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Hiroshi Ueda

Publications and source records attributed to Hiroshi Ueda.

At least 19 recordsLinked to original sources
PubMed2007

Circadian-dependent learning and memory enhancement in nociceptin receptor-deficient mice with a novel KUROBOX apparatus using stress-free positive cue task.

Using the novel apparatus KUROBOX, learning and memory behaviors, as well as various parameters of movement activity, were reevaluated in mice deficient for nociceptin/orphanin FQ receptor (NOP-/- mice) or mu-opioid receptor (MOP-/- mice). This method has the advantages that no handling procedures are required throughout the experiments performed over 3 days, positive cue paradigms are used without water or shock stress, and the method does not disturb the nocturnal habit of mice. NOP-/- mice displayed a significant enhancement of learning and memory under stress-free conditions, but there were no changes in the various physical and psychological parameters of movement activity (nest stay ratio, distance moved, speed and angle in the movement) and biological rhythm that were measured. Enhancement of nocturnal learning was observed during the first 12-h dark cycle, and enhancement of memory was observed at the beginning of the second dark cycle in NOP-/- mice. In contrast, MOP-/- mice showed no significant change in learning and memory behaviors or in physical and psychological parameters of movement activity, except for speed, MOP-/- mice showed a significant decrease in speed of movement. Thus, the KUROBOX apparatus provides a useful alternative method to evaluate learning and memory activity under the more physiological conditions. In addition, this apparatus has an advantage that various physical and psychological parameters of movement activity affecting learning and memory behavior are also evaluated at the same time.

Animals↗
PubMed2007

[Sensitive noncompetitive measurement of small molecules by open sandwich immunoassay].

Proteins having multiple epitopes can be usually measured with sandwich ELISA employing two kinds of antibodies, which permits high sensitivity as well as a wide working range of more than three orders of magnitude. On the other hand, so-called monovalent antigens with MW less than 1000 are not susceptible to sandwich assays due to their small size and have almost always been measured in competitive assays. However, while a competitive assay needs only one antibody due to the principle of ratiometric measurement, optimization of the reaction conditions is necessary to attain suitable sensitivity and working range, which are often inferior to those of sandwich assays. As an alternative immunoassay for small antigens, here we propose a noncompetitive "open sandwich" immunoassay, which is based on the principle of stabilization of the antibody variable region Fv upon binding with antigen. Using ELISA to detect labeled V(H) fragments bound to immobilized V(L) in the presence of sample in microplate wells, various small molecules with MW around 200-300 can be measured with a superior detection limit and working range compared with those achieved with the corresponding competitive assays. The results indicate a common antigen recognition mode of anti-hapten antibody and wide applicability of the assay to the sensitive and handy analysis of low molecular-weight substances in areas such as environmental analysis and clinical diagnostics.

Antibodies↗
PubMed2007

Sensitive detection of estrogenic mycotoxin zearalenone by open sandwich immunoassay.

Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium sp., and its production on corn and small grains during storage has been of considerable concern. For sensitive ZEA detection, we applied an open sandwich (OS) immunoassay that can noncompetitively detect monovalent antigens utilizing an antigen-induced enhancement of the V(H)/V(L) interaction. We cloned the V(H) and V(L) cDNAs of anti-ZEA mAb to a split-Fv phagemid pKST2, and firstly both V(H) and V(L) fragments were displayed on M13 phage p9 and p7, respectively, using an amber suppressor, TG-1, as a host. The split-Fv phage showed specific binding to immobilized ZEA, which was well inhibited by free ZEA. Then, the V(H)/V(L) interaction and its antigen-dependency were analyzed using a non-suppressor HB2151 as a host to produce V(H)-displaying phage and his/myc-tagged soluble V(L) in the culture supernatant. By capturing V(L) with an anti-myc or -his antibody and probing bound V(H)-phage, ZEA was successfully detected with a superior detection limit as well as a wider working range than those of a competitive assay. Also, essentially the same results were reproduced with purified V(H)-alkaline phosphatase and MBP-V(L) fusion proteins.

Amino Acid Sequence↗
PubMed2006

Enhanced fluorescence resonance energy transfer immunoassay with improved sensitivity based on the Fab'-based immunoconjugates.

Fluorescence resonance energy transfer (FRET) is a powerful technique to monitor protein-protein interaction. Recently, we developed homogeneous and noncompetitive immunoassay based on the enhanced FRET by leucine zipper interaction. Here we improved the assay by establishing a general method for preparation of the Fab'-based immunoconjugate. Anti-human serum albumin Fab' numbers 11 and 13 were chemically conjugated with recombinant proteins consisting of thioredoxin, flexible linker, and green fluorescent protein color variant tethered with a leucine zipper motif. Compared with single chain antibody variable region-based fusion proteins prepared by the gene fusion method in our previous study, the resultant Fab'-based immunoconjugates accomplished an assay with nearly 10 times greater sensitivity. Furthermore, the conjugation method enabled us to apply the assay generally to measurement of another high-molecular weight antigen for which antibodies prepared for sandwich immunoassay are commercially available. Because of the facility and generality of the preparation method for the immunoconjugate, the assay is expected to be applied to many antigens that require rapid diagnosis and moderate measurement range.

Fluorescence Resonance Energy Transfer↗
PubMed2006

Antigen-mediated growth control of hybridoma cells via a human artificial chromosome.

Human artificial chromosome (HAC) vectors possess several characteristics sufficient for the requirements of gene therapy vectors, including stable episomal maintenance and mediation of long-term transgene expression. In this study, we adopted an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/cytokine receptor chimera that triggers a growth signal in response to a cognate non-toxic antigen, and applied it to growth control of HAC-transferred cells by adding an antigen that differed from cytokines that may manifest pleiotropic effects. We previously constructed a novel HAC vector, 21 Delta qHAC, derived from human chromosome 21, housed in CHO cells. Here, we constructed an HAC vector harboring an ScFv-gp130 chimera responsive to fluorescein-conjugated BSA (BSA-FL) as well as a model transgene, enhanced green fluorescent protein (EGFP), in CHO cells. The modified HAC was transferred into interleukin (IL)-6-dependent hybridoma 7TD1 cells by microcell-mediated chromosome transfer, and the cells were subsequently found to show BSA-FL-dependent cell growth and sustained expression of EGFP in the absence of IL-6. The AMEGA system in combination with HAC technology will be useful for increasing the efficacy of gene therapy by conferring a growth advantage on the genetically modified cells.

Animals↗
PubMed2006

LPA-mediated demyelination in ex vivo culture of dorsal root.

Lysophosphatidic acid (LPA) causes neuropathic pain with demyelination in sensory fibers. In dorsal root (DR) ex vivo culture, the addition of 0.1 microM LPA caused a characteristic demyelination at 24h in scanning and transmission electron microscopy analyses. Moreover, direct contact between C-fibers due to loss of partition by Schwann cell in Remak bundles was observed. LPA-induced demyelination of DR was concentration-dependent in the range between 0.01 and 1M, and was abolished by BoNT/C3 and Y-27632, a RhoA and Rho kinase inhibitor, respectively. The demyelination was equivalent between the preparations with and without dorsal root ganglion. LPA also caused a down-regulation of myelin proteins, such as myelin basic protein (MBP) and myelin protein zero (MPZ) to approximately 70% of control. All these findings suggest that the demyelination observed in the neuropathic pain due to nerve injury occurs through a direct action of LPA on Schwann cells.

Animals↗
PubMed2006

Loss of spinal substance P pain transmission under the condition of LPA1 receptor-mediated neuropathic pain.

Among various machineries occurring in the experimental neuropathic pain model, there exists the loss of pain transmission through C-fiber neurons as well as the hypersensitivity through A-fibers. The current study reveals that molecular machineries underlying the latter hypersensitivity are derived from the events through LPA1 receptor and its downstream RhoA-activation following peripheral nerve injury. The loss of C-fiber responses, which are mediated by spinal substance P (SP) pain transmission was observed with the nociceptive flexor responses by intraplantar injection of SP in nerve-injured mice. The immunohistochemistry revealed that SP signal in the dorsal horn was markedly reduced in such mice. All these changes were completely abolished in LPA1-/- mice or by the pretreatment with BoNT/C3, a RhoA inhibitor. In addition, the loss of C-fiber responses and the down-regulation of spinal SP signal induced by single intrathecal LPA injection were also abolished in such treatments. All these results suggest that the loss of pain transmission through polymodal C-fiber neurons is also mediated by the LPA1 activation following nerve injury.

Animals↗
PubMed2006

Tonic inhibitory role of alpha4beta2 subtype of nicotinic acetylcholine receptors on nociceptive transmission in the spinal cord in mice.

In the spinal dorsal horn, activation of the nicotinic acetylcholine receptors (nAChR) by exogenously applied agonists is known to enhance inhibitory synaptic transmission, and to produce analgesia. However, it is still unknown whether endogenously released acetylcholine exerts a tonic inhibition on nociceptive transmission through the nAChRs in the spinal dorsal horn. Here, we report the presence of such a tonic inhibitory mechanism in the spinal dorsal horn in mice. In behavioral experiments, intrathecal (i.t.) injection of non-selective nAChR antagonist mecamylamine and alpha4beta2 subtype-selective antagonist dihydro-beta-erythroidine (DHbetaE) dose-dependently induced thermal and mechanical hyperalgesia in mice while the alpha7-selective antagonist methyllycaconitine (MLA) had no effect. Similarly, antisense knock-down of alpha4 subunit of nAChR, but not alpha7 subunit, in spinal cord induced thermal and mechanical hyperalgesia. In whole-cell patch-clamp experiments in spinal cord slice preparation from adult mice, the frequency of miniature inhibitory postsynaptic currents (mIPSCs) observed in substantia gelatinosa (SG) neurons was decreased by mecamylamine and DHbetaE, but not by MLA. The amplitudes of the mIPSCs were not affected. The nicotinic antagonists decreased the frequency of both GABAergic and glycinergic IPSCs. On the other hand, the nicotinic antagonists had no effect on the excitatory postsynaptic currents (EPSCs). Finally, acetylcholine-esterase inhibitor neostigmine-induced facilitation of IPSC frequencies in SG neurons was inhibited by mecamylamine and DHbetaE. Altogether these findings suggest that nicotinic cholinergic system in the spinal dorsal horn can tonically inhibit nociceptive transmission through presynaptic facilitation of inhibitory neurotransmission in SG via the alpha4beta2 subtype of nAChR.

Adaptation, Physiological↗
PubMed2006

Inhibition of paclitaxel-induced A-fiber hypersensitization by gabapentin.

Paclitaxel (Taxol) is a widely used chemotherapeutic agent in the treatment of several tumors. However, its use is often associated with the generation of peripheral neuropathic pain expressed as mechanical allodynia and thermal hyperalgesia. The molecular mechanism behind this debilitating side effect is obscure, and efficient drugs for its prevention are required. We sought to clarify the cellular changes in the involved nociceptor types underlying paclitaxel-induced neuropathic pain and to test for an alleviating effect of gabapentin treatment in a murine model of paclitaxel-induced neuropathic pain. We found that a single treatment with paclitaxel (4 mg/kg i.p.) led to a decrease in both thermal and mechanical nociceptive thresholds as well as a reduction in the thresholds for 250-Hz (A delta-fiber) and 2000 Hz (A beta-fiber) but not 5-Hz (C-fiber) sine wave electrical stimuli-induced paw withdrawal. The paclitaxel-induced neuropathic pain was completely abrogated by gabapentin (30 mg/kg i.p.) treatment. Furthermore, we found that mRNA and protein levels of the voltage-gated calcium channel (alpha 2)delta-1 subunit (Ca(alpha 2)delta-1), one of the putative targets for gabapentin, was up-regulated in dorsal root ganglions (DRGs), as well as increased expression of Ca(alpha 2)delta-1 protein in medium/large-sized DRG neurons by immunohistochemistry, following paclitaxel treatment. This suggests that paclitaxel induces A-fiber-specific hypersensitization, which may contribute to the functional mechanical allodynia and hyperalgesia, and that gabapentin could be a potential therapeutic agent for paclitaxel-induced neuropathic pain.

Amines↗
PubMed2006

Characterization of three different sensory fibers by use of neonatal capsaicin treatment, spinal antagonism and a novel electrical stimulation-induced paw flexion test.

In the present study, we first report an in vivo characterization of flexor responses induced by three distinct sine-wave stimuli in the electrical stimulation-induced paw flexion (EPF) test in mice. The fixed sine-wave electric stimulations of 5 Hz (C-fiber), 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber) to the hind paw of mice induced a paw-flexion response and vocalization. The average threshold for paw flexor responses by sine-wave stimulations was much lower than that for vocalization. Neonatally (P3) pretreatment with capsaicin to degenerate polymodal substance P-ergic C-fiber neurons increased the threshold to 5 Hz (C-fiber) stimuli, but not to 250 Hz (Adelta-fiber) and 2000 Hz (Abeta-fiber). The flexor responses to 5 Hz stimuli were significantly blocked by intrathecal (i.t.) pretreatment with both CP-99994 and MK-801, an NK1 and NMDA receptor antagonist, respectively, but not by CNQX, an AMPA/kainate receptor antagonist. On the other hand, the flexor responses induced by 250 Hz stimuli were blocked by MK-801 (i.t.) but not by CP-99994 or CNQX. In contrast, flexor responses induced by 2000 Hz stimuli were only blocked by CNQX treatment. These data suggest that we have identified three pharmacologically different categories of responses mediated through different primary afferent fibers. Furthermore, we also carried out characterization of the in vivo functional sensitivity of each of the sensory fiber types in nerve-injured mice using the EPF test, and found that the threshold to both 250 Hz and 2000 Hz stimulations were markedly decreased, whereas the threshold to 5 Hz stimulations was significantly increased. Thus we found opposing effects on specific sensory fiber-mediated responses as a result of nerve injury in mice. These results also suggest that the EPF analysis is useful for the evaluation of plasticity in sensory functions in animal disease models.

Animals↗
PubMed2006

Voltage-dependent N-type Ca2+ channel activity regulates the interaction between FGF-1 and S100A13 for stress-induced non-vesicular release.

1. The Ca(2+)-mediated regulation of interaction between FGF-1 and S100A13 in NG108-15 cells was studied. When the stress by depriving B27 supplement from the culture was given, cellular levels of both proteins were decreased, while their releases were significantly increased within 3 h. These stress-induced changes were all abolished by amlexanox, an anti-allergic drug. 2. These releases were significantly inhibited by the addition of EGTA or BAPTA-AM, cellular or extracellular Ca(2+)-chelating agent, respectively. The addition of omega-conotoxin GVIA, a N-type Ca(2+)-channel blocker caused a complete inhibition of the release, while increased the cytosolic levels of both proteins. However, omega-conotoxin MVIIC, the non-N-type Ca(2+)-channel blocker was ineffective. 3. In NG108-15 cells, which had been transfected with Venus-FGF-1 and CFP-S100A13, the supplement-deprivation stress caused several spike-type fluorescence resonance energy transfer (FRET) signals, suggesting that both proteins showing interaction would be immediately released. These spikes were completely abolished by the addition of omega-conotoxin GVIA. However, the addition of amlexanox caused bell-shaped FRET signals without spikes. 4. Thus, it is suggested that the interaction between FGF-1 and S100A13 responsible for stress-induced non-vesicular release is dependent of Ca(2+)-influx through N-type Ca(2+)-channels.

Aminopyridines↗
PubMed2006

Evidence for serum-deprivation-induced co-release of FGF-1 and S100A13 from astrocytes.

Since fibroblast growth factor (FGF)-1 lacks conventional amino-terminal signal peptide essential for endoplasmic reticulum (ER)-Golgi pathway, the mode of release of this polypeptide remains to be fully understood. We attempted to characterize the non-classical (non-vesicular) mode of FGF-1 release in the analyses using immunocytochemistry and immunoblot of conditioned medium (CM) from astrocytes. FGF-1 was completely released from astrocytes upon serum-deprivation stress in a Brefeldin A-insensitive manner. In the immunoprecipitation study using anti-FGF-1 IgG, S100A13 was identified to be the major protein co-eluted with FGF-1. The interaction between GST-FGF-1 and Strep-tag II-S100A13 was found to be Ca(2+)-sensitive, and to require the C-terminal 11 amino acid peptide sequence of S100A13. The overexpression of Delta88-98 mutant of S100A13 selectively inhibited the serum-deprivation stress-induced release of FGF-1, but not the release of S100A13 mutant from C6 glioma cells. However, amlexanox, anti-allergic drug whose target is S100A13, completely inhibited the stress-induced release of FGF-1 as well as S100A13. The stress-induced release of both proteins was also abolished by BAPTA-AM, an intracellular Ca(2+) chelating agent. The serum-deprivation caused Ca(2+) spikes in omega-conotoxin GVIA and thapsigargin-sensitive manner. All these results suggest that S100A13 is a cargo molecule for the serum-deprivation stress-induced non-classical release of FGF-1, and that its driving force of protein-protein interaction and release is possibly mediated by Ca(2+)-induced Ca(2+) release (CICR) coupled to N-type Ca(2+) channel activity.

Amino Acid Sequence↗
PubMed2006

NSAID zaltoprofen possesses novel anti-nociceptive mechanism through blockage of B2-type bradykinin receptor in nerve endings.

Zaltoprofen, a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs), was shown to have more powerful inhibitory effects to bradykinin (BK)-nociception than other NSAIDs. However, the molecular mechanisms underlying this potent analgesia are not yet fully understood. Here we attempted to clarify the molecular mechanism underlying zaltoprofen-induced analgesia on BK-induced nociception by a novel algogenic-induced paw flexion (APF) test in mice. The intraplantar (i.pl.) injection of zaltoprofen at 1nmol showed strong analgesic action on BK (i.pl.)-induced nociceptive flexor responses, whereas loxoprofen or its active metabolite loxoprofen-SRS did not. Zaltoprofen also inhibited the nociception induced by [Tyr8]-BK, a specific agonist of B2-type BK receptor, but did not affect the nociception by [Lys-des-Arg9]-BK, a specific agonist of B1-type BK receptor. However, zaltoprofen did not affect the substance P-induced nociception, which is mediated by common post-receptor signaling through nociceptive fibers with BK-ones. All these results suggest that NSAID zaltoprofen possesses novel anti-nociceptive mechanism, which inhibits B2-type BK receptor function in nerve endings.

Analgesics↗
PubMed2006

Nasal pressure recordings to detect obstructive sleep apnea.

Obstructive sleep apnea (OSA) is a common disease. Given the costs of in-laboratory polysomnography (PSG), alternative ambulatory methods for accurate diagnosis are desirable. The objective of this study was to evaluate the performance of a simple device (SleepCheck) to identify patients with sleep apnea. A total of 30 consecutive patients with suspected OSA syndrome referred to the sleep clinic were prospectively evaluated with standard PSG and SleepCheck simultaneously during an in-laboratory, supervised full-night diagnostic study. The PSG apnea and hypopnea index (AHI) was evaluated according to standard criteria, and SleepCheck assessed the respiratory disturbance index (RDI) based on nasal cannula pressure fluctuations. Compared to the full-night PSG, SleepCheck systematically overscored respiratory events (the mean difference between SleepCheck RDI and PSG AHI was 27.4+/-13.3 events per hour). This overscoring was in part related to normal physiologic decreases in flow during rapid eye movement sleep or after an arousal. However, there was reasonable correlation between AHI and RDI (r=0.805). Receiver operating characteristic curves with threshold values of AHI of 10 and 20/h demonstrated areas under the curves (AUCs) of 0.915 and 0.910, respectively. Optimum combinations of sensitivity and specificity for these thresholds were calculated as 86.4/75.0 and 88.9/81.0, respectively. Overall, the SleepCheck substantially overscored apneas and hypopneas in patients with suspected OSA. However, after correction of the bias, the SleepCheck had reasonable accuracy with an AUC, sensitivity, and specificity similar to other ambulatory type 4 devices currently available.

Adult↗
PubMed2006

The role of interface framework residues in determining antibody V(H)/V(L) interaction strength and antigen-binding affinity.

While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V(H))/antibody light chain variable region fragment (V(L)) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V(H)/V(L) interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V(H)/V(L) interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V(H) and V(L), facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V(H)/V(L) interaction. The phagemid library, encoding V(H) gene 7 and V(L) amber codon gene 9, was used to transform TG-1 (sup+), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V(H)-displaying phages and soluble V(L) fragment was used to evaluate the V(H)/V(L) interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V(H)/V(L) interaction strength, and a marked positive correlation between the antigen-binding affinity and the V(H)/V(L) interaction, especially in the presence of a set of particular V(L) residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.

Amino Acid Sequence↗
PubMed2005

Endocrine disrupting chemical atrazine causes degranulation through Gq/11 protein-coupled neurosteroid receptor in mast cells.

We studied the effects of representative endocrine-disrupting chemicals on beta-hexosaminidase release from mast cells and their putative neurosteroid receptor involvement. Some endocrine-disrupting chemicals, such as amitrol, benzophenon, bisphenol A, pentachlorophenol, and tetrabromophenol A did not cause hexosaminidase release from RBL-2H3 cells, but they blocked the release by dehydroepiandrosterone sulfate, a representative neurosteroid agonist. On the contrary, atrazine, which is a widely used herbicide, caused a rapid and concentration-dependent degranulation in the range between 10 nM and 1 microM in RBL-2H3 and peritoneal mast cells. Atrazine-induced degranulation was also evaluated by Alexa 488-annexin V binding to the phosphatidylserine, which is externalized during degranulation, and these actions were blocked by BSA-conjugated (membrane-impermeable) progesterone (PROG-BSA). The atrazine-induced beta-hexosaminidase release was characterized by various inhibitors including antisense-oligodeoxynucleotide for Galpha(q/11), pertussis toxin, phospholipase C inhibitor U-73122, inositol 1,4,5-triphosphate receptor inhibitor xestospongin C and Ca(2+) channel blocker lanthanum chloride. These analyses revealed that the degranulation is mediated by putative metabotropic neurosteroid receptor, G(q/11), phospholipase C and Ca(2+) mobilization from intracellular stores. Having documented progesterone receptor-modulation of atrazine-induced mast cell degranulation in vitro, this response was evaluated in mice. Atrazine caused pain responses when injected in the foot pads of mice, and they were antagonized by local administration of PROG-BSA or diphenhydramine. Atrazine also caused PROG-BSA-reversible plasma extravasation. All these findings strongly suggest that herbicide atrazine exerts inflammatory activity through activation of putative G(q/11)-coupled neurosteroid receptor and phospholipase C.

Animals↗