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M Itoh

Publications and source records attributed to M Itoh.

At least 433 records · Page 24Linked to original sources
PubMed1997

Expression of a glutamate transporter subtype, EAAT4, in the developing human cerebellum.

A glutamate transporter subtype, EAAT4, is closely related to removal of glutamate from the synaptic cleft. Immunohistochemistry for EAAT4 demonstrated the specific distribution and localization of its expression in the developing human cerebellum. Purkinje cells showed faint EAAT4 immunostaining at 17 gestational weeks (GW), which became increasingly intense from 23 GW to the infantile period. In the late fetal to early infantile periods, Purkinje cells showed marked immunoreactivity. After the late infantile period, EAAT4 immunoreactivity was the same in extent as in the adult pattern. Its intracellular localization also changed with development. EAAT4 immunoreactivity was demonstrated in the short processes of Purkinje cells in the early embryonic period, in the cell bodies and dendrites in the late fetal to early infantile periods, and then in the spines after the late infantile period. In the adult cerebellum, immunoreactivity was detected strongly in the spines of Purkinje cells and weakly in the cell bodies. No immunoreactivity was found in the axons or axon terminals of the cells. Thus, the glutamate transporter exhibits developmental changes in its distribution in the cerebellum and its localization in Purkinje cells. EAAT4 immunoreactivity may be related to the dendritic arborization of cells in the molecular layer.

Adolescent↗
PubMed1997

Regional cerebral glucose utilization is modulated by the dosage of apolipoprotein E type 4 allele and alpha1-antichymotrypsin type A allele in Alzheimer's disease.

Twenty Alzheimer's disease (AD) patients with defined apolipoprotein E (APOE), alpha1-antichymotrypsin (ACT) and presenilin-1 (PS-1) intronic genotypes were examined to quantify the regional cerebral metabolic rate of glucose (rCMRglc) using positron emission tomography (PET) and 18F-2-fluoro-2-deoxy-D-glucose (FDG). The frontal rCMRglc was significantly increased in patients with the APOE epsilon4 allele in a dose-dependent fashion. In contrast, the temporo-parietal rCMRglc was significantly reduced in ACT type A allele (ACT*A) carriers compared with those in non-ACT*A carriers. The PS-1 type 1 intronic allele had no significant effects on rCMRglc in any cerebral region. These results suggest that both the APOE and ACT genes may play a distinct role in the progression of AD as monitored by imaging studies of cerebral glucose utilization.

Aged↗
PubMed1997

Nociceptin gene expression in rat dorsal root ganglia induced by peripheral inflammation.

Nociceptin has been suggested to be involved in nociceptive processing in the spinal dorsal horn. To investigate whether the biosynthesis of nociceptin is altered in animals with nociceptive hypersensitivity, we examined effects of peripheral inflammation on the expression of prepronociceptin mRNA, especially in the dorsal root ganglion of the rat. Although little or no expression of prepronociceptin mRNA was observed in the dorsal root ganglia of naive animals, carrageenan-elicited inflammation markedly induced its expression, peaking within 30 min, a time when nociceptive hypersensitivity was not yet apparent. The results suggest that peripheral inflammation induced nociceptin expression in primary sensory neurons, which may be associated with the production of nociceptive hypersensitivity.

Animals↗
PubMed1997

Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell-cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/alpha, beta catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with alpha catenin, but not to that with beta catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and alpha catenin was approximately 0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of approximately 10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with alpha catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.

Actins↗
PubMed1997

Recombinant human TSH receptor expressed in E. coli.

We expressed the extracellular domain (20-408 aa, (T) of human TSH receptor (TSHR) in E. coli to detect TSHR autoantibodies (TRAb) and, moreover, we expressed the two portions (20-218 aa (5') and 217-408 aa (3')) of the extracellular domain thought to distinguish thyroid stimulating antibodies (TSAb) from blocking antibodies (TSBAb), using pGEX.3X as the expression vector. Using Western blotting analysis of the sera from patients with autoimmune thyroid disease, sera from Graves' patients and patients with idiopathic myxedema who had TSBAb reacted with the fusion protein (T), but none of the control sera reacted with it. We further evaluated whether or not the positive sear for T recognized fusion proteins (5') or (3'). The sera from Graves' patients reacted with both fusion proteins (5') and (3'). The sera from patients with idiopathic myxedema did not react with either of fusion proteins (5') or (3'). These findings suggest that these recombinant TSHR proteins could be used as antigens to detect TRAb, and differentiate TSABb from patients with idiopathic myxedema.

Antibodies, Blocking↗
PubMed1997

Detection of 14q32.33 translocation and t(11;14) in interphase nuclei of chronic B-cell leukemia/lymphomas by in situ hybridization.

Abnormalities of chromosome 14 involving band q32.33 are among the most commonly observed cytogenetic alterations in B-cell malignancies. To assess the incidence and pathogenetic implications of 14q32.33 translocation in chronic B-cell leukemia/lymphomas, we performed fluorescence in situ hybridization (FISH) analysis with variable region (V(H)) and gamma constant region (Cgamma) gene probes in 37 patients with these disorders. Chromosome 14q32.33 translocation was detected in 2 of 18 patients with chronic lymphocytic leukemia (CLL), 1 of 2 with CLL of mixed cell types (CLL/PL), 1 of 2 with pro-lymphocytic leukemia (PLL), 5 of 6 with leukemic mantle-cell lymphoma (MCL), 2 of 7 with splenic B-cell leukemia/lymphoma of possible marginal zone origin (SBLL) and 2 with leukemic follicular lymphoma (FL). To further characterize 14q32.33 translocations in these patients, we developed a new procedure using double-color FISH with PRAD1, BCL2, V(H) and Cgamma gene probes. Chromosome t(11;14) was detected in 1 patient with CLL/PL, 1 with PLL and 5 with MCL. Chromosome t(14;18) was detected in 2 patients with FL. In a PLL patient with t(11;14), the cosmid CPP29 containing the PRAD1 gene and its 5'-flanking region split and co-localized with both Cgamma and V(H) gene probes, thus spanning the breakpoint. In CLL and SBLL patients, donor chromosomes were other than chromosomes 2, 11, 18 and 19, suggesting the involvement of a novel oncogene(s) in the pathogenesis of these diseases. Interphase FISH rapidly detected 14q32.33 translocation, t(11;14) and t(14;18) in B-cell malignancies with low mitotic activity at the single-cell level, facilitating the correlation of the molecular features of these translocations with clinical characteristics.

Adolescent↗
PubMed1997

An alternative form of nucleolysin binds to a T-cluster DNA in the silencer element of platelet factor 4 gene.

The cDNA of a T-cluster binding protein (TCBP) has been cloned using the Southwestern method. The cDNA sequence of TCBP reveals that it has 78% homology to that of nucleolysin, a factor involved in apoptosis in cytolytic lymphocyte target cells. In particular, the 0.8kb sequences of the 5'-half of both cDNAs were identical. However, nucleolysin has a lysosome-targeting motif at the carboxy terminus, while TCBP has a hydrophobic sequence instead. Southern blot experiments have revealed that both cDNA sequences existed in the same YAC clone derived from chromosome 10. This strongly suggests that the TCBP cDNA is an alternatively spliced product of the nucleolysin/TCBP gene. The TCBP mRNA is ubiquitously expressed, not only in megakaryocytic cells but also in other hematopoietic cells. However, when HEL cells were induced to differentiate to megakaryocytes by DMSO, TCBP mRNA was reduced, while PF4 gene expression increased simultaneously. Gel mobility shift analysis demonstrated that recombinant TCBP specifically bound to the T-cluster and the proximal T-rich region of the PF4 promoter. Co-transfection experiments showed that TCBP reduced the gene expression from the PF4 promoter. On the other hand, TCBP did not affect expression from the PF4 promoter in which the T-cluster and the T-rich region had been removed. These results indicate that TCBP may participate in the regulation of PF4 gene expression by binding to the T-cluster and the T-rich sequence.

Amino Acid Sequence↗
PubMed1997

Metabolism of selenite labelled with enriched stable isotope in the bloodstream.

The metabolism of selenium (Se) in the bloodstream of rats was studied using HPLC-ICP-MS with an enriched Se stable isotope, and the results were used as Se-specific indicators for Se nutritional status. Concentration of endogenous Se in plasma depended on dietary Se, while changes in concentrations and distributions of exogenous Se revealed its metabolic pathway. Namely, selenite was taken up by red blood cells and reduced to selenide, and then reappeared in plasma in a form bound selectively to albumin within 10 min, disappeared from plasma again within 30 min after injection. Then, the concentration of labelled Se started to increase slowly as selenoprotein P and extracellular glutathione peroxidase, and attained a maximum level at about 6 h after injection. The isotope ratio of endogenous to exogenous Se concentrations in plasma after 48 h post-injection was proposed to represent the Se-specific indicator in plasma reflecting the nutritional status of Se.

Animals↗
PubMed1997

Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice.

The severe combined immune deficiency (SCID) mouse was reported as an animal model for human immune deficiency. Through the course of several studies, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene came to be considered a candidate for the SCID-responsible gene. We isolated an ORF of the murine DNA-PKcs gene from SCID mice and their parent strain C.B-17 mice and determined the DNA sequences. The ORF of the murine DNA-PKcs gene contained 4128-aa residues and had 78.9% homology with the human DNA-PKcs gene. A particularly important finding is that a T to A transversion results in the substitution of termination codon in SCID mice for the Tyr-4046 in C.B-17 mice. No other mutation was detected in the ORF of the gene. The generality of this transversion was confirmed using four individual SCID and wild-type mice. The substitution took place in the phosphatidylinositol 3-kinase domain, and the mutated gene encodes the truncated products missing 83 residues of wild-type DNA-PKcs products. Furthermore, the quantity of DNA-PKcs transcript in wild-type and SCID cells was almost equal. These observations indicate that the DNA-PKcs gene is the SCID-responsible gene itself and that the detected mutation leads to the SCID aberration.

Amino Acid Sequence↗
PubMed1997

Simple and rapid preparation of plasmid template by a filtration method using microtiter filter plates.

We developed a new simple high-throughput plasmid DNA extraction procedure, based on a modified alkaline lysis method, using only one 96-well microtiter glassfilter plate. In this method, cell harvesting, lysis by alkaline and plasmid purification are performed on only one microtiter glassfilter plate. After washing out RNAs or other contaminants, plasmid DNA is eluted by low-ion strength solution, although precipitated chromosomal DNA is not eluted. The plasmid prepared by this method can be applied to sequencing reactions or restriction enzyme cleavage.

Animals↗
PubMed1997

High efficiency selection of full-length cDNA by improved biotinylated cap trapper.

We report here an improved protocol for the preparation of full-length cDNA libraries that improves the previously reported method (Carninci, P., Kvam, K., Kitamura, A. et al. 1996, Genomics, 137, 327-336), that allows long cDNAs to be cloned more efficiently. One potential disadvantage of the original biotinylated CAP trapper protocol is the exposure of mRNA to chemical and enzymatic attacks during the biotinylation of the cap structure, before the first-strand cDNA synthesis (and selection of full-length cDNA by biotinylated cap). Here, we show that the biotinylation of the cap structure is very specific and effective even if biotinylation is performed on the mRNA/cDNA hybrid produced by the first-strand cDNA synthesis reaction. Consequently, mRNA remains protected from chemical and enzymatic degradation during the overnight biotinylation step, thus making it possible to select full-length cDNAs of longer average size. We herein report the efficiency and specificity of the new version of the protocol for cap structure biotinylation and capture of full-length cDNA.

Animals↗
PubMed1997

Telomerase activity is detected in pancreatic cancer but not in benign tumors.

Activation of telomerase and stabilization of telomeres are considered to be necessary for immortalization of human tumor cells. In the present study, telomerase activity was detected in 41 (95%) of 43 pancreatic cancer specimens but was detectable in none of 11 benign pancreatic tumors and only one of 3 pancreatitis samples. Low levels of telomerase activity were detected in 5 (14%) of 36 adjacent "normal" pancreatic tissues. These five telomerase-positive "normal" specimens were obtained from patients that also had pancreatic cancer and may reflect occult microinvasion. Telomerase activity was examined in 12 ex vivo brushing samples of the pancreatic duct, and 8 of 8 with pancreatic cancer had detectable telomerase activity, whereas 0 of 4 of benign lesions (cystadenoma and pancreatitis) did. These findings suggest that telomerase activity in cells derived from pancreatic ducts may be useful in the diagnosis of cancer and that telomerase activity may be a critical or rate-limiting step in pancreatic carcinogenesis.

Chronic Disease↗
PubMed1997

Wild-type, but not mutant-type, p53 enhances nuclear accumulation of the NS3 protein of hepatitis C virus.

By using vaccinia virus-T7 hybrid expression system, subcellular localization of the NS3 protein of hepatitis C virus was studied. Full-size NS3 (NS3F) and a carboxy-terminally truncated form (NS3 deltaC) were localized in the cytoplasm and the nucleus when expressed alone. However, NS3F and NS3 deltaC, but not amino- and carboxy-terminally truncated form (NS3 deltaN deltaC), were each co-localized with wild-type p53 almost exclusively in the nucleus upon co-expression. The wild-type p53-induced nuclear accumulation of NS3F was inhibited only partially by NS4A. When co-expressed with mutant-type p53, NS3F and NS3 deltaC were each co-localized with it exclusively in the cytoplasm. Taken together, the present results suggest that wild-type p53 enhances nuclear accumulation of NS3F and NS3 deltaC through the involvement of their amino-terminal sequences even in the presence of NS4A, and that mutant-type p53 inhibits their nuclear, and enhances their cytoplasmic, accumulation.

Base Sequence↗
PubMed1997

Performance study of a miniature gamma ray scintillation vivo probe for tumor localization.

UNLABELLED: We have developed a miniature gamma-ray endoscopic probe consisting of dual BGO detector probes for tumor detection inside the body cavities. The dual detector system was coupled with random coincidence to decrease the distant background radiation and to improve its spatial resolution for tumor localization. METHOD: The performance of the probe was investigated with a point source and a water phantom. A solution of positron emitting 18F isotope was used as the source. Clinical trials of the probe were done to localize tumors on the skin surface of four subjects carrying tumors close to the body surfaces, into whom 67Ga-citrate and 18F-FDG radiopharmaceuticals were injected. RESULTS: Measurements indicated that the spatial resolution of the dual detector probes is around 1.5 times better than the single detector probe, and both single and dual detector endoscopic probe systems are capable of localizing a tumor on a large photon background. CONCLUSION: The endoscopic probe may be easier to insert inside body cavities due to the small crystal size and the flexible light guides. A single detector probe with higher sensitivity may be useful in searching for tumors over a wide intracavity area but a dual detector probe can be used for precise tumor localization. The detector probe may also be suitable for intraoperative observation.

Adult↗
PubMed1997

Performance evaluation of a large axial field-of-view PET scanner: SET-2400W.

The SET-2400W is a newly designed whole-body PET scanner with a large axial field of view (20 cm). Its physical performance was investigated and evaluated. The scanner consists of four rings of 112 BGO detector units (22.8 mm in-plane x 50 mm axial x 30 mm depth). Each detector unit has a 6 (in-plane) x 8 (axial) matrix of BGO crystals coupled to two dual photomultiplier tubes. They are arranged in 32 rings giving 63 two-dimensional image planes. Sensitivity for a 20-cm cylindrical phantom was 6.1 kcps/kBq/ml (224 kcps/microCi/ml) in the 2D clinical mode, and to 48.6 kcps/kBq/ml (1.8 Mcps/microCi/ml) in the 3D mode after scatter correction. In-plane spatial resolution was 3.9 mm FWHM at the center of the field-of-view, and 4.4 mm FWHM tangentially, and 5.4 mm FWHM radially at 100 mm from the center. Average axial resolution was 4.5 mm FWHM at the center and 5.8 mm FWHM at a radial position 100 mm from the center. Average scatter fraction was 8% for the 2D mode and 40% for the 3D mode. The maximum count rate was 230 kcps in the 2D mode and 350 kcps in the 3D mode. Clinical images demonstrate the utility of an enlarged axial field-of-view scanner in brain study and whole-body PET imaging.

Bone Neoplasms↗
PubMed1997

Effects of dose on the methylation of selenium to monomethylselenol and trimethylselenonium ion in rats.

Mechanisms and metabolic significance in rats of methylation to the reduced form of selenium (Se), i.e., selenide (Se2-), were studied by dose- and time-related experiments with injection of selenite. Urinary Se-metabolites were determined by HPLC using an inductively coupled argon plasma-mass spectrometer as an in-line detector (HPLC/ICP-MS method). Although only monomethylselenon (MMSe) has been detected in urine of normal rats even in those fed a Se-excess diet, the three types of Se-metabolites - MMSe, trimethylselenonium ion (TMSe), and inorganic Se, were detected in urine of Wistar rats injected with selenite (0, 0.1, 0.3, 0.5 and 1.0 mg Se/kg body weight) into the tail vein. The amount of the three Se-metabolites was plotted against the total urinary Se concentration and shown to change dose- and time-dependently. The monomethylated metabolite, i.e., MMSe, increased in urine rapidly at first and was slowly followed by linear dose-dependent excretion of the trimethylated metabolite, TMSe. The new methylation pathway of MMSe leading to TMSe was assumed to be induced or activated when the dose of Se exceeds the limit of the normal capacity for monomethylation. Progressive methylation reactions were suggested to be regulated enzymatically.

Animals↗
PubMed1997

Hemangiopericytoma of the chest wall.

We treated a 79-year-old woman with hemangiopericytoma of the chest wall. This is a very rare entity, with only a total of 12 cases including our case reported in Japan. There is a predominance of women (10 female patients and two male patients) and a predilection for the right side (9 in the right side and 3 in the left side).

Aged↗