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Biomedical subjects

M Tohyama

Publications and source records attributed to M Tohyama.

At least 73 records · Page 4Linked to original sources
PubMed1999

150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death.

To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochrome c antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.

Apoptosis↗
PubMed1999

Comparison between the decrease of dopamine transporter and that of L-DOPA uptake for detection of early to advanced stage of Parkinson's disease in animal models.

Early diagnosis of Parkinson's disease (PD) is important for the potential application of neuroprotective therapies. The purpose of this study was to assess the detection of the early changes of PD by either imaging the dopamine transporter (DAT) or uptake of L-3,4-dihydroxyphenylalanine (L-DOPA). An early to advanced stage model of PD was induced in rats by stereotaxic injection of 1-10 microg 6-hydroxydopamine (6-OHDA) into the substantia nigra pars compacta. Using adjacent sections of the same animals, the binding of [I-125]beta-CIT, which labels DAT and the uptake of [C-14]L-DOPA, were evaluated 4 weeks after induction of the lesion. Any decrease in dopaminergic neurons was evaluated by in situ hybridization histochemistry (ISH) by detection of DAT mRNA-positive neurons. In addition, the expression levels of DAT, dopa decarboxylase (DDC), and vesicular monoamine transporter (VMAT2) in each neuron were studied with ISH. Our results show a decrease in both [I-125]beta-CIT binding and [C-14]L-DOPA uptake in parallel with a decrease in DA neurons from early to advanced stage models of PD. The decrease in [C-14]L-DOPA uptake was smaller than that in [I-125]beta-CIT binding in the same animal (P < 0.0001). Expression levels of DAT, DDC, and VMAT2 mRNAs were also decreased with the progression of the disease. Although ISH failed to detect the origin of the discrepancy between [I-125]beta-CIT and [C-14]L-DOPA levels, it was concluded that [C-14]L-DOPA levels underestimated the decrease of dopaminergic neurons and that [I-125]beta-CIT levels more precisely reflected the decrease.

Animals↗
PubMed1999

Cloning of a bovine orphan transporter and its short splicing variant.

We have isolated a cDNA (bv7-3) encoding a member of the Na+,Cl(-)-dependent transporter family and its short splicing variant (bv7-3s) by screening a bovine retina cDNA library. Sequence analysis revealed that bv7-3 encodes a protein of 729 amino acids and is a bovine homologue of the rat orphan transporter v7-3-2. bv7-3s contains 265 amino acids, sharing 252 N-terminal amino acids with bv7-3. Both mRNAs for bv7-3 and bv7-3s were detected in nervous system by Northern blot analysis. In immunofluorescence analysis in transfected HEK 293T cells, myc-tagged bv7-3 was mainly detected on the plasma membrane, whereas myc-tagged bv7-3s showed a pattern of intracellular membrane staining.

Alternative Splicing↗
PubMed1999

Expressed-sequence-tag approach to identify differentially expressed genes following peripheral nerve axotomy.

Gene expression profiles in the rat hypoglossal nucleus after axotomy were demonstrated using expressed-sequence-tag (EST) approach. To demonstrate the gene-expression profiles after axotomy, nerve-transected hypoglossal nuclei were dissected and collected from about 1000 rats, with which a cDNA library was constructed. More than 750 clones were sub-cloned and sequenced from the library. The clones which hit frequently are likely to be associated with mitochondrial respiratory chain, cytoskeletal protein and protein synthesis. One hundred three clones from among the sequenced clones were further processed for histological screening using unilateral-hypoglossal nerve-transected brain sections by in situ hybridization histochemistry. In situ hybridization study revealed that 26% of clones examined showed upregulated expression of mRNA in response to axotomy. They included genes encoding proteins associated with glucose, lipid and protein metabolism, cytoskeleton, neurotransmission and immune reaction. The present EST analysis may have an advantage in targeting genes which are associated with nerve injury with a good efficacy, as compared with other methods such as differential display and subtraction.

Animals↗
PubMed1999

Expression of major urinary protein genes in the nasal glands associated with general olfaction.

Gene expression of major urinary protein (MUP) mRNAs was examined in the mouse nasal tissue. By polymerase chain reaction, we identified two cDNA segments encoding MUP 4 and MUP 5 genes in the nose. The expression level of both MUP 4 and 5 mRNAs in the nasal tissue was very high and exceeded that of the liver. Liver MUPs are excreted into the urine and are known to play an important role in pheromonal communication. We showed that nose and liver MUPs were composed of different subtypes of MUPs and that nose MUP mRNAs was detected in prepubescent periods when liver MUP mRNAs had not yet been transcripted. In situ hybridization revealed that nose MUP mRNAs are localized in the lateral wall and nasal septum and their expression pattern is identical to that of rat odorant-binding protein (OBP)-I. We also identified cDNA of mouse OBP-II gene from the nasal tissue and showed that the expression pattern of MUP gene was identical to that of OBP-II gene in the nose. These histological data indicate that nose MUPs are favorable for catching odorant molecules rather than pheromones, and may share their function with OBPs.

Amino Acid Sequence↗
PubMed1999

Regional distribution of presenilin-1 messenger RNA in the embryonic rat brain: comparison with beta-amyloid precursor protein messenger RNA localization.

The messenger RNA expression of presenilin-1, an important gene responsible for early-onset familial Alzheimer's disease, was investigated in the embryonic rat brain with in situ hybridization histochemistry using an oligonucleotide probe specific to the messenger RNA. It was also compared with that of beta-amyloid precursor protein messenger RNA. Presenilin-1 and beta-amyloid precursor protein messenger RNA were abundantly expressed throughout the central nervous system in the embryonic day 13, 17 and 20 rat brain. Presenilin-1 messenger RNA was strongly expressed in both neuroepithelium and differentiating fields. In contrast, beta-amyloid precursor protein messenger RNA was preferentially expressed in differentiating fields, while low expression of beta-amyloid precursor protein messenger RNA was seen in neuroepithelium. Although the expression patterns of these two messenger RNAs were basically similar, there seemed to be a tendency that presenilin-1 messenger RNA was preferentially expressed in immature neurons, while beta-amyloid precursor protein messenger RNA was preferentially expressed in mature neurons, suggesting that presenilin-1 is expressed earlier than beta-amyloid precursor protein and that presenilin-1 is involved in beta-amyloid precursor protein processing. These data raise the possibility that presenilin-1 and beta-amyloid precursor protein co-operatively play pivotal roles in rat neurogenesis.

Amyloid beta-Protein Precursor↗
PubMed1999

Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response.

Missense mutations in the human presenilin-1 (PS1) gene, which is found on chromosome 14, cause early-onset familial Alzheimer's disease (FAD). FAD-linked PS1 variants alter proteolytic processing of the amyloid precursor protein and cause an increase in vulnerability to apoptosis induced by various cell stresses. However, the mechanisms responsible for these phenomena are not clear. Here we report that mutations in PS1 affect the unfolded-protein response (UPR), which responds to the increased amount of unfolded proteins that accumulate in the endoplasmic reticulum (ER) under conditions that cause ER stress. PS1 mutations also lead to decreased expression of GRP78/Bip, a molecular chaperone, present in the ER, that can enable protein folding. Interestingly, GRP78 levels are reduced in the brains of Alzheimer's disease patients. The downregulation of UPR signalling by PS1 mutations is caused by disturbed function of IRE1, which is the proximal sensor of conditions in the ER lumen. Overexpression of GRP78 in neuroblastoma cells bearing PS1 mutants almost completely restores resistance to ER stress to the level of cells expressing wild-type PS1. These results show that mutations in PS1 may increase vulnerability to ER stress by altering the UPR signalling pathway.

Alzheimer Disease↗
PubMed1999

Accumulation of murine amyloidbeta42 in a gene-dosage-dependent manner in PS1 'knock-in' mice.

The establishment of an animal model with a missense mutation of presenilin-1 (PS1) is an initial step toward understanding the molecular pathogenesis of familial Alzheimer's disease (FAD) and developing therapeutic strategies for the disease. We previously described a Japanese family with FAD caused by the I213T mutation of PS1, in which typical signs and symptoms of Alzheimer's disease were observed at the age of 45 +/- 4.2 years [Hardy, J. (1997) Trends. Neurosci., 20, 154-159; Kamino, K et al. (1996) Neurosci. Lett., 208, 195-198]. Here, we report the establishment of 'knock-in' mice with the I213T PS1 missense mutation. Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analyses showed that the mutated PS1 allele was expressed at the same level as the endogenous PS1 allele, demonstrating that the PS1 missense mutation was successfully introduced into the mouse PS1 locus, and therefore that the situation mimics that in FAD patients bearing PS1 missense mutations. Amyloid beta (Abeta) 42(43) peptide, but not Abeta40 peptide, accumulated in 'knock-in' mice at the age of 16-20 weeks. A clear gene-dosage effect on the increase of Abeta42(43) was observed in 'knock-in' mice: the percentage increase of Abeta42(43) in mice with mutations in both alleles was twice as high as that in mice with a single allele. These results indicate that the level of the mutated PS1 gene expression is likely to be critically involved in the production of highly amyloidogenic Abeta42(43), and confirm that PS1 mutation has an important effect on amyloid precursor protein (APP) processing, in proportion to the level of the expression of the mutant gene.

Alleles↗
PubMed1999

Differential regulation of adenine nucleotide translocators by hypertonicity in the brain.

To determine the gene(s) induced by hypertonicity in the brain, we performed a differential display analysis using RNA isolated from isotonic and hypertonic rat astrocytes. One cDNA rapidly up-regulated by hypertonicity was isolated, and the DNA sequence revealed that it was identical to adenine nucleotide translocator (ANT)2. ANT2 protein exchanges intramitochondrial ATP for cytoplasmic ADP. Among three ANT isoforms, only ANT2 mRNA was up-regulated markedly from 1 to 4 h after exposure to hypertonicity. Induction of the mRNA did not require de novo protein synthesis. Furthermore, ADP translocase activity in mitochondria of astrocytes was increased significantly by hypertonicity. To see the localization and regulation of ANT2 mRNA in the brain, we performed in situ hybridization of rat brain after intraperitoneal injection of a high concentration of NaCl. Although there were only weak signals in the control, intense hybridization signals were seen in hypertonic rat whole brain. Microscopic examination showed that ANT2 signals were present in the neurons, as well as glial cells. These results suggest that ANT2 may play a role in brain cells to adapt to the hypertonic environment.

Adenosine Diphosphate↗
PubMed1999

Neuroprotective role of Na+/myo-inositol cotransporter against veratridine cytotoxicity.

Na+/myo-inositol cotransporter has been shown to protect cells from the perturbing effects of hypertonic stress by the accumulation of myo-inositol. Here we report a regulatory mechanism for the cotransporter. Induction of myo-inositol cotransporter mRNA was observed after exposure to veratridine, a voltage-gated sodium channel opener. The veratridine-elicited induction was inhibited when Na+ was eliminated from the bath, although calcium chelation failed to modify the gene expression. Veratridine evoked an accumulation of Na+ in the cells, which paralleled the abundance of the mRNA. These results strongly suggested that an increase in Na+ influx due to sodium channel opening affected transcription of the cotransporter gene. Activity of the myo-inositol cotransporter was also up-regulated after veratridine exposure. To clarify the possible roles of myoinositol accumulation under veratridine exposure, we next examined the neurotoxic effects of veratridine when myo-inositol uptake was blocked. Neither 30 microM veratridine nor 500 microM 2-O,C-methylene myo-inositol, a competitive inhibitor of myo-inositol, elicited apparent cytotoxicity. However, a combination of these agents markedly increased cytotoxicity in culture, suggesting that an adequate amount of myo-inositol was necessary when the cells were stimulated with veratridine.

Carrier Proteins↗
PubMed1999

A novel presenilin-2 splice variant in human Alzheimer's disease brain tissue.

Mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes account for the majority of cases of early-onset familial Alzheimer's disease (AD). Alternative splicing forms of the PS-1 and PS-2 gene products have previously been reported in fibroblast and brain tissue from both familial and sporadic AD patients, as well as from normal tissues and cell lines. We demonstrate here unusual alternative splicing of the PS-2 gene that leads to the generation of mRNA lacking exon 5 in human brain tissue. This product was more frequently detected in brain tissue from sporadic AD patients (70.0%; 21 of 30) than from normal age-matched controls (17.6%; three of 17). In cultured neuroblastoma cells, this splice variant was generated in hypoxia but not under other forms of cellular stress. Hypoxia-mediated induction of this splice variant was blocked by pretreatment of neuroblastoma cells with the protein synthesis inhibitor cycloheximide or antioxidants such as N-acetylcysteine and diphenyl iodonium, suggesting that hypoxia-mediated oxidant stress might, at least in part, underlie the alternative splicing of PS-2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N-terminal part of PS-2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.

Aged↗
PubMed1999

Chemokine responses and accumulation of inflammatory cells in the lungs of mice infected with highly virulent Cryptococcus neoformans: effects of interleukin-12.

We examined the mechanisms involved in the development of lung lesions after infection with Cryptococcus neoformans by comparing the histopathological findings and chemokine responses in the lungs of mice infected with C. neoformans and assessed the effect of interleukin (IL) 12 which protects mice from lethal infection. In mice infected intratracheally with a highly virulent strain of C. neoformans, the yeast cells multiplied quickly in the alveolar spaces but only a poor cellular inflammatory response was observed throughout the course of infection. Very little or no production of chemokines, including MCP-1, RANTES, MIP-1alpha, MIP-1beta and IP-10, was detected at the mRNA level using RT-PCR as well as at a protein level in MCP-1, RANTES and MIP-1alpha. In contrast, intraperitoneal administration of IL-12 induced the synthesis of these chemokines and a marked cellular inflammatory response involving histiocytes and lymphocytes in infected mice. Our findings were confirmed by flow cytometry of intraparenchymal leukocytes obtained from lung homogenates which showed IL-12-induced accumulation of inflammatory cells consisting mostly of macrophages and CD4+ alphabeta T cells. On the other hand, C-X-C chemokines including MIP-2 and KC, which attract neutrophils, were produced in infected and PBS-treated mice but treatment with IL-12 showed a marginal effect on their level, and neutrophil accumulation was similar in PBS- and IL-12-treated mice infected with C. neoforman. Our results demonstrate a close correlation between chemokine levels and development of lung lesions, and suggest that the induction of chemokine synthesis may be one of the mechanisms of IL-12-induced protection against cryptococcal infection.

Animals↗
PubMed1999

[A case of breast cancer with liver metastases responding remarkably to combination therapy of mitoxantrone (MIT), doxifluridine (5'-DFUR) and medroxyprogesterone acetate (MPA)].

A 60-year-old woman with her right breast cancer showing simultaneous and multiple liver metastases was initially treated with CEFT [cyclophosphamide (CPA), epirubicin (epi-ADM), 5-fluorouracil (5-FU), tamoxifen (TAM)]. After one treatment course the primary lesion did not decrease while tumor markers and liver lesion size increased. Therefore, the foul-smelling primary lesion was resected followed by treatment with mitoxantrone (MIT), 10 mg intravenously every 4 weeks as well as daily/oral administrations of doxifluridine (5'-DFUR) and medroxyprogester-one acetate (MPA). Following MIT administration, the tumor markers decreased markedly, so treatment was continued. After the third course, therapy was continued on an outpatient basis. During treatment WBC reduction to about 3,000/microliter was the only adverse reaction. After 10 courses, the tumor markers were normal, and after 15 courses there were no liver metastases on abdominal CT. Generally, patients with resistance to standard anthracycline chemotherapy are difficult to treat. Those with liver metastasis especially have a poor response which results in a poor prognosis. However, therapy with MIT, 5'-DFUR and MPA may be useful in previously treated, advanced and recurrent breast cancer. Furthermore, this therapy can be done on an outpatient basis, which presumably improves the quality of life (QOL).

Administration, Oral↗
PubMed1999

Activation of Akt kinase inhibits apoptosis and changes in Bcl-2 and Bax expression induced by nitric oxide in primary hippocampal neurons.

Emerging data indicate that growth factors such as insulin-like growth factor-1 (IGF-1) prevent neuronal death due to nitric oxide (NO) toxicity. On the other hand, growth factors can promote cell survival by acting on phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target, serine-threonine kinase Akt, in various types of cells. Here, we examined the mechanism by which IGF-1 inhibits neuronal apoptosis induced by NO in primary hippocampal neurons. IGF-1 was capable of preventing apoptosis and caspase-3-like activation induced by a NO donor, sodium nitroprusside or 3-morpholin-osydnonimine. Incubation of neurons with a P13-kinase inhibitor, wortmannin or LY294002, blocked the effects of IGF-1 on NO-induced neurotoxicity and caspase-3-like activation. In addition, the P13-kinase inhibitors blocked the effect of IGF-1 on down-regulation in Bcl-2 and upregulation in Bax expression induced by NO. Adenovirus-mediated overexpression of the activated form of Akt significantly inhibited NO-induced cell death, caspase-3-like activation, and changes in Bcl-2 and Bax expression. Moreover, expression of the kinase-defective form of Akt almost completely blocked the effects of IGF-1. These findings suggest that activation of Akt is necessary and sufficient for the effect of IGF-1 and is capable of preventing NO-induced apoptosis by modulating the NO-induced changes in Bcl-2 and Bax expression.

Adenoviridae↗
PubMed1998

A sequence-specific splicing activator, tra2beta, is up-regulated in response to nerve injury.

Tra2beta is the first mammalian protein which is proved to activate mRNA splicing in sequence-specific manner. Following hypoglossal nerve injury, the expression of Tra2beta mRNA was elevated in injured motoneurons transiently. The up-regulation of Tra2beta mRNA was observed from post-operative day 3 to 21. In addition to the nerve injury in PNS, a brain lesion in CNS also enhanced the expression of Tra2beta mRNA. The present study could be the first observation showing that an expression of the sequence-specific splicing activator is enhanced in neuronal cells in response to nerve injury, and indicates that Tra2beta may participate in the control of injury-specific splicing patterns in order to express molecules which are necessary for regeneration.

Animals↗
PubMed1998

Molecular cloning and distinct developmental expression pattern of spliced forms of a novel zinc finger gene wiz in the mouse cerebellum.

In the course of a study conducted to identify the mouse homologue of Drosophila eyes absent (eya), we isolated a novel mouse cDNA fragment which show little homology to eya but encodes a protein with Krüppel (C2H2)-type zinc finger motifs. By further screening using this cDNA fragment as a probe, we obtained the short and long forms of full-length cDNAs, which were apparently alternatively spliced products from one gene. Since both mRNAs encode proteins with widely-interspaced zinc finger motifs, we termed this gene wiz and refer to the short and long wiz transcripts as wizS and wizL, respectively. In situ hybridization studies using the probe against the region common to wizS and wizL showed that these mRNAs were expressed abundantly in the granule cell layers of the mouse cerebellum, the olfactory bulb, and the dentate gyrus, whereas the same technique using the probe against only wizL could not detect positive signals in the developing cerebellum, indicating that there is no expression of wizL mRNA there. Northern blot and in situ hybridization analyses demonstrated that the extracerebellar regions expressed both wizS and wizL mRNAs from the midgestational period to adulthood. The finding that two types of wiz transcripts (wizS and wizL) are expressed with different developmental patterns might indicate separate transcription functions in the cerebellar granule cells and the extracerebellar regions.

Alternative Splicing↗
PubMed1998

Roles of Bcl-2 and caspases in hypoxia-induced neuronal cell death: a possible neuroprotective mechanism of peptide growth factors.

We examined whether apoptosis is involved in hypoxic cell death using primary cultures of rat cortical neurons and whether the cell death is associated with changes in Bcl-2 and Bax expressions and activities of caspases. Hypoxic insult accelerates apoptosis, as shown by apoptotic nuclei and by chromatin degradation of internucleosomal fragments. This apoptotic process is accompanied by a rapid and sustained down-regulation of Bcl-2, whereas levels of Bax are unchanged. Furthermore, hypoxic insult activates sequentially caspase-1-like and caspase-3-like proteases, following down-regulation of Bcl-2 expression. Peptide inhibitors of either caspase-1 or caspase-3 protect against neuronal death, although they do not prevent hypoxia-induced down-regulation of Bcl-2. Furthermore, treatment of cortical neurons with either insulin-like growth factor-1 (IGF-1) or basic fibroblast growth factor (bFGF), growth factors which are implicated to prevent neuronal loss in ischemic brain, partly prevented neuronal death accompanied by inhibition of alterations in Bcl-2 protein levels and caspase-3-like activities. These results suggest that hypoxia induces neuronal death by down-regulation of Bcl-2 protein levels followed by sequential activation of the caspases, and the protection from neuronal cell death of these growth factors under hypoxic conditions derives at least partly from their capability to prevent down-regulation of the anti-apoptotic protein levels.

Animals↗