Biomedical subjects

E Ito

Publications and source records attributed to E Ito.

At least 55 records · Page 3Linked to original sources

PubMed2000

A core-promoter region functions bi-directionally for human opioid-receptor-like gene ORL1 and its 5'-adjacent gene GAIP.

We report the genomic structure and functional activities of the promoter regions of the human opioid-receptor-like gene ORL1 and its 5'-adjacent gene GAIP (G alpha interacting protein). The transcription and alternative splicing of human ORL1 are controlled by two alternate promoters, located approximately 10 kb apart. The two promoter regions lack a TATA-box and are GC rich. Promoter 1A initiates, from a single transcription start point (TSP), two transcripts: one consisting of exons 1A, 1B, 2, etc., the other without exon 1B. A potential ATG codon upstream of the initiation codon of ORL1 starts a new open-reading frame encoding a theoretical polypeptide of 205 amino acid residues. The promoter 1B transcribes, from multiple TSPs, only one mRNA starting with exon 1B. Two different repeat sequence polymorphisms are found in the ORL1 promoter regions. Luciferase reporter gene assays with promoter regions and a series of deletion mutants have mapped the core-promoter 1A and 1B within two short fragments. DNA sequencing and a database search reveal that the human GAIP gene is located upstream of ORL1 and is oriented in the opposite direction. The transcription and alternative splicing of GAIP are also under the control of alternate promoters. The first exons of ORL1 and GAIP are separated by only 83 bp. This 83 bp fragment, together with short surrounding sequences from both first exons, functions bi-directionally as a core-promoter for both genes. The transcription and alternative splicing of human ORL1 and GAIP are cell-type specific. While GAIP is expressed in both NT2 precursor cells and differentiated NT2 neuronal cells, ORL1 is only expressed in differentiated NT2 neurons. Since ORL1 is a G protein-coupled receptor and GAIP interacts with G protein alpha subunits, their physical linkage in the genome and co-operative transcriptional regulation may play a significant role in ORL1 receptor signal transduction.

Alternative Splicing↗

PubMed2000

Transcription factor BACH1 is recruited to the nucleus by its novel alternative spliced isoform.

The transcription factor Bach1 is a member of a novel family of broad complex, tramtrack, bric-a-brac/poxvirus and zinc finger (BTB/POZ) basic region leucine zipper factors. Bach1 forms a heterodimer with MafK, a member of the small Maf protein family (MafF, MafG, and MafK), which recognizes the NF-E2/Maf recognition element, a cis-regulatory motif containing a 12-O-tetradecanoylphorbol-13-acetate-responsive element. Here we describe the gene structure of human BACH1, including a newly identified promoter and an alternatively RNA-spliced truncated form of BACH1, designated BACH1t, abundantly transcribed in human testis. The alternate splicing originated from the usage of a novel exon located 5.6 kilobase pairs downstream of the exon encoding the leucine zipper domain, and produced a protein that contained the conserved BTB/POZ, Cap'n collar, and basic region domains, but lacked the leucine zipper domain essential for NF-E2/Maf recognition element binding. Subcellular localization studies using green fluorescent protein as a reporter showed that full-length BACH1 localized to the cytoplasm, whereas BACH1t accumulated in the nucleus. Interestingly, coexpression of BACH1 and BACH1t demonstrated interaction between the molecules and the induction of nuclear import of BACH1. These results suggested that BACH1t recruits BACH1 to the nucleus through BTB domain-mediated interaction.

Alternative Splicing↗

PubMed2000

Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni.

During summer and autumn, the box jellyfish (sea wasp) Carybdea rastoni is one of the most bothersome stinging pests to swimmers and bathers on the Japanese coast. Two labile but potent hemolytic toxins from the tentacles of Carybdea rastoni were isolated in their active forms using newly developed purification methods. The molecular masses of the isolated C. rastoni toxin-A and toxin-B (CrTX-A and CrTX-B) are 43 and 46 kDa, respectively, as calculated from SDS-PAGE. In the present study, we sequenced the full-length cDNA (1600 bp), which encodes both CrTX-A and CrTX-B. The deduced 450 amino acid sequence of the CrTXs, showed no significant homology with any known protein. This report presents the first complete sequence of a proteinaceous jellyfish toxin. Furthermore, it was revealed that CrTX-A was primarily localized in the nematocyst, whereas CrTX-B was detected only in the tentacle. Because the nematocyst is the organ responsible for the cnidarian sting, the remainder of the study focused on the toxicity of CrTX-A. We found that CrTX-A was fatally toxic to mice at 20 microg/kg (i.v.) and crayfish at 5 microg/kg (i.p.). Subcutaneously injected CrTX-A (0.1 microg) caused inflammation of mouse skin. These results showed that CrTX-A is responsible for the cutaneous inflammation observed in humans stung by C. rastoni.

Animals↗

PubMed2000

Time-sharing contributions of A- and D-type K+ channels to the integration of high-frequency sequential excitatory post synaptic potentials at a model dendrite in rats.

A- and D-type K+ channels (KA and KD channels) have unique physiological properties that play important roles in the integration of excitatory post synaptic potentials (EPSPs) in neuronal dendrites. These functions were analyzed using a computer program, NEURON, to simulate high-frequency sequential synaptic inputs, that can induce long-term potentiation (LTP). We paid close attention to the stability of the reduction of sequential EPSPs. When either KA or KD channels were included in models, the EPSP reduction ratios were less stable than containing both KA and KD channels. When both KA and KD channels were present in the model, the variance of EPSP reduction ratios was significantly smaller in comparison with that in the presence of either KA or KD channels alone. We thus concluded that the co-existence of KA and KD channels is necessary to produce stable EPSPs during the high-frequency synaptic stimulation necessary for induction of LTP.

Animals↗

PubMed2000

Cloning and expression of human B cell-specific transcription factor BACH2 mapped to chromosome 6q15.

The transcription factor Bach2, a member of the BTB-basic region leucine zipper (bZip) factor family, binds to a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element and the related Maf-recognition element (MARE) by forming homodimers or heterodimers with Maf-related transcription factors. Bach2 regulates transcription by binding to these elements. To understand the function in hematopoiesis, we isolated a cDNA clone for human Bach2 (BACH2) encoding a protein of 841 amino acid residues with a deduced amino acid sequence having 89.5% identity to mouse homolog. Among human hematopoietic cell lines, BACH2 is expressed abundantly only in some B-lymphocytic cell lines. RT-PCR analysis of hematopoietic cells revealed that BACH2 mRNA is expressed in primary B-cells. Enforced expression of BACH2 in a human Burkitt cell line, RAJI that does not express endogenous BACH2, resulted in marked reduction of clonogenic activity, indicating that BACH2 possesses an inhibitory effect on cell proliferation. By fluorescent in situ hybridization, the BACH2 gene was localized to chromosome 6q15. Because deletion of the long arm of chromosome 6 (6q) is one of the commonest chromosomal alterations in human B-cell lymphoma, we examined for the loss of heterozygosity (LOH) of the BACH2 gene in human B-cell non-Hodgkin's lymphomas (NHL). Among 25 informative cases, five (20%) showed LOH. These results indicate that BACH2 plays important roles in regulation of B cell development.

Adult↗

PubMed2000

A thermodynamical study of the clockwork hypothesis proposed by E. Schrödinger.

In the present study, the 'clockwork' hypothesis proposed by Schrödinger was examined from the viewpoint of thermodynamics. Firstly, noticing a unidirectional transfer of entropy in a heat engine, the logic was briefly explained about a close relation between this entropy transfer and an irreversible cycle performed by a working body. Next, paying attention to two fundamental differences between a heat engine and a biological system, we considered an isolated system Asigma consisting of three one-component systems (Ai, A, Ao) and noted a case that the same molecules as the component ones flowed quasistatically into Ai from the outside. Then, the unidirectional flows of the molecules, energy and entropy, which were induced by the above inflow in Asigma, were formulated on the basis of the equilibrium thermodynamics for an open system. Furthermore, it was clarified that the fundamental equation for these flows is the Schrödinger inequality and that the necessary-sufficient condition for this inequality is the existence of an irreversible cycle performed by A. Here A corresponds to a working body in a heat engine. It was, thus, concluded that the 'clockwork' hypothesis by Schrödinger is considered to be reasonable for a biological system composed of various irreversible subsystems.

Algorithms↗

PubMed2000

Bimodal effects of acetylcholine on synchronized calcium oscillation in rat cultured cortical neurons.

To understand the plastic changes in neural network dynamics, we analyzed the modulating effects of cholinergic agonists on synchronized Ca(2+) oscillation in the rat primary cultured cortical neurons. At 5-10 days after establishment of the culture, neurons exhibited synchronized Ca(2+) oscillation. This Ca(2+) oscillation was derived from glutamatergic and gamma-aminobutyric acid (GABA)ergic synaptic inputs from neighboring neurons. Application of acetylcholine and nicotine increased, decreased, or did not change the frequency of synchronized Ca(2+) oscillation, depending on the colonies in the culture. The presence of cholinergic neurons in the culture was confirmed by immunocytochemical staining. Our study provided evidence for the first time that cholinergic neurons exert excitatory influences on GABAergic neurons as well as glutamtergic neurons, resulting in bimodal effects on the frequency of synchronized Ca(2+) oscillation in the cortical neural network.

Acetylcholine↗

PubMed2000

Chromosomal instability syndrome of total premature chromatid separation with mosaic variegated aneuploidy is defective in mitotic-spindle checkpoint.

Skin fibroblast cells from two unrelated male infants with a chromosome-instability disorder were analyzed for their response to colcemid-induced mitotic-spindle checkpoint. The infants both had severe growth and developmental retardation, microcephaly, and Dandy-Walker anomaly; developed Wilms tumor; and one died at age 5 mo, the other at age 3 years. Their metaphases had total premature chromatid separation (total PCS) and mosaic variegated aneuploidy. Mitotic-index analysis of their cells showed the absence of mitotic block after the treatment with colcemid, a mitotic-spindle inhibitor. Bromodeoxyuridine-incorporation measurement and microscopic analysis indicated that cells treated with colcemid entered G1 and S phases without sister-chromatid segregation and cytokinesis. Preparations of short-term colcemid-treated cells contained those cells with chromosomes in total PCS and all or clusters of them encapsulated by nuclear membranes. Cell-cycle studies demonstrated the accumulation of cells with a DNA content of 8C. These findings indicate that the infants' cells were insensitive to the colcemid-induced mitotic-spindle checkpoint.

Abnormalities, Multiple↗

PubMed2000

Two different types of nonthrombogenic surfaces: PEG suppresses platelet adhesion ATP-independently but HEMA-St block copolymer requires ATP consumption of platelets to prevent adhesion.

Poly(ethylene glycol) (PEG) and a hydrophobic-hydrophilic microdomain structured block copolymer comprising poly(2-hydroxyethyl methacrylate) and polystyrene (HEMA-St) have been reported to show good blood compatibility owing to inhibition of platelet activation. By using a computer-assisted novel technique to analyze platelet behavior on the surfaces, we found two different mechanisms to prevent platelet adhesion. Platelets were prevented from adhesion and spreading on the microdomain surface and retained cell movement for a long time. The platelet movement velocity was not significantly different between PEG-grafted surfaces and HEMA-St block copolymer-cast surfaces. However, platelet motion was qualitatively different. Platelets on HEMA-St block copolymer-cast surfaces moved with rolling, spinning, and vibrating, whereas platelet movement was limited to oscillatory vibration on PEG-grafted surfaces. When platelets were treated with NaN(3), an adenosine triphosphate (ATP) synthesis inhibitor, before contacting the surfaces, platelets movement velocity was decreased only on HEMA-St block copolymer-cast surfaces. Such an inhibitory effect was hardly observed with platelets on PEG-grafted surfaces. We propose two different mechanisms to prevent platelet adhesion onto surfaces. One is ATP-independent as observed with PEG, and the other is ATP-dependent for HEMA-St block copolymer, where platelets consume ATP to prevent adhesion.

Adenosine Triphosphate↗

PubMed2000

Complement receptor 3-like immunoreactivity in the light green cells and the canopy cells of the pond snail, Lymnaea stagnalis.

We observed CR3-like immunoreactivity in the central nervous system (CNS) and its surrounding peripheral nerves of the pond snail, Lymnaea stagnalis. In the CNS of L. stagnalis, the immunoreactivity presenting meshwork-like structure was detected in some neurosecretory cells, which are the light green cells (LGCs) and the canopy cells (CCs), both controlling the body growth. The immunoreactivity was also observed along the edges of median lip nerves. The immunoreactive regions in the median lip nerves appeared to form the axonal plates, from which the LGCs and the CCs release molluscan insulin-related peptides (MIPs) into the blood. By contrast, no immunoreactivity was detected in other neurosecretory cells or their release sites, for example the caudodorsal cells and the cerebral commissure, which release ovulation hormones. The present findings, therefore, suggested that CR3 expresses only in the neurosecretory cells releasing MIPs in L. stagnalis.

Animals↗

PubMed2000

Nitric oxide suppresses fictive feeding response in Lymnaea stagnalis.

Fictive feeding activity was monitored in the buccal ganglia of semi-intact preparations of the pond snail, Lymnaea stagnalis, to examine the effects of nitric oxide (NO) released from motoneurons innervating the esophagus on the feeding response. The present results suggest that first; even the low concentration of constitutive NO precisely regulates the feeding rhythm by suppressing high frequency feeding responses; second, that the high concentration of NO released after activation of the feeding central pattern generator following appetitive stimulation of the lips suppresses the feeding rate, resulting in recurrent inhibition. This is the first direct evidence that NO can function to suppress rhythmic activity in the brain.

Animals↗

PubMed2000

An alternatively spliced transcript of the rat nociceptin receptor ORL1 gene encodes a truncated receptor.

Opioid receptor-like protein ORL1, the receptor for the neuropeptide nociceptin (also named orphanin FQ), has two alternatively spliced isoforms in the rat. This alternative splicing event is generated by retaining of intron 3, 81 bases in length, in the mRNA region encoding the second extracellular loop of ORL1. A full-length rat ORL1 receptor has 367 amino acid residues. However, as revealed by sequencing of rat ORL1 genomic DNA and cDNA, the insertion of the unspliced intron 3 brings in an in-frame stop codon and, therefore, creates a truncated open-reading frame encoding only the N-terminal half of ORL1 (from the N-terminus to an alternate extracellular tail C-terminal to the fourth transmembrane domain). The two alternatively spliced transcripts are differentially expressed in tissues. In transfected mammalian cells, the full-length ORL1 displays high-affinity and selective binding for nociceptin, and inhibits the production of cyclic AMP. In contrast, the truncated ORL1 binds nociceptin and other opioid peptides very poorly and non-selectively (affinity in micromolar range), and it does not mediate any inhibitory effects on cyclic AMP production. Apparently, this truncated ORL1 does not function as a receptor for nociceptin or other ligands tested. Such alternative splicing to create a truncated ORL1 receptor might be an endogenous mechanism to negatively regulate nociceptin/ORL1 functions.

Alternative Splicing↗

PubMed2000

Distribution and developmental changes in GABA-like immunoreactive neurons in the central nervous system of pond snail, Lymnaea stagnalis.

We examined three-dimensionally the arrangement of gamma-aminobutyric acid (GABA)-like immunoreactive neurons in the central nervous system (CNS) of the pond snail, Lymnaea stagnalis, by a combination of immunohistochemistry and confocal laser scanning microscopy on whole-mount preparations. GABA-like immunoreactivity was detected in all ganglia of the adult CNS. The following distribution of immunoreactive cell bodies was noted in the adult snail. Buccal ganglia: one cell body and five pairs of cell bodies, cerebral ganglia: one pair of cell bodies, pedal ganglia: two single cell bodies, two pairs of cell bodies, and three pairs of cell clusters, and pleural ganglia: one pair of cell bodies. In the asymmetrical parietal ganglia, three cell bodies were located in the left parietal ganglion; three cell bodies and three cell clusters were located in the right parietal ganglion. In the single visceral ganglion, a few scattered individual cell bodies and a cell cluster were GABA-like immunoreactive. Our results showed that the occurrence of GABA is widely spread in the CNS of adult L. stagnalis. GABA-like immunoreactivity in the CNS was not detected in the embryo but was observed after hatching, although the number of stained cells was less than in the adult, with the exception of those in the cerebral ganglia where their number decreased with maturation. Our results provide detailed maps of the central GABA-like immunoreactive neurons in juveniles, immatures, and adults of L. stagnalis.

Aging↗

PubMed2000

Development of key neurons for learning stimulates learning ability in Lymnaea stagnalis.

The pond snails, Lymnaea stagnalis, change their ability of conditioned taste aversion (CTA) during their development, for example, stage 29 embryos can acquire the CTA, whereas immature snails come to use a long-term memory to maintain the conditioned response. We thus examined the relationships between the learning ability and the development of key neurons (cerebral giant cells: CGCs) for this CTA. The immunoreactivity of serotonin, which is a main neurotransmitter employed in the feeding circuitry, was first observed in the CGCs at the stage 29. After hatching, the CGCs developed their neuropile faster than other cells in the buccal and cerebral ganglia, resulting in their early innervation at the immature stage. The present results, therefore, indicate that the development of key neurons for learning stimulates the developmental changes in learning ability.

Animals↗

PubMed2000

Methylation of the hMLH1 promoter in familial gastric cancer with microsatellite instability.

Microsatellite instability (MSI), which is recognized as an important mechanism in tumorigenesis, has been reported in familial gastric cancers (FGC). However, genetic defects responsible for this phenotype, that is, mutations in mismatch-repair genes such as hMLH1 and hMSH2, have not been detected in most FGC cases. Earlier studies have shown that the promoter region of the hMLH1 gene was methylated in some sporadic colorectal and endometrial cancers. To determine how FGC acquire MSI, we examined the MSI status, hMLH1-protein expression and methylation status of the hMLH1-promoter region in FGC cases. Out of 9 cancers, 6 from 8 FGC kindreds showed MSI at one or more loci; no germline mutations in the hMLH1 or hMSH2 genes were detected; 4 cancers exhibiting MSI displayed aberrant hMLH1 expression: complete loss in one, decreased level in another, and partially staining pattern in the remaining 2. Methylation in the hMLH1-promoter region was found in these 4 cases. In contrast, the cancers displaying hMLH1-protein expression were not methylated in the hMLH1-promoter region. Our data show a significant association between the absence of hMLH1 expression and methylation of its promoter in FGC cases with MSI. This suggests that the mechanism of inactivation of hMLH1 is epigenetic and that there are other genes responsible for FGC.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Induction of apoptosis and growth inhibition of cultured human endometrial adenocarcinoma cells (Sawano) by an antitumor lipoprotein fraction of rice bran.

OBJECTIVE: A lipoprotein fraction extracted from rice bran (RBF) has been proved to be nontoxic to normal cells and to possess the ability to derange energy metabolism and induce apoptosis in Ehrlich ascites tumor cells. This study is designed to investigate the therapeutic effects of RBF as an antitumor substance on endometrial adenocarcinoma cells (Sawano). METHODS: Cultured human endometrial adenocarcinoma cells were treated with medium only (untreated), DMSO, or RBF at 100, 200, and 300 microg/ml. Mitotic indexes were counted by Giemsa stain and apoptotic index by fluorescent TUNEL stain for confocal laser scanning microscopy. DNA fragments were examined by agarose gel electrophoresis. RESULTS: Characteristic apoptotic morphology was observed in Giemsa-stained cells and further confirmed by confocal laser scanning microscopy in RBF-treated cells. Growth of Sawano cells was inhibited by RBF in a dose- and time-dependent manner. Apoptotic indexes (AI) showed an increasing tendency, and mitotic indexes (MI) showed a downward tendency. In 300 microg/ml RBF-treated Sawano cells, AI even reached 41.62 +/- 2. 23% and MI decreased to 0.79 +/- 0.08% at 96 h. Both showed significant differences from to controls and other RBF-treated groups. DNA agarose electrophoresis revealed DNA fragmentation in a ladder pattern. CONCLUSIONS: RBF can induce apoptosis of cultured human endometrial adenocarcinoma cells (Sawano) and inhibit its cell proliferation as an antitumor substance.

Adenocarcinoma↗

PubMed2000

First report on the distribution of orally administered microcystin-LR in mouse tissue using an immunostaining method.

The purpose of this study was to investigate the distribution of microcystin-LR (MCLR) orally administered to mice using an immunostaining method. MCLR was orally dosed at 500 microg/kg to aged Balb/C and ICR mice and their lethality was 23.9%. The former was more sensitive to MCLR than the latter, suggesting that oral toxicity by MCLR is related to the animal strains tested, although the pathological and immunostaining changes were essentially the same in both strains. According to this method the distribution of MCLR and related compounds were indicated as the red staining. Particularly, livers of dead aged mice were intensively stained. The main route of absorption was considered to be the small intestine because the villi contained a large amount of MCLR in both surface epithelial cells and lamina propria, resulting in erosion. The absorbed MCLR was contained in blood plasma and moved to the liver, lung, and heart, and finally to capillaries of the whole body. Excretion of MCLR was shown in the mucous from goblet cells in both the small intestine and large intestine.

Animals↗

PubMed2000

Bleeding from the small intestine caused by aplysiatoxin, the causative agent of the red alga Gracilaria coronopifolia poisoning.

The cause of death by aplysiatoxin poisoning was bleeding from the small intestine in mice. The pathological changes related to the cause and progression of bleeding were studied morphologically. Bleeding from the capillaries was observed 60 min after i.p. treatment at 250 microg/kg, and this was preceded by dilatation of the lymphatic vessel and congestion of capillaries in the lamina propria from 10 min after the injection. At 100 microg/kg i.v., the target vessels were in the lung, where fibrin deposition was observed in the dilated pulmonary artery, and blood flowed out through a gap in the artery. Then, in the small intestine, similar changes appeared to have occurred, and bleeding was induced in two characteristic ways, one through deposition of fibrin in the lumen and the other via distension of the capillary wall.

Animals↗