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Hiroshi Ueda

Publications and source records attributed to Hiroshi Ueda.

At least 37 records · Page 2Linked to original sources
PubMed2005

Exogenous gene expression and growth regulation of hematopoietic cells via a novel human artificial chromosome.

A number of gene delivery systems are currently being developed for potential use in gene therapy. Here, we demonstrate the feasibility of 21deltaqHAC, a newly developed human artificial chromosome (HAC), as a gene delivery system. We first introduced a 21deltaqHAC carrying an EGFP reporter gene and a geneticin-resistant gene (EGFP-21deltaqHAC) into hematopoietic cells by microcell-mediated chromosome transfer. These HAC-containing hematopoietic cells showed resistance to geneticin, expressed EGFP and retained the ability to differentiate into various lineages, and the EGFP-21deltaqHAC was successfully transduced into primary hematopoietic cells. Hematopoietic cells harboring the EGFP-21deltaqHAC could still be detected at two weeks post-transplantation in immunodeficient mice. We also showed effective expansion of hematopoietic cells by introducing the 21deltaqHAC containing ScFvg, a gp130-based chimeric receptor that transmits growth signals in response to specific-antigen of this receptor. All of these results demonstrate the usefulness of HAC in gene therapy.

Animals↗
PubMed2005

Kyotorphin has a novel action on rat cardiac muscle.

Several endogenous peptides for G-protein-coupled receptors have been found to play physiological roles in muscle contraction in addition to their well-demonstrated actions in other tissues. To further identify such peptides, we screened over 400 peptides using an isometric tension assay of rat papillary muscle. Here, we report that kyotorphin, which is known as an analgesic dipeptide, has a cardiac effect. Although kyotorphin had no effect on the twitch tension itself, it inhibited beta-adrenergic agonist isoprenaline-induced increases in twitch tension in a dose-dependent manner. Leu-Arg, a selective antagonist of kyotorphin, reversed this inhibitory effect. The inhibitory effect was also reversed by naloxone, an opioid receptor antagonist. These results suggest that kyotorphin may release opioid peptides from rat cardiac muscle and have an indirect regulatory role in beta-adrenergic action through cross-talk with opioid receptors.

Animals↗
PubMed2005

The role of firefly luciferase C-terminal domain in efficient coupling of adenylation and oxidative steps.

The N-terminal domain (N-domain) of the firefly luciferase from Photinus pyraris has weak luminescence activity, and shows a unique light emitting profile with very long rise time of more than several minutes. Through a sensitive assay of the reaction intermediate luciferyl-adenylate (LH2-AMP), we found that the slow increase in the N-domain luminescence faithfully reflected the concentration of dissociated LH2-AMP. No such correlation was observed for wild-type or mutant enzymes with short rise time, except one with longer rise time. The results suggest that the C-terminal domain plays an indispensable role in efficiently coupling adenylation and oxidative steps.

Adenosine Monophosphate↗
PubMed2005

Effects of various mu- and delta-opioid ligands on food intake in the meat-type chick.

The present study was designed to examine the effects of mu- and delta-opioid receptor ligands on feeding behavior in meat-type chicks. Intracerebroventricular (ICV) injection of naltrexone (mu- and delta-antagonist), beta-funaltrexamine (beta-FNA; mu-antagonist), ICI-174,864 (ICI; delta-antagonist), or naloxonazine (NAL; mu1-antagonist) significantly decreased deprivation-induced feeding at 30 min postinjection. Co-injection of beta-FNA, but not NAL, significantly blocked the depressive effect of [D-Ala2, N-MePhe4, Gly5-ol]-enkephalin (mu-opioid agonist) under ad libitum conditions. Central injection of ICI attenuated significant effects of [D-Pen(2,5)]-enkephalin (delta-opioid agonist) on feeding behavior in ad libitum fed chicks. Co-injection of beta-FNA, but not ICI, significantly attenuated the orexigenic effect of [D-Ala2, D-Leu3]-enkephalin (mu- and delta-opioid agonist). These results suggest that the endogenous opioid peptides, which act on the mu- and/or delta-opioid receptor, have an important role in feeding behavior in the central nervous system of meat-type chicks.

Animals↗
PubMed2005

Molecular mechanisms of neuropathic pain-phenotypic switch and initiation mechanisms.

Many known painkillers are not always effective in the therapy of chronic neuropathic pain manifested by hyperalgesia and tactile allodynia. The mechanisms underlying neuropathic pain appear to be complicated and to differ from acute and inflammatory pain. Recent advances in pain research provide us with a clear picture for the molecular mechanisms of acute pain, and substantial information is available concerning the plasticity that occurs under conditions of neuropathic pain. The most important changes responsible for the mechanisms of neuropathic pain are found in the altered gene/protein expression in primary sensory neurons. After damage to peripheral sensory fibers, up-regulated expression of the Ca(v)alpha(2)delta-(1) channel subunit, the Na(v)1.3 sodium channel, and bradykinin (BK) B1 and capsaicin TRPV1 receptors in myelinated neurons contribute to hyperalgesia; while the down-regulation of the Na(v)1.8 sodium channel, B2 receptor, substance P (SP), and even mu-opioid receptors in unmyelinated neurons is responsible for the phenotypic switch in pain transmission. Clarification of the molecular mechanisms for such complicated plasticity would be extremely valuable when considering the therapeutic design of pain relieving drugs. Although many reports deal with the changes in expression of key molecules related to neuropathic pain, the initiation and the mechanisms that follow remain to be determined. The current study using lysophosphatidic acid (LPA) receptor knockout mice revealed that LPA produced by nerve injury initiates neuropathic pain and demyelination following partial sciatic nerve ligation (PSNL). A single injection of LPA was found to mimic PSNL in terms of neuropathic pain and its underlying mechanisms. This discovery may lead to the subsequent discovery of LPA-induced secondary genes, which would be therapeutic targets for neuropathic pain.

Animals↗
PubMed2005

Morphine-induced overexpression of prepro-nociceptin/orphanin FQ in cultured astrocytes.

The effects of morphine on the gene expression of prepro-nociceptin/orphanin FQ (ppN/OFQ) in various primary cultured brain cells from embryonic day 17, rats were studied by use of real-time RT-PCR method. The basal level of ppN/OFQ mRNA in terms of ratio to the beta-actin in astrocytes was equivalent to that in neurons, but 10-times higher than that in microglia. The addition of 1 microM morphine significantly enhanced the ppN/OFQ mRNA levels in cultured astrocytes, but not neurons or microglia. The enhancement was observed as early as 1h after the addition of morphine, reached maximum at 6h. There was a concentration-dependency between 30 nM to 1 microM. The morphine-induced enhancement was abolished by naloxone, an antagonist of mu opioid peptide receptor (MOP), wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, and PD98059, a MEK inhibitor, but not by 1,10-phenanthroline, a metalloprotease inhibitor and U73122, a phospholipase C inhibitor. These profiles contrast to the data with morphine-induced enhancement of brain-derived growth factor (BDNF) gene expression in microglia, where 1,10-phenanthroline abolished the expression. Furthermore, the ELISA analysis revealed that the immunoreactive ppN/OFQ or N/OFQ level was also increased by morphine. The present findings suggest that astrocytes could play roles in the neuronal plasticity during morphine chronic treatments by enhancing gene expression of anti-opioid peptide, N/OFQ.

Analgesics, Opioid↗
PubMed2005

Pre-injury administration of morphine prevents development of neuropathic hyperalgesia through activation of descending monoaminergic mechanisms in the spinal cord in mice.

The present study examined whether pre-injury administration of morphine can prevent partial sciatic nerve injury-induced neuropathic pain in mice. We observed that pre-injury administration of subcutaneous (s.c.) and intracerebroventricular (i.c.v.) morphine dose-dependently prevented the development of both thermal and mechanical hyperalgesia at 7 days following nerve injury in mice. The pre-injury morphine (s.c.)-induced analgesia was significantly blocked by pretreatment with naloxone injected s.c. or i.c.v., but not i.t., suggesting that systemic morphine produced the pre-emptying effects mainly by acting at the supra-spinal sites. Since it is believed that activation of descending monoaminergic mechanisms in spinal cord largely contributes to the supra-spinal analgesic effects of morphine, we investigated the involvement of serotonergic and noradrenergic mechanisms in spinal cord in the pre-injury morphine-induced analgesic effects. We found that pre-injury s.c. morphine-induced analgesic effect was significantly blocked by i.t. pretreatment with serotonergic antagonist, methysergide and noradrenergic antagonist, phentolamine. In addition, pre-injury i.t. injection of serotonin uptake inhibitor, fluoxetine and alpha2-adrenergic agonist, clonidine significantly prevented the neuropathic hyperalgesia. We next examined whether pre-injury morphine prevented the expression of neuronal hyperactivity markers such as c-Fos and protein kinase C gamma (PKCgamma) in the spinal dorsal horn. We found that pre-injury administration of s.c. morphine prevented increased expressions of both c-Fos and PKCgamma observed following nerve injury. Similar results were obtained with i.t. fluoxetine and clonidine. Altogether these results suggest that pre-injury administration of morphine might prevent the development of neuropathic pain through activation of descending monoaminergic pain inhibitory pathways.

Journal Article↗
PubMed2005

Insulin receptor-protein kinase C-gamma signaling mediates inhibition of hypoxia-induced necrosis of cortical neurons.

Ischemic stress causes neuronal death and functional impairment. Evidence has suggested that cells in the ischemic core first lose viability due to the decline in blood flow and cellular energy metabolism and then die by necrosis. Although inhibition of necrosis could be a potent therapeutic target for brain ischemia, known neurotrophic factors are ineffective for neuronal necrosis. We previously reported that insulin, but not brain-derived neurotrophic factor or insulin like-growth factor-1, inhibited neuronal necrosis under serum-free starvation stress. Although insulin receptors are abundant in the central nervous system as well as in peripheral tissues, neurons are not dependent upon insulin for their glucose supply, indicating that insulin receptors have other roles in the central nervous system. In the present study, by using hypoxia-reperfusion stress, we showed that cortical neurons rapidly died by necrosis as evaluated by propidium iodide staining and transmission electron microscopic analysis. As expected, insulin treatment significantly inhibited neuronal necrosis, although this effect was blocked by pretreatment with an antisense oligonucleotide for the insulin receptor. Furthermore, an inhibitor of protein kinase C (PKC) eliminated the insulin-induced antinecrotic effect. The addition of insulin induced significant translocation of only the PKC-gamma isoform, whereas antisense oligonucleotide treatment for this isoform abolished the insulin-induced inhibition of necrosis. Together, these results suggest that insulin mediates inhibition of neuronal necrosis through a novel mechanism involving PKC-gamma activation.

Animals↗
PubMed2005

Morphine-induced chemotaxis and brain-derived neurotrophic factor expression in microglia.

The addition of morphine at 1 mum induced morphological changes of cultured microglia such that they changed from having globular or bipolar rod-like shapes to being flat and lamellipodial, with membrane ruffling at the edge, which was stained with phalloidin. The membrane ruffling was clearly colocalized with Rac. Morphine also induced chemotaxis in Boyden chamber analysis at concentrations of 1 mum or more in microglia and the microglial cell line EOC 2. All of these changes were abolishable by naloxone, antisense oligodeoxynucleotide for mu-opioid receptor (MOR), pertussis toxin (PTx), and wortmannin, but not genistein or 1,10-phenanthroline. The addition of morphine to microglia stimulated the gene expression of brain-derived neurotrophic factor (BDNF) as early as the 1 hr point, and this lasted for >12 hr. Morphine induced BDNF gene expression and ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation, and these were abolishable by naloxone, wortmannin, PD98059, genistein, and 1,10-phenanthroline. The addition of conditioned medium derived from the culture of morphine-treated microglia also increased the phosphorylation of ERK1/2. All of these findings suggest that morphine induces significant changes in both morphology and gene expression at relatively high concentrations, but the underlying signaling pathways downstream of MOR and G(i/o) appear to be different from each other. Phosphoinositide 3-kinase gamma activation and Rac activation are involved in chemotaxis, whereas indirect pathways through ERK1/2 phosphorylation induced by unknown growth factors generated through an MOR-mediated metalloprotease activation are linked to the enhanced BDNF gene expression.

Animals↗
PubMed2005

Response of neuropeptide Y-induced feeding to mu-, delta- and kappa-opioid receptor antagonists in the neonatal chick.

It is known that opioid antagonists reduce the orexigenic effect of neropeptide Y (NPY) in mammals. We studied the effect of three opioid antagonists on NPY-induced feeding in male broiler chicks. Beta-funaltrexamine (beta-FNA), naloxonazine (NAL), ICI-174,864 (ICI) or nor-binaltorphimine (nor-BNI), antagonists of mu-, mu1-, delta- or kappa-receptors, and NPY were co-injected in chicks. Food intake was measured 30 min after treatment. Co-injection of beta-FNA or NAL was effective in reducing NPY-induced feeding, whereas ICI and nor-BNI had little effect on NPY-induced feeding. These data suggest that the mu-opioid receptor, especially the mu1-opioid has some relation to NPY-induced feeding, and implies that an endogenous ligand, such as beta-endorphin, participates in the orexigenic effect of NPY in neonatal chicks.

Animals↗
PubMed2005

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells.

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation-dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin-V-FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation-dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK-886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.

Animals↗
PubMed2005

Seasonal changes in expression of genes encoding five types of gonadotropin-releasing hormone receptors and responses to GnRH analog in the pituitary of masu salmon.

Five types of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, R2, R3, R4, and R5, are expressed in the brain and pituitary of masu salmon (Oncorhynchus masou). In the present study, seasonal changes in the expression of these five genes were examined in the pituitary to elucidate their roles in GnRH action during growth and sexual maturation. In addition, the seasonal variation of these genes in response to GnRH was examined in a GnRH analog (GnRHa) implantation experiment. Pituitary samples were collected 1 week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single pituitaries was determined by real-time PCR assays. Among the five genes, R4 was predominantly expressed in the pituitaries. In the immature fish, the amount of GnRH-R mRNA varied with seasons and subtypes. In the pre-spawning period, R1 and R4 mRNAs in both sexes and R2 and R3 mRNAs in the females increased 4- to 20-fold and then decreased in the spawning season. The effects of GnRHa treatment were significantly different in both sexes. In the females, GnRHa tended to elevate the expression of all the subtypes of GnRH-R genes in various stages during the experimental period, whereas it had almost no apparent effects in the males. These results indicate that the expression of the five GnRH-R genes is seasonally variable and may be related to the responses of the pituitary hormone genes to GnRH, and the regulation of GnRH-R genes by GnRH is different in both sexes.

Aging↗
PubMed2005

Novel type of Gq/11 protein-coupled neurosteroid receptor sensitive to endocrine disrupting chemicals in mast cell line (RBL-2H3).

1 Agonistic neurosteroids, including pregnenolone, dehydroepiandrosterone and its sulfate (DHEAS), caused rapid degranulation in measurements of beta-hexosaminidase (beta-HEX) release from a mast cell line, RBL-2H3. This degranulation was blocked by BSA-conjugated progesterone (PROG-BSA) or 17beta-estradiol, both of which are antagonistic neurosteroids. 2 DHEAS-induced beta-HEX release was blocked by U-73122 or xestospongin C, but not by PTX or EGTA. DHEAS-induced beta-HEX release was also abolished by G(q/11)-AS, but not by G(q/11)-MS. Pharmacological analyses revealed that the neurosteroids stimulated a putative membrane receptor through activation of the novel G(q/11) and phospholipase C. 3 While representative endocrine-disrupting chemicals (EDCs) did not show any degranulation or nocifensive actions by themselves, they blocked the DHEAS-induced degranulation. 4 The binding of a PROG-BSA-fluorescein isothiocyanate conjugate (PROG-BSA-FITC) to cells was inhibited by neurosteroids and EDCs. 5 In the algogenic-induced biting and licking responses test, DHEAS caused agonistic nocifensive actions in a dose-dependent manner between 1 and 10 fmol (i.pl.). DHEAS-induced nocifensive actions were abolished by PROG-BSA or nonylphenol. 6 Taken together, these results suggest that a G(q/11)-coupled neurosteroid receptor may regulate the neuroimmunological activity related to sensory stimulation and that some EDCs have antagonistic actions for this receptor.

Animals↗
PubMed2005

Importance of terminal residues on circularly permutated Escherichia coli alkaline phosphatase with high specific activity.

While the construction of fusion or tagged proteins is a useful method to obtain bifunctional proteins such as enzymes with specific binding activities, the region of the protein amenable to the fusion is limited to either the N- or C-terminus of the polypeptide, which often hampers its utility. Here we propose circular permutation as a method for tethering other protein(s) at a site(s) other than the two termini. As the effect of circular permutation on the activity of practically important proteins remains to be established, Escherichia coli alkaline phosphatase was subjected to circular permutation with its novel termini at the loops near the active site, and the original termini were linked by a flexible linker. While a permutant with the termini at original residues 407 and 408 was not active, a permutant with termini at residues 90 and 94 showed significant activity. Also, the addition of a randomized residue at positions 91 and 93 as well as outer peptide epitopes yielded several mutants with specific activity comparable to the wild-type enzyme with similar outer peptides. In addition, the mutants retained specific binding activity to anti-epitope antibodies, showing their potential utility in competitive immunoassay.

Alkaline Phosphatase↗
PubMed2005

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor genes in the brain of masu salmon from immaturity to spawning.

Seasonal variation in the expression of five subtypes of gonadotropin-releasing hormone receptor (GnRH-R) genes, designated as msGnRH-R1, -R2, -R3, -R4, and -R5, was examined in the brain of masu salmon (Oncorhynchus masou). In addition, responses of these genes to GnRH were examined in a GnRH analog (GnRHa) implantation experiment. Brain samples were collected one week after the implantation every month from immaturity through spawning. The absolute amount of GnRH-R mRNA in single forebrains was determined by real-time PCR assays. Among the five genes, R4 and R5 were dominantly expressed in both sexes. R1, R4, and R5 mRNAs showed similar changes throughout the experimental period in both sexes. Levels tended to be high in winter and low in the pre-spawning season, followed by elevations in the spawning period. The mRNA levels had weak to moderate negative correlations with the plasma level of estradiol-17beta (E2) in females. The effects of GnRHa on msGnRH-R mRNAs were not apparent for all the subtypes. These results indicate that the msGnRH-R1, -R4, and -R5 genes are synchronously expressed during sexual maturation. There was a trend toward decreased levels of their expression prior to the spawning period and then increased levels at spawning, possibly causing GnRH target neurons to sensitize to a GnRH stimulus. Furthermore, E2 may be involved in msGnRH-R gene expression in the brain of female masu salmon during sexual maturation.

Aging↗
PubMed2004

An antigen-mediated selection system for mammalian cells that produce glycosylated single-chain Fv.

Selection and production of specific antibodies are limiting the development of high-throughput immunoassays such as antibody chips. In this study, we propose an antigen-mediated selection of antibody producers (ASAP) system in mammalian cells. As a model system, transgenes encoding anti-fluorescein ScFv fused to cytokine receptors were introduced to IL-3-dependent cell lines. Addition of fluorescein-conjugated BSA induced growth signal through the ScFv/receptor chimeras, leading to selective expansion of the transduced cells. Cre recombinase was then used to excise the receptor gene flanked by two loxP recognition sites in the introns, resulting in secretion of his-myc-tagged ScFv to the culture medium. When the first loxP site was used in the exon as a linker between ScFv and receptor, enhanced antigen-mediated cell proliferation and production of unexpectedly glycosylated ScFv were achieved. ASAP is the first mammalian selection/production system of recombinant human ScFvs, without need for subcloning and with the advantage of glycosylated product.

Animals↗
PubMed2004

Improved growth response of antibody/receptor chimera attained by the engineering of transmembrane domain.

Structure-based design of antibody/cytokine receptor chimeras has permitted a growth signal transduction in response to non-natural ligands such as fluorescein-conjugated BSA as mimicry of cytokine-cytokine receptor systems. However, while tight on/off regulation is observed in the natural cytokine receptor systems, many chimeras constructed to date showed residual growth-promoting activity in the absence of ligands. Here we tried to reduce the basal growth signal intensity from a chimera by engineering the transmembrane domain (TM) that is thought to be involved in the interchain interaction of natural cytokine receptors. When the retroviral vectors encoding the chimeras with either the wild-type erythropoietin receptor (EpoR) TM or the one bearing two mutations in the leucine zipper motif were transduced to non-strictly interleukin-6-dependent 7TD1 cells, a tight antigen-dependent on/off regulation was attained, also demonstrating the first antigen-mediated genetically modified cell amplification of non-strictly factor-dependent cells. The results clearly indicate that the TM mutation is an effective means to improve the growth response of the antibody/receptor chimera.

Animals↗