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H Takeshima

Publications and source records attributed to H Takeshima.

At least 37 records · Page 2Linked to original sources

Abnormal formation of sarcoplasmic reticulum networks and triads during early development of skeletal muscle cells in mitsugumin29-deficient mice.

Recently, we detected a novel membrane protein, mitsugumin29 (MG29), in the triads in rabbit skeletal muscle cells and suggested important roles for this membrane protein in the formation of the sarcoplasmic reticulum (SR) networks and triads in muscle cells. In the present study, we examined the development of skeletal muscle cells in MG29-deficient mice to try to determine the roles played by MG29 in the formation of the SR networks and triads. Ultrastructural observations revealed some morphological abnormalities in these mice, such as incomplete formation of the SR networks, an irregular running of the transverse tubule and a partial defect in the triads at the A-I junctional region. These ultrastructural abnormalities occurred during early myogenesis and were preserved until the adult stage. The possible roles for MG29 in the formation of SR networks and triads in skeletal muscle cells are discussed in the light of these observations.

Animals↗

Modulation of synaptic transmission by nociceptin/orphanin FQ and nocistatin in the spinal cord dorsal horn of mutant mice lacking the nociceptin/orphanin FQ receptor.

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are two neuropeptides derived from the same precursor protein that exhibit opposing effects on spinal neurotransmission and nociception. Here, we have used whole-cell, patch-clamp recordings from visually identified neurons in spinal cord dorsal horn slices of genetically modified mice to investigate the role of the N/OFQ receptor (N/OFQ-R) in the modulatory action of both peptides on excitatory glutamatergic and inhibitory glycinergic and gamma-aminobutyric acid (GABA)-ergic synaptic transmission. In wild-type mice, N/OFQ selectively suppressed excitatory transmission in a concentration-dependent manner but left inhibitory synaptic transmission unaffected. In contrast, NST reduced only inhibitory but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated excitatory synaptic transmission. N/OFQ-mediated inhibition of excitatory transmission was completely absent in N/OFQ-R receptor-deficient (N/OFQ-R(-/-)) mice and significantly reduced in heterozygous (N/OFQ-R(+/-)) mice, whereas the action of NST on inhibitory neurotransmission was completely retained. To test for the relevance of these results for spinal nociception, we investigated the effects of intrathecally injected N/OFQ in the mouse formalin test, an animal model of tonic pain. N/OFQ (3 nmol/mouse) induced significant antinociception in wild-type mice, but had no antinociceptive effects in N/OFQ-R(-/-) mice. These results indicate that the inhibitory action of N/OFQ on excitatory glutamatergic synaptic transmission and its spinal antinociceptive action are mediated via the N/OFQ receptor, whereas the action of NST is independent of this receptor.

Analgesics, Opioid↗

Pronociceptive effects of nociceptin/orphanin FQ (13-17) at peripheral and spinal level in mice.

The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) is reported to be metabolized by aminopeptidase N and endopeptidase 24.15. In the present study, N/OFQ C-terminal fragments elicited nociceptive responses in the peripheral nociceptors and in the spinal cord, whereas N-terminal fragments had no significant nociception. The nociceptive effect of N/OFQ (13-17) was most potent and remained unchanged in N/OFQ peptide receptor (NOPR) gene knockout mice, indicating that N/OFQ (13-17)-induced nociception is mediated through a novel mechanism independent of the activation of NOPR. This finding was further confirmed by in vitro guanosine 5'-O-(3-[35S]thio)triphosphate binding experiments, in which N/OFQ (13-17) showed no significant binding activity in baculovirus/sf21 cells expressing NOPR together with G protein alpha(i)1-, beta1-, and gamma2-subunits, whereas N/OFQ showed stimulation in a concentration-dependent manner. On the other hand, although a typical bell-shaped dose-response relationship was observed with a wide range of N/OFQ doses in both peripheral and central nociception tests, N/OFQ (13-17) did not show bell-shaped dose-response relationship in the central nociception test. This finding indicates that N/OFQ (13-17), in contrast to N/OFQ, lacks the postsynaptic antinociceptive actions modulating substance P signaling in the spinal cord. Together, our results suggest that C-terminal fragments of N/OFQ have potent nociceptive actions, and N/OFQ (13-17) could have the potential to mediate its actions through a novel mechanism independent of the activation of NOPR in the nociceptors and in spinal synapses.

Animals↗

[A case of transitional cell carcinoma of the urinary bladder with high serum level of CEA and CA19-9].

An 85-year-old male with asymptomatic gross hematuria was diagnosed with invasive bladder tumor, transitional cell carcinoma grade 3. Serum levels of CEA and CA19-9 were elevated and histological examination revealed expression of both markers in the cytoplasm of cancer cells. Out of therapeutic options, intra-arterial chemotherapy and radiotherapy were selected because of his age. During the treatment, serum levels of CEA and CA19-9 decreased along with reduction of tumor size. These serum markers have been reported to be elevated in 10 to 60% of patients with bladder tumor and are useful markers for evaluation of the treatment as suggested in the present case.

Aged↗

[Junctophilins: molecular components contributing junctional membrane complexes between the cell-surface membrane and endoplasmic/sarcoplasmic reticulum].

The junctional membrane complex between the plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ER/SR) is an important structural foundation for functional crosstalk between ionic channels. In cardiac myocytes, functional coupling between cell-surface and intracellular Ca(2+) channels produces Ca(2+) transients for contraction. Junctophilins, a novel family of junctional membrane complex proteins, contribute to the stabilization of the junctional membrane complex by anchoring the ER/SR and interacting with the PM. Mutant mice lacking the cardiac-type junctophilin exhibited embryonic lethality due to heart failure, and the mutant cardiac myocytes showed deficiency of the junctional membrane complexes and abnormal Ca(2+) signaling.

English Abstract↗

Increased susceptibility to fatigue of slow- and fast-twitch muscles from mice lacking the MG29 gene.

Mitsugumin 29 (MG29), a major protein component of the triad junction in skeletal muscle, has been identified to play roles in the formation of precise junctional membrane structures important for efficient signal conversion in excitation-contraction (E-C) coupling. We carried out several experiments to not only study the role of MG29 in normal muscle contraction but also to determine its role in muscle fatigue. We compared the in vitro contractile properties of three muscles types, extensor digitorum longus (EDL) (fast-twitch muscle), soleus (SOL) (slow-twitch muscle), and diaphragm (DPH) (mixed-fiber muscle), isolated from mice lacking the MG29 gene and wild-type mice prior to and after fatigue. Our results indicate that the mutant EDL and SOL muscles, but not DPH, are more susceptible to fatigue than the wild-type muscles. The mutant muscles not only fatigued to a greater extent but also recovered significantly less than the wild-type muscles. Following fatigue, the mutant EDL and SOL muscles produced lower twitch forces than the wild-type muscles; in addition, fatiguing produced a downward shift in the force-frequency relationship in the mutant mice compared with the wild-type controls. Our results indicate that fatiguing affects the E-C components of the mutant EDL and SOL muscles, and the effect of fatigue in these mutant muscles could be primarily due to an alteration in the intracellular Ca homeostasis.

Animals↗

Enhanced spinal nociceptin receptor expression develops morphine tolerance and dependence.

The tolerance and dependence after chronic medication with morphine are thought to be representative models for studying the plasticity, including the remodeling of neuronal networks. To test the hypothesis that changes in neuronal plasticity observed in opioid tolerance or dependence are derived from increased activity of the anti-opioid nociceptin system, the effects of chronic treatments with morphine were examined using nociceptin receptor knock-out (NOR(-/-)) mice and a novel nonpeptidic NOR antagonist, J-113397, which shows a specific and potent NOR antagonist activity in in vitro [(35)S]GTPgammaS binding assay and in vivo peripheral nociception test. The NOR(-/-) mice showed marked resistance to morphine analgesic tolerance without affecting morphine analgesic potency in tail-pinch and tail-flick tests. The NOR(-/-) mice also showed marked attenuation of morphine-induced physical dependence, manifested as naloxone-precipitated withdrawal symptoms after repeated morphine treatments. Similar marked attenuation of morphine tolerance was also observed by single subcutaneous (10 mg/kg) or intrathecal (1 nmol) injection of J-113397, which had been given 60 min before the test in morphine-treated ddY mice. However, the intracerebroventricular injection (up to 3 nmol) did not affect the tolerance. On the other hand, morphine dependence was markedly attenuated by J-113397 that had been subcutaneously given 60 min before naloxone challenge. There was also observed a parallel enhancement of NOR gene expression only in the spinal cord during chronic morphine treatments. Together, these findings suggest that the spinal NOR system develops anti-opioid plasticity observed on morphine tolerance and dependence.

Animals↗

Characterization of a calcium-regulation domain of the skeletal-muscle ryanodine receptor.

A negatively charged region of the N-terminal portion of the skeletal ryanodine receptor (RyR), located between residues 1872-1923, is involved in Ca (2+)-dependent regulation of the Ca(2+)-release channel. This region is divergent between the skeletal (RyR1) and cardiac (RyR2) isoforms of the channel, and is known as D3. Ca(2+) exerts important regulatory functions on the RyR, being involved in both activation and inactivation functions of the channel, i.e. the effects occurring at micromolar and millimolar Ca(2+) concentrations respectively. To characterize the role of D3 in the Ca(2+)-dependent regulation of the Ca(2+)-release channel, we studied the functional consequences of deleting the D3 region from RyR1 (DeltaD3-RyR1) using a heterologous expression system, [(3)H]ryanodine binding assays and single-channel recordings in lipid bilayers. Deletion of the D3 region selectively affected Ca(2+)-dependent regulation of RyR1, but did not alter [(3)H]ryanodine binding or the effect of other modulators on the RyR. Compared with full-length RyR1 (wt-RyR1), the Ca(2+)-dependence curve of DeltaD3-RyR1 is broader, reflecting increased sensitivity to Ca(2+) activation and decreased sensitivity to Ca(2+) inactivation. In addition, DeltaD3-RyR1 was more resistant to inhibition by Mg(2+). Comparison of the effect of caffeine on wt-RyR1 and DeltaD3-RyR1 suggested that D3 is an important region of RyR that participates in Ca(2+)-dependent activation and inactivation of the Ca(2+)-release channel.

Animals↗

A region of the sulfonylurea receptor critical for a modulation of ATP-sensitive K(+) channels by G-protein betagamma-subunits.

To determine the interaction site(s) of ATP-sensitive K(+) (K(ATP)) channels for G-proteins, sulfonylurea receptor (SUR2A or SUR1) and pore-forming (Kir6.2) subunits were reconstituted in the mammalian cell line, COS-7. Intracellular application of the G-protein betagamma2-subunits (G(betagamma)(2)) caused a reduction of ATP-induced inhibition of Kir6.2/SUR channel activities by lessening the ATP sensitivity of the channels. G(betagamma)(2) bound in vitro to both intracellular (loop-NBD) and C-terminal segments of SUR2A, each containing a nucleotide-binding domain (NBD). Furthermore, a single amino acid substitution in the loop-NBD of SUR (Arg656Ala in SUR2A or Arg665Ala in SUR1) abolished the G(betagamma)(2)-dependent alteration of the channel activities. These findings provide evidence that G(betagamma) modulates K(ATP) channels through a direct interaction with the loop-NBD of SUR.

ATP-Binding Cassette Transporters↗

Distribution of nociceptin/orphanin FQ precursor protein and receptor in brain and spinal cord: a study using in situ hybridization and X-gal histochemistry in receptor-deficient mice.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-like heptadecapeptide agonist for the opioid receptor homolog, N/OFQ receptor. To explore the precise distribution of the peptide-receptor system, the authors examined the brain and spinal cord from receptor-deficient mice bearing the targeted mutation (morc(m1)), a lacZ insertional mutation in the N/OFQ receptor gene. Precursor protein N/OFQ (preproN/OFQ) mRNA was detected by using in situ hybridization, and the N/OFQ receptor was detected by using X-gal histochemistry. The N/OFQ receptor reflected by lacZ expression was observed at high levels in the dentate gyrus, lateral septum, subparafascicular thalamic nucleus, medial preoptic area, median preoptic nucleus, ventromedial preoptic nucleus, anterior hypothalamic area, paraventricular hypothalamic nucleus, ventromedial hypothalamic nucleus, auditory brainstem nuclei, pontine dorsal tegmentum, and nucleus of the solitary tract. In situ detection of the N/OFQ receptor mRNA by digoxigenin-labeled riboprobes coupled with tyramide signal amplification in normal and wild-type mice resulted in the regional distribution paralleling the lacZ expression in these regions. PreproN/OFQ mRNA was expressed at high levels in the subparafascicular thalamic nucleus, central gray, central tegmental field, auditory brainstem nuclei, caudal spinal trigeminal nucleus, and spinal dorsal horn. Furthermore, variable levels of expression of the peptide and receptor were seen in distinct sites of the brain and spinal cord. These data indicate a correspondence of the peptide and the receptor in local distribution at limbic, hypothalamic, and brainstem sites. Together with concurrent physiologic and behavioral studies in mutant mice, the results suggest functional roles for the N/OFQ system, including the central regulation of learning and memory, hearing ability, water balance, food intake, and blood pressure.

Animals↗

Characterization of human junctophilin subtype genes.

Junctophilin (JP) subtypes, namely JP-1, 2, and 3, have been currently identified in excitable cells and constitute a novel family of junctional membrane complex proteins. Our studies have suggested that JPs take part in the formation of junctional membrane complexes by spanning the membrane of the intracellular Ca(2+) store and interacting with the cell-surface membrane. In this report we describe the primary structures, genomic organization, and tissue distribution of human JP subtypes. By cloning and analyzing human genomic DNA segments, the protein-coding sequence interrupted with four introns was defined in each JP gene. The deduced human JP subtypes shared characteristic structural features with their rabbit and mouse counterparts. Genomic mapping demonstrated that JP genes do not cluster on the human genome. RNA blot hybridization indicated that tissue-specific expression patterns of JP genes in human are essentially the same as those in mouse; skeletal muscle contained both JP-1 and JP-2 mRNAs, the heart predominantly expressed JP-2 mRNA, and the brain specifically contained JP-3 mRNA. In the light of this, we propose intramolecular domains of JP subtypes based on the structural and functional characteristics.

Amino Acid Sequence↗

Mutant mice lacking ryanodine receptor type 3 exhibit deficits of contextual fear conditioning and activation of calcium/calmodulin-dependent protein kinase II in the hippocampus.

As it is known that ryanodine receptor type 3 is expressed in the hippocampus, we examined the contribution of this receptor to contextual fear conditioning behavior and to the activation of Ca(2+)/calmodulin-dependent protein kinase II using mice lacking the receptor. Ryanodine receptor type 3-deficient mice exhibited impairments of performance in the contextual fear conditioning test, passive avoidance test, and Y-maze learning test. Both the activities of Ca(2+)/calmodulin-dependent protein kinase IIbeta and Ca(2+)/calmodulin-dependent protein kinase IIalpha were significantly increased in the experimental group compared to the control group in the hippocampus, but not in the cingulate cortex on the testing day 24 h after contextual fear training. However, the activities of Ca(2+)/calmodulin-dependent protein kinase IIbeta and alpha were almost the same in the experimental and control groups in the hippocampus on the training day. Ryanodine receptor type 3-deficient mice did not show the increment of Ca(2+)/calmodulin-dependent protein kinase IIbeta and alpha activities in the hippocampus on the testing day. In addition, these mutant mice showed the reduction of fear response in the elevated plus-maze test. The present results suggest that calcium-induced calcium release through the activation of ryanodine receptor type 3 in the hippocampus is important to the expression of the performance of contextual learning through the elevation of Ca(2+)/calmodulin-dependent protein kinase IIbeta and alpha activities.

Animals↗

Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene.

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the "A" fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.

Animals↗

Application of advances in molecular biology to the treatment of brain tumors.

Recent advances in molecular biology have substantially improved our understanding of the molecular genetics of primary brain neoplasms. Soon each histopathologic category of glioma will be further divided into subgroups according to similar genetic background, gene expression profile, and similarity of biologic responses to radiotherapy or chemotherapy. Identification of key molecules that are specifically altered in neoplastic cells will provide candidate molecular targets for tumor treatment. Novel therapeutic tools for targeting tumor cells, such as viral vectors for gene therapy, have been created. In the near future, the accumulation of new knowledge in brain tumor biology and genetics, combined with rational drug design, will revolutionize the treatment of malignant gliomas, which are among the most lethal human cancers.

Apoptosis↗

Molecular cloning of cDNA encoding a drosophila ryanodine receptor and functional studies of the carboxyl-terminal calcium release channel.

Ryanodine is a plant alkaloid that was originally used as an insecticide. To study the function and regulation of the ryanodine receptor (RyR) from insect cells, we have cloned the entire cDNA sequence of RyR from the fruit fly Drosophila melanogaster. The primary sequence of the Drosophila RyR contains 5134 amino acids, which shares approximately 45% identity with RyRs from mammalian cells, with a large cytoplasmic domain at the amino-terminal end and a small transmembrane domain at the carboxyl-terminal end. To characterize the Ca(2+) release channel activity of the cloned Drosophila RyR, we expressed both full-length and a deletion mutant of Drosophila RyR lacking amino acids 277-3650 (Drosophila RyR-C) in Chinese hamster ovary cells. For subcellular localization of the expressed Drosophila RyR and Drosophila RyR-C proteins, green fluorescent protein (GFP)-Drosophila RyR and GFP-Drosophila RyR-C fusion constructs were generated. Confocal microscopic imaging identified GFP-Drosophila RyR and GFP-Drosophila RyR-C on the endoplasmic reticulum membranes of transfected cells. Upon reconstitution into the lipid bilayer membrane, Drosophila RyR-C formed a large conductance cation-selective channel, which was sensitive to modulation by ryanodine. Opening of the Drosophila RyR-C channel required the presence of microM concentration of Ca(2+) in the cytosolic solution, but the channel was insensitive to inhibition by Ca(2+) at concentrations as high as 20 mM. Our data are consistent with our previous observation with the mammalian RyR that the conduction pore of the calcium release channel resides within the carboxyl-terminal end of the protein and further demonstrate that structural and functional features are essentially shared by mammalian and insect RyRs.

Amino Acid Sequence↗

Comparison of Ca(2+) sparks produced independently by two ryanodine receptor isoforms (type 1 or type 3).

The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo-3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling.

Animals↗

Role of nociceptin systems in learning and memory.

This article summarizes our recent finding that the nociceptin system is involved in the regulation of learning and memory. The nociceptin-knockout mice show greater learning ability in the water maze task, an enhanced latent learning in the water finding task, better memory in the passive avoidance task, and further, larger long-term potentiation in the hippocampal CA1 region than wild-type mice. Nociceptin itself induces an impairment of passive avoidance task in wild-type mice, which is reversed by naloxone benzoylhydrazone (NalBzoH). Thus, the nociceptin system seems to play negative roles in learning and memory, and NalBzoH may act as a potent antagonist for the nociceptin receptor.

Animals↗