detected in the SCN. The number of SP-immunoreactive fibers gradually increased between postnatal days (P) 15 and 30. At P30, the distribution pattern of SP-immunoreactive fibers in the SCN was similar to that in the adult rat. The number of NPY- and 5-HT-immunoreactive fibers increased greatly between P10 and P15, and the increase in NPY- and 5-HT-immunoreactive fibers continued until P20. CTB was injected into the unilateral eyeball of the rat at various postnatal stages. In neonates, several labeled retinal fibers already existed in the ventral part and ventro-lateral edge of the SCN. The number and density of labeled retinal fibers in the SCN gradually increased between P10 and P20. Between P20 and P30, a decrease in the labeling was observed in the dorsolateral part of the SCN. The adult pattern of labeled retinal fibers was achieved between P20 and P30. The development of SP-immunoreactive fibers was delayed about 10 days relative to that of NPY-, 5-HT-immunoreactive fibers and retinal fibers.

Animals↗
PubMed1995

Mac1 discriminates unusual CD4-CD8- double-negative T cells bearing alpha beta antigen receptor from conventional ones with either CD4 or CD8 in murine lung.

Pulmonary intraparenchymal leukocytes were purified from normal mice. By flow cytometry, 20-30% of the lymphocytes were positive for the expression of Mac1, a cell-surface antigen largely restricted to macrophages, neutrophils and natural killer (NK) cells. Sorted Mac1+ lung lymphocytes were large and had abundant cytoplasm with few azurophilic granules. Because Mac1+ lymphocytes did not contain any asiallo GM1+ cells, they are not likely to be NK cells. By a two-color flow cytometric analysis, Mac1+ lymphocytes were demonstrated to be TCR-alpha beta intermediate+, TCR-gamma delta-, CD3intermediate+, CD4-, CD8-, Thy1-, CD5-, and B220-. These Mac1+ alpha beta T cells were not found in other organs such as spleen, thymus, liver, bone marrow and intestine of mice uninfected and infected with Mycobacterium bovis BCG. There was a considerable population of this unusual subset of alpha beta T cells in the lungs of congenitally athymic nude mice. In the Mac1+ alpha beta T-cell population, the proportions of V beta 8+ T cells and of forbidden T-cell clones expressing V beta 6 TCR were not much different from that in the conventional T-cell population. These results indicated that extrathymically developed alpha beta T cells reside in considerable proportions in the lung and that Mac1 clearly discriminates these cells from conventional ones. Interestingly, the proportion of these cells increased in the lungs of mice infected with M. bovis BCG, which raises a possibility that these cells may play some role in the host defense against mycobacterial infection.

Animals↗
PubMed1995

Expression and phosphorylation of rat c-met/hepatocyte growth factor receptor during rat liver regeneration.

Hepatocyte growth factor receptor is identified as a heterodimeric tyrosine kinase encoded by the c-met gene. This study was designed to determine how the c-met/hepatocyte growth factor receptor participates in the intracellular events involved in rat liver regeneration induced by administration of carbon tetrachloride. Expression of the rat c-met mRNA increased, peaking 24 h after carbon tetrachloride administration almost in parallel with MET protein expression. Histochemical studies demonstrated that expression of the rat c-met was enhanced in cells surrounding the damaged areas, and also that the distribution of cells expressing MET was almost in accordance with that of cells expressing proliferating cells nuclear antigen. The MET protein underwent intense tyrosine phosphorylation peaking at 12 h after carbon tetrachloride administration, and prior to DNA synthesis. Phospholipase C gamma and phosphatidylinositol 3-kinase, intracellular signal transducing molecules containing Src homology 2 domain, were associated with the MET protein following tyrosine phosphorylation in vivo. These observations suggest that expression and tyrosine phosphorylation of MET protein associated with signal transducing molecules may provide a mechanism whereby hepatocyte growth factor exerts its action on hepatocyte growth during rat liver regeneration induced by carbon tetrachloride administration.

Animals↗
PubMed1995

Changes in growth inhibitory factor mRNA expression compared with those in c-jun mRNA expression following facial nerve transection.

We investigated growth inhibitory factor (GIF) mRNA expression within the rat facial nucleus with the aid of in situ hybridization. We found that GIF mRNA was expressed abundantly in the facial motoneurons of sham operated animals, and that this gene expression decreased after transection of the facial nerve. This decrease of GIF mRNA was first detected on the third day and was maintained for at least five weeks after transection of the nerve. Changes in c-jun, an immediate early gene, were also investigated with this model, and it was found that c-jun mRNA started to increase in the facial nucleus on the first day and that this increase was maintained for at least 5 weeks. These results suggest that the facial motoneurons, when their axons are transected, continuously respond to the injury and that GIF mRNA is actively suppressed to reduce the inhibition of neurite outgrowth in order to regenerate the axons.

Animals↗
PubMed1995

Cloning and expression of a bovine glutamate transporter.

We have isolated a 3845 base-pair cDNA (BNGLUAS) encoding a bovine glutamate transporter (bovine GLAST) by screening a bovine retina cDNA library with an oligonucleotide probe corresponding to conserved regions of known glutamate transporters. The cDNA sequence predicted a protein of 542 amino acids and displayed 96% and 97% amino acid identity with the rat GLAST/GluT-1 and human GLAST, respectively. Expression of the bovine GLAST in Xenopus oocytes revealed Na(+)-dependent [14C]L-glutamate uptake and electrogenic glutamate uptake.

ATP-Binding Cassette Transporters↗
PubMed1995

Differential expressions of the eph family of receptor tyrosine kinase genes (sek, elk, eck) in the developing nervous system of the mouse.

To examine the roles of the eph subfamily of receptor tyrosine kinase (RTK), we isolated mouse cDNAs for sek, elk, and eck and localized their mRNAs in the developing mouse, with particular reference to the CNS development, by in situ hybridization. sek mRNA is most abundantly expressed throughout development; sek was detected in the germinal layer of the embryonic CNS during mid- to late-gestation and was widely expressed in the early postnatal brain. elk was expressed in the mantle layer of the embryonic CNS and showed a distribution complementary to that of sek. Differential expression of sek and elk was also observed in the early postnatal cerebellum; sek was expressed in the Purkinje cells, while elk was detected in the granule cells. eck was moderately expressed in the germinal layer of the embryonic CNS at mid-gestation, but its expression decreased as development proceeded. These spatio-temporally different patterns of gene expression suggest that these RTKs have distinct roles in mouse development despite their structural homology.

Amino Acid Sequence↗