Biomedical subjects

M Tohyama

Publications and source records attributed to M Tohyama.

At least 55 records · Page 3Linked to original sources

PubMed2000

Molecular cloning of a novel membrane glycoprotein, pal, specifically expressed in photoreceptor cells of the retina and containing leucine-rich repeat.

We have isolated a novel retina-specific gene in a screen for genes of which expression is not apparent neonatally in rat retina but is abundant postnatally on day 14 (P14). This gene, named Pal, encodes a putative type I transmembrane protein containing five leucine-rich repeats (LRRs), a single C2-type Ig-like domain, and a single fibronectin type III domain and is considered to be a new member of the LRR and Ig superfamily. No expression of Pal was found in rat retina at P1, but it was detected at P7 and markedly increased with subsequent development. These expression patterns of Pal appeared to be correlated with the development of the photoreceptor outer segments, because in the adult rat retina it was specifically localized in these segments. Ultrastructually, Pal immunoreactivity was distributed diffusely on the disk membrane in the lamellar regions. On the basis of its structural features and localization pattern, Pal may act as a receptor for a certain trophic factor or for an adhesion molecule participating in morphogenesis. The human homolog of Pal was mapped to chromosome 10q23.2-23.3 using fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed2000

Regulatory mechanisms of TRAF2-mediated signal transduction by Bcl10, a MALT lymphoma-associated protein.

To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c-Jun N-terminal kinase activation and induced nuclear factor kappaB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosis in vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Changes in mRNA for post-synaptic density-95 (PSD-95) and carboxy-terminal PDZ ligand of neuronal nitric oxide synthase following facial nerve transection.

When the axon of motoneurons is transected, the number of synaptic boutons contacting the cell body is decreased, and the recovery of synapses depends on muscle reinnervation. Post-synaptic density-95 (PSD-95) is a protein which is located at the post-synaptic density (PSD) and it plays a pivotal role in regulating synaptic plasticity and synaptogenesis. In addition, PSD-95 binds with neuronal nitric oxide synthase (nNOS), which is competitively inhibited by carboxy-terminal PDZ ligand of nNOS (CAPON) and, thereby, nNOS activity is thought to be regulated by PSD-95 and CAPON. We investigated the changes in mRNA for PSD-95, CAPON and nNOS in the facial motor nucleus of adult rats following axotomy, by in situ hybridization, in combination with the time course of muscle reinnervation, by retrograde tracing and nNOS protein expression, by examining nicotinamide adenine nucleotide phosphate diaphorase (NADPH-d) activity. Signals of mRNA for PSD-95 and CAPON were initially expressed in the facial motoneurons, transiently decreased following axotomy and gradually recovered to the control level. When reinnervation of the axotomized nerve into muscle was observed, mRNA expression of PSD-95 and CAPON started to recover in the facial motoneurons. It was also found that mRNA and protein expression of nNOS started to increase in the axotomized facial motoneurons just prior to the recovery of mRNA expression of PSD-95 and CAPON. These results suggest that PSD-95 and CAPON are involved in synaptogenesis and/or recovery of synaptic function in motoneurons after axotomy.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Changes in mRNA of protein inhibitor of neuronal nitric oxide synthase following facial nerve transection.

Protein inhibitor of neuronal nitric oxide synthase (PIN) is reported as the protein inhibiting neuronal nitric oxide synthase (nNOS) activity by preventing dimerization of nNOS. It was also reported that PIN inhibits the activity of all nitric oxide synthase (NOS) isozymes. We examined the effects of facial nerve transection on PIN mRNA and NOS expression by in situ hybridization for PIN mRNA and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) staining. PIN mRNA was initially expressed and transiently increased from 3 to 5 days and returned to the basal level at 7 days after axotomy in the motoneurons of the facial nucleus. NADPH-d-positive motoneurons were found from 7 days post-operation in the facial nucleus. These results suggest that PIN may interact with NOS from 7 days post-operation.

Animals↗

PubMed2000

Effect of controlling lactate concentration and periodic change in DO concentration on fermentation characteristics of a mixed culture of Lactobacillus delbrueckii and Ralstonia eutropha for PHB production.

A mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly beta-hydroxybutyrate (PHB) by Ralstonia eutropha in one fermentor. Based on the experimental studies on the effect of lactate concentration on the cell growth of both microorganisms, the lactate concentration was controlled at less than 5 g/l using an on-line enzymatic lactate and glucose sensors with the FIA (flow injection analysis) system, and by manipulating the glucose feeding rate. Since L. delbrueckii prefers anaerobic conditions while R. eutropha prefers aerobic conditions, we studied the effect of DO concentration on fermentation characteristics of each microorganism. For the mixed culture, we considered the control scheme of a two inputs and three outputs multivariable system. It was experimentally shown that the periodic fermentation resulted in superior PHB yield with relatively high productivity as compared with the cases where DO concentration was controlled to be constant either at less than 1 ppm or 3 ppm.

Journal Article↗

PubMed2000

Two cis-acting elements in the 3' untranslated region of alpha-CaMKII regulate its dendritic targeting.

Dendritic localization of the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (alphaCaMKII) mRNA in CNS neurons requires its 3' untranslated region (3'UTR). We investigated this targeting mechanism by identifying two cis-acting elements in the 3'UTR. One is a 30-nucleotide element that mediated dendritic translocation. A homologous sequence in the 3'UTR of neurogranin, transcripts of which also reside in dendrites, also funtioned in cis to promote its dendritic transport. Other putative elements in the alphaCaMKII mRNA inhibit its transport in a resting state. This inhibition was removed in depolarized neurons, and such activity-dependent derepression was a primary requirement for their dendritic targeting.

Animals↗

PubMed2000

A novel strategy for cancer therapy by mutated mammalian degenerin gene transfer.

Mammalian degenerin (MDEG) is a member of the amiloride-sensitive sodium ion channel family, and its site-directed active mutant (MDEG-G430F) induces massive Na+ influx into cells, leading to cell ballooning and cell bursting. We attempted a novel therapeutic approach for gastric cancers by transferring MDEG-G430F into cancer cells using tumor-specific promoters. In carcinoembryonic antigen (CEA)-producing gastric cancer cells, the level of cell death observed when MDEG-G430F was used with a CEA promoter was similar to that observed when using a potent nonspecific promoter such as the cytomegalovirus promoter. In an in vivo study, fusogenic liposome complexes containing MDEG-G430F driven by the CEA promoter were injected intraperitoneally into CEA-producing gastric cancer cells in a mouse peritoneal dissemination model. Although all 15 of the control mice were dead by 50 days postinoculation, 13 of the 15 mice treated with MDEG-G430F survived. These results indicate that transferring MDEG-G430F into cancer tissues using tumor-specific promoters can achieve striking and selective cancer cell death irrespective of the transcriptional efficiency of the promoters used in vivo, and suggest that this approach is a promising new strategy for cancer gene therapy.

Acid Sensing Ion Channels↗

PubMed2000

A pathway of neuronal apoptosis induced by hypoxia/reoxygenation: roles of nuclear factor-kappaB and Bcl-2.

As a model of the reperfusion injury found in stroke, we have exposed neurons to hypoxia followed by reoxygenation. Neurons treated with hypoxia/reoxygenation (H/R) respond by activating nuclear factor-kappaB (NFkappaB), releasing cytochrome c from their mitochondria, and ultimately dying. Further supporting an apoptotic mechanism, expression of the antiapoptotic Bcl-2 and Bcl-x proteins was increased following H/R. In this model, adenoviral-mediated transduction of lkappaB expression inhibited NFkappaB activation and significantly accelerated cytochrome c release and caspase-dependent neuronal death. At the same time, expression of mutated lkappaB prevented the increased expression of endogenous Bcl-2 and Bcl-x. In the presence of mutated lkappaB, singular overexpression of only Bcl-2 by adenoviral-mediated transduction significantly inhibited cytochrome c release, caspase-3-like activation, and cell death in response to H/R. These findings suggest a pathway where NFkappaB activation induces overexpression of Bcl-2 and Bcl-x, which function to prevent apoptotic cell death following H/R treatments.

Aerobiosis↗

PubMed2000

Interleukin-10 gene transfer improves the survival rate of mice inoculated with Escherichia coli.

OBJECTIVE: To determine whether administration of recombinant adenovirus vectors encoding the interleukin (IL)-10 protein (AxCAmIL-10) decreases the mortality of septic mice. DESIGN: Prospective, randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Adult male C57B/6 mice. INTERVENTIONS: Untreated mice and those injected intraperitoneally with 1 x 10(9) pfu of AxCAmIL-10 were used as control 1 and 2, respectively. Double-capsules without Escherichia coli were intraperitoneally embedded in another group (control 3). Mice embedded with capsules containing E. coli were divided into the following groups: simultaneous administration of 0.5 mL of saline (group 1), and administration of AxCAmIL-10 3 hrs before embedding (group 2) or 1 hr after embedding (group 3). Histopathologic changes together with expression concentrations of IL-10 and tumor necrosis factor (TNF) in various organs and plasma were examined 18 hrs after each treatment. Observation periods were 5-8 days. Survival rates were compared between these groups. MEASUREMENTS AND MAIN RESULTS: The plasma IL-10 concentrations were increased in control 2, group 2, and group 3 but not in control 1, control 3, or group 1, indicating successful adenovirus gene transfer. Plasma TNF values were significantly reduced in groups 2 and 3 as compared with group 1, with no significant differences in endotoxin concentrations. Survival rates were significantly better in groups 2 and 3 than in group 1 (p < .05). CONCLUSION: These findings suggested that IL-10 has a favorable effect on survival of septic mice via inhibition of TNF production or endotoxin stimulation.

Adenoviridae↗

PubMed2000

150-kDa oxygen-regulated protein (ORP150) functions as a novel molecular chaperone in MDCK cells.

To assess the participation of the 150-kDa oxygen-regulated protein (ORP150) in protein transport, its function in Madin-Darby canine kidney (MDCK) cells was studied. Exposure of MDCK cells to hypoxia resulted in an increase of ORP150 antigen and increased binding of ORP150 to GP80/clusterin (80-kDa glycoprotein), a natural secretory protein in this cell line. In ORP150 antisense transformant MDCK cells, GP80 was retained within the endoplasmic reticulum after exposure to hypoxia. Metabolic labeling showed the delay of GP80 maturation in antisense transformants in hypoxia, whereas its matured form was detected in wild-type cells, indicating a role of ORP150 in protein transport, especially in hypoxia. The affinity chromatographic analysis of ORP150 suggested its ability to bind to ATP-agarose. Furthermore, the ATP hydrolysis analysis showed that ORP150 can release GP80 at a lower ATP concentration. These data indicate that ORP150 may function as a unique molecular chaperone in renal epithelial cells by facilitating protein transport/maturation in an environment where less ATP is accessible.

Adenosine Triphosphate↗

PubMed1999

Stress-associated endoplasmic reticulum protein 1 (SERP1)/Ribosome-associated membrane protein 4 (RAMP4) stabilizes membrane proteins during stress and facilitates subsequent glycosylation.

Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61alpha and Sec61beta, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.

Amino Acid Sequence↗

PubMed1999

Exposure of cultured primary rat astrocytes to hypoxia results in intracellular glucose depletion and induction of glycolytic enzymes.

Based on the neurotrophic properties of astrocytes in response to ischemia, the current work focuses on the mechanism for cultured astrocytes to adapt to a hypoxic environment. Intracellular glucose levels in primary cultured rat astrocytes exposed to hypoxia fell by 30% within 24 h, in parallel with a decrease in glycogen stores. Glycolytic metabolism was crucial for cell survival during hypoxia, as 2-deoxyglucose resulted in rapid ATP depletion and cell death. The mechanism for maintaining glucose levels under these conditions appeared to be mobilization of glycogen stores, rather than increased extracellular uptake of glucose, as gluconolactone (an inhibitor of beta1-4 amyloglucosidase) induced a rapid fall in cellular ATP in cultures subjected to hypoxia, whereas cytochalasin B was without affect. Addition of cycloheximide diminished the viability of astrocytes in hypoxia, suggesting an obligatory role of de-novo gene expression to respond to hypoxia. Consistently, the results of differential display suggested the induction of glycolytic enzymes, including aldolase A (EC 4.1.2.13), hexokinase II (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), and triosephosphate isomerase (EC 5.3.1.1) in the hypoxic culture. Marked induction of these glycolytic enzymes in hypoxic astrocytes was confirmed by Northern blot analysis. These data provide a theoretical basis to understand the ability of astrocytes to tolerate ischemic condition.

Animals↗

PubMed1999

Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina.

The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15-P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.

Aging↗

PubMed1999

Kainic acid-induced seizure upregulates Na(+)/myo-inositol cotransporter mRNA in rat brain.

A major organic osmolyte, myo-inositol protects cells from perturbing effects of high intracellular concentrations of electrolytes. Myo-inositol is accumulated into cells through Na(+)/myo-inositol cotransporter (SMIT). In order to investigate the regulation of SMIT in generalized seizure, we employed Northern blot analysis and in situ hybridization to study the changes in SMIT mRNA expression in kainic acid-injected rats. Northern blot analysis demonstrated that SMIT mRNA began to increase in the brain 2 h after onset of seizure, and peaked at 12 h. In situ hybridization revealed rapid increase of SMIT mRNA (2 h of seizure) in the CA3 hippocampal pyramidal cells and in the dentate granular cells. Then, at 4-6 h SMIT mRNA expression was observed in the other limbic structure such as amygdala and piriform cortex. Finally, in neocortex and in CA1 pyramidal cells, SMIT mRNA was slowly increased and peaked at 12 h. Microautoradiogram demonstrated that cells expressed SMIT mRNA were mainly neurons. These results suggest that SMIT mRNA is upregulated by kainic acid-induced seizure primarily in structures involved in seizure activity.

Animals↗

PubMed1999

IPP isomerase, an enzyme of mevalonate pathway, is preferentially expressed in postnatal cortical neurons and induced after nerve transection.

To understand the molecular mechanisms underlying the developmental processes of the cerebral cortex, we screened genes whose mRNA expression was up-regulated in neonatal in the rat cortex to a greater extent than in adult by differential display and obtained five genes. Among these genes, we focused on pyrophosphate (isopentenyl diphosphate, dimethylallyl diphosphate: IPP) isomerase gene, the product of which is known as an enzyme of the mevalonate pathway. Rat IPP isomerase was recently cloned and the gene expression was shown to be dependent on the activation of the mevalonate pathway. Its expression and roles in the brain, however, have not been investigated hitherto. In the present study, Northern blots and in situ hybridization analysis showed that at embryonic stage weak signals for IPP mRNA were diffusely detected in the CNS, and the signal in the cortex became intense at postnatal day 1 and maximized in almost all neurons of all layers at postnatal day 7 with a subsequent reduction. At 8 weeks, the expression of IPP isomerase mRNA in neurons decreased, while it was detected in the oligodendrocytes in the regions containing abundant nerve fibers. These findings suggested that IPP isomerase contributes to postnatal neuronal maturation and myelination. We also demonstrated that IPP isomerase mRNA is induced after nerve axotomy, which suggests a relationship between neuronal regeneration and IPP isomerase. Taken together, these results suggest that elevation of IPP isomerase mRNA levels in neurons contributes to construction of nerve fibers both during the postnatal period in the cortex and their regeneration.

Animals↗

PubMed1999

Expression of norepinephrine and serotonin transporter mRNAs in the rat superior cervical ganglion.

We investigated the gene expression of three monoamine transporters (norepinephrine transporter, NET; serotonin transporter, SERT; and dopamine transporter, DAT) in the rat superior cervical ganglion (SCG). Most of principal ganglion neurons abundantly expressed NET mRNA. In addition, about 30% of principal ganglion neurons also expressed SERT mRNA. However, DAT mRNA expression was not observed there. These results suggest that serotonin as well as norepinephrine works as a neurotransmitter in a subset of principal ganglion neurons.

Animals↗

PubMed1999

Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NFkappaB activation in primary hippocampal neurons.

Emerging data indicate that tumor necrosis factor (TNF) exerts a neuroprotective effect in response to brain injury. Here we examined the mechanism of TNF in preventing neuronal death in primary hippocampal neurons. TNF protected neurons against hypoxia- or nitric oxide-induced injury, with an increase in the anti-apoptotic proteins Bcl-2 and Bcl-x as determined by Western blot and reverse transcriptase-polymerase chain reaction analysis. Treatment of neurons with an antisense oligonucleotide to bcl-2 mRNA or that to bcl-x mRNA blocked the up-regulation of Bcl-2 or Bcl-x expression, respectively, and partially inhibited the neuroprotective effect induced by TNF. Moreover, adenovirus-mediated overexpression of Bcl-2 significantly inhibited hypoxia- or nitric oxide-induced neuronal death. To examine the possible involvement of a transcription factor, NFkappaB, in the regulation of Bcl-2 and Bcl-x expression in TNF-treated neurons, an adenoviral vector capable of expressing a mutated form of IkappaB was used to infect neurons prior to TNF treatment. Expression of the mutant NFkappaB completely inhibited NFkappaB DNA binding activity and inhibited both TNF-induced up-regulation of Bcl-2 and Bcl-x expression and neuroprotective effect. These findings indicate that induction of Bcl-2 and Bcl-x expression through NFkappaB activation is involved in the neuroprotective action of TNF against hypoxia- or nitric oxide-induced injury.

Animals↗

PubMed1999

The cell death-promoting gene DP5, which interacts with the BCL2 family, is induced during neuronal apoptosis following exposure to amyloid beta protein.

DP5, which contains a BH3 domain, was cloned as a neuronal apoptosis-inducing gene. To confirm that DP5 interacts with members of the Bcl-2 family, 293T cells were transiently co-transfected with DP5 and Bcl-xl cDNA constructs, and immunoprecipitation was carried out. The 30-kDa Bcl-xl was co-immunoprecipitated with Myc-tagged DP5, suggesting that DP5 physically interacts with Bcl-xl in mammalian cells. Previously, we reported that DP5 is induced during neuronal apoptosis in cultured sympathetic neurons. Here, we analyzed DP5 gene expression and the specific interaction of DP5 with Bcl-xl during neuronal death induced by amyloid-beta protein (A beta). DP5 mRNA was induced 6 h after treatment with A beta in cultured rat cortical neurons. The protein encoded by DP5 mRNA showed a specific interaction with Bcl-xl. Induction of DP5 gene expression was blocked by nifedipine, an inhibitor of L-type voltage-dependent calcium channels, and dantrolene, an inhibitor of calcium release from the endoplasmic reticulum. These results suggested that the induction of DP5 mRNA occurs downstream of the increase in cytosolic calcium concentration caused by A beta. Moreover, DP5 specifically interacts with Bcl-xl during neuronal apoptosis following exposure to A beta, and its binding could impair the survival-promoting activities of Bcl-xl. Thus, the induction of DP5 mRNA and the interaction of DP5 and Bcl-xl could play significant roles in neuronal degeneration following exposure to A beta.

Alzheimer Disease↗