Biomedical subjects

Hiroshi Ueda

Publications and source records attributed to Hiroshi Ueda.

At least 55 records · Page 3Linked to original sources

PubMed2004

Intracerebroventricular administration of chicken motilin does not induce hyperphagia in meat-type chicks.

The effect of chicken motilin on food intake was investigated in meat-type chicks under ad libitum feeding, refeeding, and fasting conditions. We found that the intracerebroventricular injection of chicken motilin (0.1 and 0.2 microg) tended to increase food intake under ad libitum feeding and refeeding conditions at 60 min postinjection, but the differences were not significant (P>.05). On the other hand, central administration of chicken motilin (0.2 and 0.4 microg) showed a tendency to suppress feeding of fasted chicks as well as the result of high dose (5.0 microg) under ad libitum feeding conditions. Therefore, the results presented here suggest that central motilin alone does not induce hyperphagia in meat-type chicks.

Animals↗

PubMed2004

Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system.

A protein-synthesizing system based on a minimal set of purified components was used to investigate the roles molecular chaperones play in the folding of newly synthesized polypeptides. After we ascertained that this system lacks intrinsic chaperones, the effect of adding chaperones in a co-translational or post-translational manner was directly evaluated. An aggregation-prone single-chain antibody was used as the model nascent chain. The participation of the trigger factor or the DnaK system during translation efficiently increased the level of functional protein that was generated. In addition, both systems also acted as chaperones after translation had been stopped. In contrast, the GroEL/ES system showed little or no co- or post-translational assistance in folding.

Cell-Free System↗

PubMed2004

Reversal of antigen-dependent signaling by two mutations in antibody/receptor chimera: implication of inverse agonism in cytokine receptor superfamily.

Understanding the receptor activation mechanism is essential for the rational design of pharmacologically active ligand molecules. However, the activation mechanism of most cytokine receptors remains still unclear, and while agonism and antagonism have been described for ligand-mimetic peptides, there has been no report of inverse agonism that has been characterized for G protein-coupled receptors (GPCRs). To explore the activation mechanism of cytokine receptors, here we tried to investigate how agonism and antagonism could be altered by randomizing antibody variable region of an antibody/cytokine receptor chimera recognizing hen egg lysozyme (HEL) as an agonist. Based on our previous finding that the co-expression of V(H)-gp130 and V(L)-erythropoietin receptor (EpoR) chimeras transduced strict and efficient HEL-dependent cell growth signal, a V(H)-gp130 library encoding four randomized CDR2 residues was retrovirally infected to IL-3-dependent Ba/F3 cells already transfected with V(L)-EpoR. The selection without IL-3 resulted in a clonal expansion of the transduced cells, and interestingly some of which showed HEL dose-dependent growth suppression. Our results clearly indicate that agonism and antagonism of the antibody/cytokine receptor chimera can be readily switched by a subtle modification of the ligand binding domain as well as that of GPCRs, also implying the existence of inverse agonism in cytokine receptor superfamily.

Animals↗

PubMed2004

Galphaq/11 signaling induces apoptosis through two pathways involving reduction of Akt phosphorylation and activation of RhoA in HeLa cells.

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.

Apoptosis↗

PubMed2004

Gi2 signaling enhances proliferation of neural progenitor cells in the developing brain.

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.

Animals↗

PubMed2004

Initiation of neuropathic pain requires lysophosphatidic acid receptor signaling.

Lysophosphatidic acid (LPA) is a bioactive lipid with activity in the nervous system mediated by G-protein-coupled receptors. Here, we examined the role of LPA signaling in the development of neuropathic pain by pharmacological and genetic approaches, including the use of mice lacking the LPA(1) receptor. Wild-type animals with nerve injury develop behavioral allodynia and hyperalgesia paralleled by demyelination in the dorsal root and increased expression of both the protein kinase C gamma-isoform within the spinal cord dorsal horn and the alpha(2)delta(1) calcium channel subunit in dorsal root ganglia. Intrathecal injection of LPA induced behavioral, morphological and biochemical changes similar to those observed after nerve ligation. In contrast, mice lacking a single LPA receptor (LPA(1), also known as EDG2) that activates the Rho-Rho kinase pathway do not develop signs of neuropathic pain after peripheral nerve injury. Inhibitors of Rho and Rho kinase also prevented these signs of neuropathic pain. These results imply that receptor-mediated LPA signaling is crucial in the initiation of neuropathic pain.

ADP Ribose Transferases↗

PubMed2004

Enzymatic labeling of a single chain variable fragment of an antibody with alkaline phosphatase by microbial transglutaminase.

Functional cross-linking of a single chain Fv fragment of anti-hen egg-white lysozyme antibody (scFv) and alkaline phosphatase (AP) was explored using microbial transglutaminase (MTG) from Streptomyces mobaraensis. A specific peptidyl linker for MTG was genetically fused to the N-terminus of each protein and the resultant proteins were obtained separately by bacterial expression. The recombinant peptide-tagged scFv and AP were site-specifically cross-linked by MTG through the extra peptidyl linkers in vitro, which mainly yielded the heterodimer (i.e., scFv-AP conjugate). The enzymatic cross-linking reaction had little influence on either the antigen-binding ability of the scFv moiety or the enzymatic activity of the AP moiety of the conjugate, allowing use within an enzyme-linked immunosorbent assay. The results obtained suggest that the enzymatic approach with MTG facilitates the posttranslational construction of functional fusion proteins.

Alkaline Phosphatase↗

PubMed2004

Synergistic effects of salmon gonadotropin-releasing hormone and estradiol-17beta on gonadotropin subunit gene expression and release in masu salmon pituitary cells in vitro.

Effects of salmon gonadotropin-releasing hormone (sGnRH) and estradiol-17beta (E2) on gene expression and release of gonadotropins (GTHs) were examined in masu salmon (Oncorhynchus masou) using primary pituitary cell cultures at three reproductive stages, initiation of sexual maturation in May, pre-spawning in July, and spawning in September. Amounts of GTH subunit mRNAs were determined by real-time polymerase chain reaction, and levels of GTH released in the medium were determined by RIA. In control cells, the amounts of three GTH subunit mRNAs (alpha2, FSHbeta, and LHbeta) peaked in July prior to spawning. FSH release spontaneously increased with gonadal maturation and peaked in September, whereas LH release remained low until July and extensively increased in September. Addition of E2 to the culture extensively increased the amounts of LHbeta mRNA in May and July in both sexes. It also increased the alpha2 mRNA in July in the females. In contrast, sGnRH alone did not have any significant effects on the amounts of three GTH subunit mRNAs at all stages, except for the elevation of alpha2 and FSHbeta mRNAs in July in the females. Nevertheless, synergistic effects by sGnRH and E2 were evident for all three GTH subunit mRNAs. In May, sGnRH in combination with E2 synergistically increased the amounts of LHbeta mRNA in the males and alpha2 mRNA in the females. However, in July the combination suppressed the amounts of alpha2 and FSHbeta mRNAs in the females. sGnRH alone stimulated LH release at all stages in both sexes, and the release was synergistically enhanced by E2. Synergistic stimulation of FSH release was also observed in May and July in both sexes. These results indicate that a functional interaction of sGnRH with E2 is differently involved in synthesis and release of GTH. The synergistic interaction modulates GTH synthesis differentially, depending on subunit, stage, and gender, whereas it potentiates the activity of GnRH to release GTH in any situation.

Animals↗

PubMed2004

Kinetics and binding sites for interaction of the prefoldin with a group II chaperonin: contiguous non-native substrate and chaperonin binding sites in the archaeal prefoldin.

Prefoldin is a jellyfish-shaped hexameric co-chaperone of the group II chaperonins. It captures a protein folding intermediate and transfers it to a group II chaperonin for completion of folding. The manner in which prefoldin interacts with its substrates and cooperates with the chaperonin is poorly understood. In this study, we have examined the interaction between a prefoldin and a chaperonin from hyperthermophilic archaea by immunoprecipitation, single molecule observation, and surface plasmon resonance. We demonstrate that Pyrococcus prefoldin interacts most tightly with its cognate chaperonin, and vice versa, suggesting species specificity in the interaction. Using truncation mutants, we uncovered by kinetic analyses that this interaction is multivalent in nature, consistent with multiple binding sites between the two chaperones. We present evidence that both N- and C-terminal regions of the prefoldin beta sub-unit are important for molecular chaperone activity and for the interaction with a chaperonin. Our data are consistent with substrate and chaperonin binding sites on prefoldin that are different but in close proximity, which suggests a possible handover mechanism of prefoldin substrates to the chaperonin.

Archaeal Proteins↗

PubMed2004

Homogeneous sandwich immunoassay based on the enzymatic complementation induced by single-chain Fv fragments.

We describe a novel homogeneous sandwich immunoassay based on beta-galactosidase (beta-gal) complementation (the crab-claw sandwich enzymatic complementation immunoassay, CS-ECIA). We chose a high-molecular-weight antigen human serum albumin (HSA) as a model and constructed two chimeric proteins, in which a pair of single-chain Fvs (scFvs) recognizing distant epitopes of HSA was fused to either an N-terminal deletion mutant of beta-gal (deltaalpha) or a C-terminal deletion mutant of beta-gal (deltaomega). Upon simple mixing of the reagents with the sample, the two chimeric proteins became associated through binding separate epitopes on HSA that allowed reassociation of the two mutant enzymes. The resulting enzymatic complementation was measured as an increase in beta-gal activity using a luminescent substrate. With this CS-ECIA, a HSA concentration of 10-1000 pg/mL could be determined. In addition, the assay was easy to operate and required less time, handling, and sample volume than conventional sandwich enzyme-linked immunoassays. The assay will have general utility by substituting scFvs with other pairs of scFvs recognizing any polyvalent antigens.

Antigens↗

PubMed2004

Selection of genetically modified cell population using hapten-specific antibody/receptor chimera.

Efficient selection of the genetically modified cell population is a critical step to obtain the cells with desired properties. In this study, we propose an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/receptor chimera that triggers a growth signal in response to a non-toxic hapten dimer. An anti-fluorescein single-chain Fv fused to the extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of gp130 was expressed together with a model transgene, enhanced green fluorescent protein (EGFP) downstream of IRES sequence, by retroviral infection to IL-3-dependent Ba/F3 cells. Addition of fluorescein dimers connected by various oligo-DNA linkers induced selective growth of transfectants, thus leading to efficient expansion of EGFP-positive cell population. Also, digestion of the oligonucleotides by specific restriction endonuclease completely suppressed cell growth. Because these hapten dimers are not harmful for normal cells, the approach will be especially useful for reversible in vitro or in vivo expansion of genetically modified cell population employed for cell therapy and tissue engineering.

Animals↗

PubMed2004

Loss of peripheral morphine analgesia contributes to the reduced effectiveness of systemic morphine in neuropathic pain.

It is well known that the analgesic potency of morphine is reduced in neuropathic pain. In this study, we demonstrate that the decreased effectiveness of systemic morphine in neuropathic pain might be caused by the loss of morphine analgesia at the periphery. When given s.c. or i.t., the dose-response curves for morphine analgesia in Hargreaves thermal test were shifted rightward in partial sciatic nerve-injured mice compared with control sham-operated mice. The dose-response curves for i.c.v. morphine analgesia, however, were unchanged in nerve-injured mice, indicating no decrease in morphine potency at the supraspinal level. On the other hand, the dose-dependent analgesia produced by intraplantar (i.pl.) morphine in sham-operated mice almost completely disappeared in nerve-injured mice. With the more sensitive algogenic-induced nociceptive flexion test, significant reduction in the analgesic potency of systemic morphine was observed for bradykinin (BK) nociception in nerve-injured mice, and the analgesic effect of i.pl. morphine against BK nociception in sham-operated mice disappeared in nerve-injured mice. In immunohistochemical experiments, we found that, under normal state, mu-opioid receptors (MOPs) were mainly expressed in small-diameter unmyelinated dorsal root ganglion (DRG) neurons and colocalized with bradykinin B2 receptors. When we examined MOP expression in the DRG of nerve-injured mice, we observed a drastic decrease in MOP expression. Altogether, these data suggest that the lower potency of systemic morphine in neuropathic pain could be at least partly caused by the decreased MOP expression in DRG and subsequent loss of peripheral morphine analgesia in such a condition.

Analgesia↗

PubMed2004

The cognition-enhancer nefiracetam inhibits both necrosis and apoptosis in retinal ischemic models in vitro and in vivo.

The retinal ischemic-reperfusion stress (130 mm Hg, 45 min) caused neuronal damage throughout all cell layers and reduced the thickness of retinal layer by 30% at 7 days after the stress of mouse retina. The intravitreous injection of 100 pmol of nefiracetam, a cognition-enhancer, completely prevented the damage when it was given 30 min before and 3 h after the stress. Partial prevention was observed when it was given 24 h after the stress, or low dose (10 pmol) nefiracetam was given 30 min before the stress. However, aniracetam had no effect. In the retinal cell line N18-RE-105, the ischemic-reperfusion stress by 2 h culture under the serum-free condition with low oxygen (less of 0.4% O(2)) and low glucose (1 mM) caused necrosis or apoptosis in the low-density (0.5 x 10(4) cell/cm(2))or high-density (5 x 10(4) cell/cm(2)) culture, respectively. The necrosis showed membrane disruption, loss of electron density, and mitochondrial swelling, whereas apoptosis showed nuclear fragmentation and condensation in transmission electron microscopical analyses and in experiments using specific cell death markers. Nefiracetam inhibited both necrosis and apoptosis, whereas brain-derived neurotrophic factor (BDNF) inhibited only apoptosis. The cell-protective actions of nefiracetam were abolished by nifedipine and omega-conotoxin GVIA, L-type and N-type calcium channel blocker, but not by PD98059 or wortmannin, extracellular signal-regulated kinase 1/2 or phosphoinositide 3-kinase inhibitor, respectively, whereas those of BDNF were abolished by PD98059 and wortmannin, but not by nifedipine and omega-conotoxin GVIA. All these findings suggest that nefiracetam inhibit necrosis and apoptosis occurred in the ischemic/hypoxic neuronal injury through an increase in Ca(2+) influx.

Animals↗

PubMed2004

Central administration of leucine, but not isoleucine and valine, stimulates feeding behavior in neonatal chicks.

Branched-chain amino acids (BCAAs) are essential amino acids that play a major role in brain energy metabolism. This study was done to elucidate whether central injection of BCAAs influences feeding behavior in chicks. We found that the intracerebroventricular injection of leucine (200 microg) significantly stimulated food intake in neonatal chicks during 30 min postinjection. Additionally, the starting time of feeding and pecking rhythm after injection were significantly accelerated by leucine. In contrast, isoleucine and valine had no effect on ingestive response during experiment periods. Moreover, a metabolite of leucine (alpha-ketoisocaproic acid) at an equimolar concentration of leucine also did not increase food intake in chicks. These results suggest that leucine induces hyperphagia of neonatal chicks and it may be due to the synthesized glutamate by exogenous leucine.

Animals↗

PubMed2004

AMEGA: antigen-mediated genetically modified cell amplification.

Selection of genetically modified cells is a critical step to engineer the cells with desired properties. While antibiotic selection has been commonly used, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. To overcome this problem, a positive screening method for genetically modified cells is proposed using a pair of chimeric receptors that trigger a growth signal in response to a specific antigen. Either V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was fused to extracellular D2 domain of erythropoietin receptor (EpoR) and transmembrane/cytoplasmic domains of either EpoR or gp130. A model transgene, enhanced green fluorescent protein (EGFP) and the chimeric receptor genes that reconstituted functional Fv were retrovirally co-infected to interleukin (IL)-3-dependent Ba/F3 cells, followed by direct HEL selection in the absence of IL-3. Consequently, a single round of selection led to a single population of EGFP-positive cells. The detailed protocol of the method termed antigen-mediated genetically modified cell amplification (AMEGA) is described.

Antigens↗

PubMed2004

Desensitization of the inhibitory effect of norepinephrine on insulin secretion from pancreatic islets of exercise-trained rats.

The effect of exercise training (9 weeks of running) on norepinephrine-induced inhibition of insulin secretion was examined in rat islets. Insulin secretions from islets in the presence of glucose (> or =5.5 mmol/L) were significantly lower in trained (TR) than in control rats (CR). Norepinephrine inhibited 5.5 mmol/L glucose-stimulated insulin secretions and cyclic adenosine monophosphate (cAMP) contents in a dose-dependent manner in CR. Norepinephrine (10 micromol/L)-induced inhibition of insulin secretion was reversed by the blockade of the alpha(2)-adrenergic receptor in CR, but not in TR. Exercise training substantially shifted the dose-dependent curve for clonidine-induced inhibition of insulin secretions and that of cAMP contents to the right. Exercise training did not alter the density of the alpha(2)-adrenergic receptor either per islet or per protein of islet crude membrane. However, exercise training significantly reduced the protein expression of G alpha i-2 without change in G alpha i-2 mRNA. In CR but not in TR, norepinephrine significantly inhibited insulin secretions elicited by a combination of high glucose, a protein kinase C activator, and an adenylate cyclase activator under Ca(2+)-free conditions. Thus, exercise training appears to provoke a decreased expression of G alpha i-2 protein. This, at least in part, results in loss of the inhibitory effect of norepinephrine either on cAMP content or on insulin secretion at the post-calcium events in stimulus-secretion coupling, which, in turn, leads to the blunted inhibitory effects of norepinephrine on insulin secretion.

Adrenergic alpha-Agonists↗

PubMed2004

Feeding responses to mu-, delta- and kappa-opioid receptor agonists in the meat-type chick.

The present study was designed to examine the effect of specific opioid agonists on feeding behavior in neonatal chicks. The food intake of broiler chicks was significantly decreased by intracerebroventricular injection of DAMGO and beta-casomorphin, micro-opioid receptor agonists, at 30-min postinjection. In contrast, both delta-opioid receptor agonists (DADLE and DPDPE) stimulated the food intake of the chick. Similar to the delta-opioid receptor agonists, food intake was elevated by the kappa-opioid receptor agonist (U-50488H and U-62066) in a dose-dependent manner. These results suggest that the endogenous opioid peptides have an important role for feeding behavior in the central nervous system of chicks.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed2004

Locus-specific involvement of anti-opioid systems in morphine tolerance and dependence.

Opioid tolerance and addiction could be discussed as two types of plasticity or counteradaptation, at the cellular level and through neuronal circuits. Cellular counteradaptation mechanisms include receptor desensitization through phosphorylation and endocytosis and through altered gene expression. The former mechanisms are related to the acute tolerance mechanisms, while the latter to chronic one. From current studies, it is known that various phosphorylation steps, such as protein kinase C (PKC) and G protein-coupled receptor (GPCR) kinase (GRK) regulate endocytosis. Of interest is that there are some differences in the physiological roles between opioid receptor endocytosis and other GPCR ones. Endocytosis of the opioid receptor is conceived as a recycling and resensitization step rather than the desensitization step. PKC phosphorylation inhibits endocytosis (PKC hypothesis). Therefore the PKC inhibitor attenuates acute analgesic tolerance. The agonist, which shows high-endocytosis stimulation, therefore makes less significant tolerance liability (RAVE hypothesis). Chronic tolerance is more likely related to the mechanisms through plastic modulation of neuronal circuits, where anti-opioidergic neurons are involved. The knockout mice lacking the receptors for anti-opioidergic nociceptin/orphanin FQ (N/OFQ) or glutamatergic neurons show weak or no morphine tolerance and dependence. As their gene expression or protein expression increases during chronic morphine treatments, we propose the hypothesis that the enhanced anti-opioid system may cause a counteradaptation to show tolerance and dependence. By a novel electroporation technique to deliver the receptor into the brain of knockout mice, we succeeded in determining the specific locus for the site of anti-opioid (through GluRepsilon1 or NR2A) action. All these results suggest that enhanced anti-opioid systems may contribute to the development of morphine tolerance and dependence, and their contributions could be brain locus specific.

Animals↗