Biomedical subjects

M Tohyama

Publications and source records attributed to M Tohyama.

At least 109 records · Page 6Linked to original sources

PubMed1998

Substance P receptor (NK1) gene expression in synovial tissue in rheumatoid arthritis and osteoarthritis.

The distribution of messenger RNA coding for substance P receptor (NK1) in rheumatoid synovia and the relationship between signal intensity of NK1 mRNA and clinical parameters of disease activity were investigated. The synovia from 10 patients with rheumatoid arthritis (RA) and 7 patients with osteoarthritis was investigated by in situ hybridization histochemistry. In rheumatoid synovia, strong hybridization signal of NK1 mRNA was detected in 80% of the cells in both the lining layer and the interstitial layer. Moreover, the signal intensities of NK1 mRNA positively correlated with serum C-reactive protein levels and radiographic grade of joint destruction. These results provide histochemical evidence that rheumatoid synoviocytes strongly express NK1 gene, and the positive relation of the signal intensity of NK1 mRNA with CRP and radiographic severity suggests that the facilitation of NK1 gene expression in rheumatoid synovium relate the disease progression of RA.

Adult↗

PubMed1998

Ketamine inhibits monoamine transporters expressed in human embryonic kidney 293 cells.

BACKGROUND: Ketamine has been characterized as having psychotomimetic and sympathomimetic effects. These symptoms have raised the possibility that ketamine affects monoaminergic neurotransmission. To elucidate the relation between ketamine and monoamine transporters, the authors constructed three cell lines that stably express the norepinephrine, dopamine, and serotonin transporters and investigated the effects of ketamine on these transporters. METHODS: Human embryonic kidney cells were transfected using the Chen-Okayama method with the human norepinephrine, rat dopamine, and rat serotonin transporter cDNA subcloned into the eukaryotic expression vector. Using cells stably expressing these transporters, the authors investigated the effects of ketamine on the uptake of these compounds and compared them with those of pentobarbital. RESULTS: Inhibition analysis showed that ketamine significantly inhibited the uptake of all three monoamine transporters in a dose-dependent manner. The Ki (inhibition constant) values of ketamine on the norepinephrine, dopamine, and serotonin transporters were 66.8 microM, 62.9 microM, and 162 microM, respectively. Pentobarbital, a typical general anesthetic agent with no psychotic symptoms, did not affect the uptake of monoamines, however. Further, neither the glycine transporter 1 nor the glutamate/aspartate transporter was affected by ketamine, indicating that ketamine preferentially inhibits monoamine transporters. CONCLUSIONS: Ketamine inhibited monoamine transporters expressed in human embryonic kidney cells in a dose-dependent manner. This result suggests that the ketamine-induced inhibition of monoamine transporters might contribute to its psychotomimetic and sympathomimetic effects through potentiating monoaminergic neurotransmission.

ATP-Binding Cassette Transporters↗

PubMed1998

Expression of the 150-kd oxygen-regulated protein in human breast cancer.

Tumor cells subjected to environmental stress, such as oxygen deprivation followed by reoxygenation, redirect biosynthetic pathways to express oxygen-regulated proteins (ORPs) and heat-shock proteins (HSPs). The 150-kd oxygen-regulated protein (ORP150) is a novel endoplasmic reticulum-associated polypeptide in the HSP70 family. In view of links between expression of HSPs/ORPs and tumor properties, especially tumor invasiveness and resistance to therapeutic regimens, expression of ORP150 in human breast cancers was examined. Western and Northern blotting demonstrated elevated expression of ORP150 in breast cancer, regardless of estrogen receptor status, compared with normal breast tissue. Immunohistochemical and in situ hybridization techniques revealed that infiltrating cancer cells in the stroma expressed ORP150 more strongly than large nests of cancer cells. Furthermore, pancreatic and thyroid carcinomas also displayed greater ORP150 expression. These results suggest that ORP150 is up-regulated in tumors and, in breast tumors, may be associated with tumor invasiveness.

Adenocarcinoma, Mucinous↗

PubMed1998

Selective expression of mercurial-insensitive water channel (AQP-4) gene in Hensen and Claudius cells in the rat cochlea.

We investigated the cellular localization of mercurial-insensitive water channel (MIWC) mRNA in the rat cochlea. MIWC gene expression was detected in the supporting cells in Corti's organ. The function of these supporting cells is not clear, but the results suggest that they reabsorb water and play a role in maintaining the ionic balance of inner ear fluids.

Animals↗

PubMed1998

[A case of giant advanced breast cancer responding remarkably to chemo-endocrine therapy chiefly with doxifluridine].

A 68-year-old woman presented with advanced ulcerative breast cancer of the precordium; edema of the face, cervix and bilateral upper extremities; dyspnea from carcinomatous pleurisy; and multiple bone metastases, which suggested a terminal state. Her general condition improved with symptomatic therapies; thus, doxifluridine (5'-DFUR) and an endocrine therapeutic drug were given to reduce the primary focus, eliminate the edemas and decrease the plural effusion. When the tumor markers increased again, CEFT therapy [cyclophosphamide (CPA), epirubicin (epi-ADM), 5-fluorouracil (5-FU) and tamoxifen (TAM)] was conducted. This resulted in no adverse drug reaction, further reduction of the primary focus, and extremely improved Performance status (PS). The patient was discharged on 5'-DFUR and TAM therapy, which resulted in scarring of the ulcer, normalization of tumor marker levels, disappearance of the pleural effusion, and a reduction of metastatic bone foci. The findings suggest 5'-DFUR and endocrine therapeutic drugs can have a favorable clinical effect without impacting QOL and should be employed in patients with advanced cancer and poor general condition.

Aged↗

PubMed1997

An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer's disease.

Amyloid-beta is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimer's disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimer's disease. ERAB immunoprecipitates with amyloid-beta, and when cell cultures are exposed to amyloid-beta, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-beta on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-beta, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimer's disease.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1997

Characterization of glycine release mediated by glycine transporter 1 stably expressed in HEK-293 cells.

We constructed a cell line which stably expresses glycine transporter 1 (GlyT1) proteins. The cell line showed significant [14C]glycine uptake and could keep steep glycine concentration gradient between intracellular and extracellular space (in > out). Using this cell line, we investigated glycine release mediated by this transporter. The [14C]glycine release was enhanced by extracellular glycine and sarcosine, a selective inhibitor of the transporter, in a dose-dependent manner. In addition, the replacement of extracellular Na+ with Li+ or extracellular Cl- with acetate- markedly increased the release. Furthermore, we investigated the effects of extracellular Ca2+ and K+. The removal of these ions also showed enhancement of the release. These results suggest that glycine transporter 1 protein, which might be involved in the NMDA receptor neurotransmission, can release glycine into the extracellular space in the vicinity of synapses, and that the release might be influenced by the extracellular substrate concentration and ion composition in the synaptic cleft.

Amino Acid Transport Systems, Neutral↗

PubMed1997

Isolation of a cDNA encoding a photoreceptor cell-specific actin-bundling protein: retinal fascin.

We have isolated a novel retina-specific gene, retinal fascin, encoding a new member of actin-bundling protein gene family, from a bovine retina cDNA library. The cDNA encodes a 492 amino acid protein which shows 36-57% amino acid identity with three vertebrate fascins, echinoid fascin and Drosophila singed gene. Northern blot analysis revealed that retinal fascin mRNA was exclusively expressed in the eye and not seen in other tissues examined. In situ hybridization analysis indicated that retinal fascin mRNA signals were found only in the inner segment of the photoreceptor layer and outer nuclear layer, indicating that retinal fascin was specifically expressed in photoreceptor cells. As fascins are actin-bundling proteins important for constructing several intracellular structures, retinal fascin might play a pivotal role in photoreceptor cell-specific events, such as disk morphogenesis.

Amino Acid Sequence↗

PubMed1997

Expression of zinc transporter gene, ZnT-1, is induced after transient forebrain ischemia in the gerbil.

To elucidate the molecular mechanisms underlying neuronal death after transient forebrain ischemia, we cloned genes expressed after transient forebrain ischemia in the Mongolian gerbil by a differential display method. A gerbil homolog of rat zinc transporter, ZnT-1, which transports intracellular Zn2+ out of cells, was isolated. Its expression became detectable exclusively in pyramidal neurons of the CA1 region 12 hr after ischemia and reached a maximum from day 1 to day 2 as shown by in situ hybridization. By day 7, expression had disappeared entirely from the cells in the CA1 region, because the neurons had died. No other brain regions exhibited such a significant level of ZnT-1 mRNA expression during this period. Zn2+ was shown to accumulate in CA1 pyramidal neurons expressing ZnT-1 mRNA after the ischemia by using zinquin, a zinc-specific fluorescent dye. When primary hippocampal neurons were exposed to a high dose of Zn2+, ZnT-1 mRNA accumulated. These results suggest that the induction of ZnT-1 mRNA observed in CA1 neurons was caused by an increase in the intracellular Zn2+ concentration. It was reported recently that Zn2+ chelator blocked neuronal death after ischemia and that the influx of Zn2+ might be a key mechanism underlying neuronal death. The induction of ZnT-1 mRNA in CA1 pyramidal neurons fated to die after transient ischemia is of interest to the study of postischemic events and the molecular mechanisms underlying delayed neuronal death.

Amino Acid Sequence↗

PubMed1997

Molecular cloning of a novel polypeptide, DP5, induced during programmed neuronal death.

To study the molecular mechanisms underlying neuronal programmed cell death (PCD), we performed differential display screening for genes, the expression of which was induced during PCD in the sympathetic neuron culture model deprived of NGF. We cloned a gene encoding a novel polypeptide (DP5) which consisted of 92 amino acids. DP5 polypeptide had no homology with any other known protein and contained no motif that would indicate its putative biochemical functions. DP5 mRNA levels peaked at 15 h after nerve growth factor withdrawal, concurrent with the time at which neurons were committed to die. The induction of DP5 gene expression was blocked when cell death was rescued by treatment with cycloheximide, KCl, or the cyclic AMP analogue CPTcAMP. Overexpression of the full-length DP5 in cultured sympathetic neurons was in itself sufficient to induce apoptosis. These results suggest that DP5 plays a role in programmed neuronal death.

Amino Acid Sequence↗

PubMed1997

Cloning of a putative vesicle transport-related protein, RA410, from cultured rat astrocytes and its expression in ischemic rat brain.

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of approximately 70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with these in vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes.

Amino Acid Sequence↗

PubMed1997

Coordinate expression of alpha-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells.

Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the alpha-tropomyosin (alpha-TM) gene, exon 2a rather than exon 2b is specifically spliced in alpha-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of alpha-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the down-regulation of beta-TM-SM, alpha-TM-SM converted to alpha-TM-F1 and alpha-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high Mr type to low Mr type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express alpha-TM-SM, beta-TM-SM, and high Mr caldesmon. In situ hybridization revealed specific coexpression of alpha-TM-SM and high Mr caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.

Animals↗

PubMed1997

Distribution of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP receptor, vasoactive intestinal polypeptide (VIP), and VIP receptors in the rat superior cervical ganglion.

The distribution of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), PACAP receptor (PACAP-R), vasoactive intestinal polypeptide (VIP) and two subtypes of VIP receptors (VIP1-R and VIP2-R) was examined by in situ hybridization in the superior cervical ganglion (SCG) of the adult rat. PACAP-R mRNA was expressed intensely in virtually all principal neurons. PACAP mRNA was expressed in approximately half of the principal neurons, where the levels of expression vary extensively. Intense expression of VIP mRNA was observed only in a few principal neurons. Neither VIP1-R mRNA nor VIP2-R mRNA was detected in SCG cells. These findings suggest that PACAP, but not VIP, may function as a paracrine or autocrine regulatory factor through PACAP-R in the principal neurons of the SCG.

Animals↗

PubMed1997

Cloning and functional expression of a brain peptide/histidine transporter.

Here we report the cloning and functional characterization of a rat novel peptide/histidine transporter (PHT1), which was expressed in the brain and the retina. The cDNA encodes the predicted protein of 572 amino acid residues with 12 putative membrane-spanning domains. The amino acid sequence has moderate homology with a nonspecific peptide transporter found in the plant. When expressed in Xenopus laevis oocytes, PHT1 cRNA induced high affinity proton-dependent histidine transport activity. This transport process was inhibited by dipeptides and tripeptides but not by free amino acids such as glutamate, glycine, leucine, methionine, and aspartate. Dipeptide carnosine transport activity was also confirmed by direct uptake measurement. By in situ hybridization analysis, PHT1 mRNA was widely distributed throughout whole brain. Especially, intense hybridization signals were found in the hippocampus, choroid plexus, cerebellum, and pontine nucleus. Signals were located in both the neuronal and small nonneuronal cells in these areas. PHT1 protein could contribute to uptake of oligopeptides, which function as neuromodulators, and clearance of degraded neuropeptides and be a new member in the growing superfamily of proton-coupled peptide and nitrate transporters, although its structure, localization, and pharmacological characteristics are unique among these members.

Amino Acid Sequence↗

PubMed1997

Expression of vasoactive intestinal polypeptide receptor mRNA and secretory regulation by vasoactive intestinal polypeptide in rat submandibular and sublingual salivary glands.

Vasoactive intestinal polypeptide (VIP)-receptor mRNA was strongly expressed in the acinar cells in the submandibular gland but not in the sublingual gland. VIP-containing nerve fibres were richly distributed around acini in the submandibular gland but were rare around acini of the sublingual gland. In the submandibular gland, the chorda was stimulated at various frequencies (1-40 Hz) together with an infusion of (N-Ac-Tyr1, D-Phe2)-GRF(1-29)-NH2 (109 M), VIP antagonist, which reduced salivary flow from the submandibular gland only at high-frequency stimulation (> 20 Hz), and more markedly reduced the salivary protein concentration. When the chorda was continuously stimulated the antagonist reduced the salivary flow only during the initial 5 min. Exogenous VIP 10(-12) - 10(-8) M) infusion at the same time as chorda stimulation caused no increase in salivary flow, but the salivary protein concentration was increased in a dose-dependent manner. In the sublingual gland, neither VIP nor the VIP antagonist affected chorda-evoked salivary flow and protein concentration. Thus, endogenous VIP may play a part in the regulation of both fluid and protein secretion, especially of protein, evoked by chorda stimulation at high frequency in the submandibular gland. These phenomena occurred only in the initial phase of secretion. In the sublingual gland, it seems likely that VIP plays no part in the regulatory mechanism, at least with regard to salivary fluid secretion in the acinar cells.

Animals↗

PubMed1997

Expression of c-fos and c-jun mRNA following transient retinal ischemia: an approach using ligation of the retinal central artery in the rat.

The expression of the proto-oncogenes c-fos and c-jun was examined by in situ hybridization at various timepoints following transient retinal ischemia by means of ligation of the retinal central artery in the rat. Ischemia of 90-minute duration resulted in the degeneration of neurons in both the ganglion cell layer and the inner nuclear layer at 48 hours after reperfusion. The expression of c-fos and c-jun messenger RNA throughout the entire inner nuclear layer was transiently coinduced following 90-minute retinal ischemia with a peak at 1 hour after reperfusion. This expression was also found in the ganglion cell layer at 3 hours after reperfusion. Weak signals for c-fos and c-jun mRNA were observed at 24 hours after reperfusion and returned to near control levels by 48 hours. c-jun protein expression was detected in the ganglion cell layer, the middle of the inner nuclear layer, and optic nerve head at 3 hours, but not 1 hour, after lethal ischemia/reperfusion; however, c-fos protein expression was not detected after reperfusion. Whereas no neuronal degenerative changes were found at 7 days after 30-minute ischemic retina, c-fos and c-jun messenger RNA were also induced at 1 hour postreperfusion. To our knowledge, this study is the first report to show expression patterns of immediate-early genes after retinal ischemia/reperfusion. These results suggest that changes in expression of c-fos and c-jun after transient retinal ischemia are similar to those after transient brain ischemia, and the selective occlusion of the central retinal artery will provide a useful model for studying ischemic neuronal degeneration in vivo in the rat retina.

Animals↗

PubMed1997

Molecular cloning of mouse Doc2alpha and distribution of its mRNA in adult mouse brain.

We have previously isolated from a human brain cDNA library, a new protein having two C2-like domains which interact with Ca2+ and phospholipid, and named Doc2alpha. Doc2alpha is abundantly expressed in brain, where it is highly concentrated on the synaptic vesicle fraction, and is implicated in Ca2(+)-dependent exocytosis. We have isolated here a mouse Doc2alpha cDNA and determined the localization of its mRNA in adult mouse brain. The amino acid sequence of the mouse Doc2alpha cDNA is 92% identical with that of the human counterpart. Northern blot analysis and in situ hybridization on adult mouse brain sections have revealed that Doc2alpha is predominantly expressed in mouse brain, where it is expressed in neuronal cells, but not in non-neuronal cells. Doc2alpha is highly expressed in the olfactory bulb, cerebral cortex, hippocampus, amygdaloid complex, and ventromedial hypothalamus nucleus, but not in the cerebellum, caudate-putamen, or ventral thalamus. These results indicate that Doc2alpha is expressed heterogeneously in mouse brain, where it is predominantly expressed in neuronal cells, and suggest that Doc2alpha plays a specific role in the area where it is expressed.

Amino Acid Sequence↗

PubMed1997

Regional distribution of GABA transporter 1 (GAT1) mRNA in the rat brain: comparison with glutamic acid decarboxylase67 (GAD67) mRNA localization.

The mRNA expression of GABA transporter 1 (GAT1) was investigated in the rat brain by in situ hybridization histochemistry using an oligonucleotide probe specific to the mRNA. The pattern of GABA transporter 1 mRNA expression was well coincident with that of GABAergic terminals detected by using glutamic acid decarboxylase (GAD) as a marker. In addition, we compared the GAT1 mRNA expression with that of glutamic acid decarboxylase67 (GAD67) mRNA positive neurons. Although the expression patterns of both mRNAs were similar in most nuclei, the number of GABA transporter 1 mRNA positive cells were higher than that of GAD67 mRNA positive cells in some nuclei, such as the inferior colliculus, cerebellar nuclei and cerebral cortex. Furthermore the subthalamic nucleus and inferior olive nucleus, which lacked GAD67 mRNA expression, showed intense GABA transporter 1 mRNA expression. These data suggest that GABA transporter 1 mRNA is expressed not only in GABAergic neurons but also in non-GABAergic neurons and/or glial cells which are involved in GABAergic neurotransmission.

Animals↗