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Biomedical subjects

Hiroshi Ueda

Publications and source records attributed to Hiroshi Ueda.

105 records · Page 6Linked to original sources
PubMed2002

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells.

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF-induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF- or VEGF-stimulated BAECs and lysophosphatidic acid-stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gbetagamma expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo.

Actin Cytoskeleton↗
PubMed2002

Intracerebroventricular administration of octopamine stimulates food intake of chicks through alpha(2)-adrenoceptor.

Octopamine, known to be an important neurotransmitter in invertebrates, has been noted to have several similarities to noradrenaline (NA) in mammals. The present study was done to elucidate whether central injection of octopamine enhances the feeding behavior of chicks and to investigate the interaction of octopamine with both alpha(1)- and alpha(2)-adrenoceptors. We found that the intracerebroventricular injection of octopamine significantly stimulated food intake of neonatal chicks during 30 min postinjection, but not thereafter. Moreover, this octopamine-induced eating response was attenuated by the alpha(2)-antagonist yohimbine, but not by the alpha(1)-antagonist prazosin. These results suggest that the action of octopamine on the feeding behavior of the neonatal chick is similar to that of NA, since octopamine regulates food intake through the alpha(2)-adrenoceptor.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2002

The cognition-enhancer nefiracetam is protective in BDNF-independent neuronal cell death under the serum-free condition.

Cortical cells from embryonic mice (E17) showed a rapid cell-death under the serum-free condition. The addition of nefiracetam at 0.1-10 microM increased the survival activity, while aniracetam at the same concentrations did not. The cell death was characterized to be apoptotic, since dead cells showed nuclear condensation, fragmentation, and TUNEL-positive staining. The nefiracetam-induced anti-apoptotic activity was completely blocked by 1 microM nifedipine or omega-conotoxin GVIA, and partially by 1 microM verapamil. These results suggest that the reported anti-amnesic action of nefiracetam in ischemic animals may be partly attributed to the neuroprotective action.

Animals↗
PubMed2002

Stimulation of peripheral nociceptor endings by low dose morphine and its signaling mechanism.

In this report, we demonstrated that peripheral application of very low dose (amol ranges) of morphine induced flexor response through a substance P (SP) release at the nociceptor endings in mice. The intraplantar (i.pl.) application of morphine produced flexor response in a dose-dependent manner from 0.1 to 1000amol. The mu-opioid receptor (MOP-R) agonist [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) also produced dose-dependent flexor response in same dose ranges. Morphine-induced flexor responses were markedly inhibited by naloxone and D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) both MOP-R antagonists and by intrathecal injection of antisense oligodeoxynucleotide (AS-ODN) for MOP-R which is expected to reduce the receptor expression in sensory nerve endings. Prior incubation with capsaicin, a depletor of SP from polymodal C fibers and [(+)-(2S,3S)-(2-methoxybenzylamino)-2-phenylpiperidine] (CP-99994), a tachykinin 1 receptor antagonist, also blocked the morphine-induced flexor responses. Moreover, pertussis toxin (PTX) which inactivates G(alpha)(i/o); [(1-[6-([(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino)hexyl]-1H-pyrrole-2,5-dione)] (U-73122), an inhibitor of phospholipase C (PLC); ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), a Ca(2+) chelating agent; xestospongin C, a membrane-permeable inositol trisphosphate (InsP(3)) receptor antagonist inhibited the morphine-flexor responses. However, thapsigargin, a depletor of intracellular Ca(2+) concentration and diphenhydramine, a histamine (His) H1 receptor antagonist, were unable to block the morphine-induced flexor responses. These results suggest that extremely low doses of morphine can stimulate sensory nerve endings through activation of peripheral MOP-R and its downstream mechanisms include activation of PLC through a SP release from polymodal C fibers.

Analgesics, Opioid↗
PubMed2002

Selection of highly productive mammalian cells based on an inducible growth advantage using an antibody/receptor chimera.

In mammalian cell culture, the selection of high producers is a critical step in efficient recombinant protein production. Drug-resistance selection has been commonly used, but does not always give a pure population of high producers. In this study, we propose a novel selection method in which the growth of high producers is specifically promoted. Two plasmids encoding (i) a hybrid receptor composed of the V(H) portion of anti-hen egg lysozyme antibody HyHEL-10 and an N-terminally truncated erythropoietin receptor (V(H)-EpoR), and (ii) a V(L)-EpoR fusion derived from the same construct as in (i), were employed. The second plasmid contained enhanced green fluorescent protein (EGFP) as a model recombinant protein that was flanked by the internal ribosomal entry sequence. Both plasmids were used simultaneously to transfect an IL-3-dependent murine myeloid cell line, 32D. The transfectants, after antigen selection in the absence of IL-3, showed a clear antigen-induced dose-dependent proliferation. In addition, a high EGFP expression level was observed by flow cytometry in comparison with the cells before antigen selection. The results clearly demonstrate the advantage of our method over conventional drug-resistance selection. We propose the term AMEGA (Antigen MEdiated Genetically-modified cell Amplification) for such an approach.

Journal Article↗
PubMed2002

Downregulation of P2X3 receptor-dependent sensory functions in A/J inbred mouse strain.

There is large variability in the various pain responses including those to tissue injury among inbred mouse strains. However, the determinant factors for the strain-specific differences remain unknown. The P2X3 sensory-specific ATP-gated channel has been implicated as a damage-sensing molecule that evokes a pain sensation by receiving endogenous ATP from injured tissue. In this study, to clarify the contribution of the sensory P2X3 signalling to strain-specific differences in tissue injury pain, we examined whether the P2X3-mediated in vivo and in vitro responses in dorsal root ganglion (DRG) neurons are changed in the A/J inbred mouse strain, which is known to be resistant to tissue injury pain caused by formalin. Here we found that A/J mice exhibited a low magnitude of nocifensive behaviour induced by the P2X agonist alpha,beta-methylene ATP (alpha beta meATP) into the hindpaw compared with C57BL/6 J mice. This behaviour was blocked by P2X3 antisense oligodeoxynucleotides. The low magnitude of the in vivo pain sensation could be observed similarly in the in vitro response; the increase in the intracellular Ca(2+) increase by alpha beta meATP in capsaicin-sensitive DRG neurons from A/J mice was significantly lower than that from C57BL/6 J mice. In A/J DRG neurons the P2X3 protein level was significantly lower compared with C57BL/6 J DRG neurons. The change in P2X3 protein was selective because P2X2 protein was expressed equally in both strains. The present study suggests that the downregulation of sensory P2X3 could be one of the molecular predispositions to low sensitivity to tissue injury pain in the A/J inbred mouse strain.

Adenosine Triphosphate↗
PubMed2002

Importance of a CDR H3 basal residue in V(H)/V(L) interaction of human antibodies.

Although the cooperativity of the V(H) and V(L) domains of an antibody in antigen binding has been extensively studied, the interaction between the V(H) and V(L) domains had not received sufficient attention. To systematically investigate the relationship between the amino acid sequence and V(H)/V(L) interaction strength, we here used a set of anti-bovine serum albumin antibodies having a single human framework for V(H) (V3-23/DP-47 and JH4b) and Vk (O12/O2/DPK9 and Jk1), but with different V(H)/V(L) interaction strengths. By phage display of a V(H) mini-library and analysis of the interaction of amino acids with immobilized V(L) fragments, the residue at H95 (Kabat numbering) at the beginning of seven CDR H3 residues was found to play a key role in determining the V(H)/V(L) interaction. On saturation mutagenesis of H95, Gly showed the strongest interaction, while Asp, Asn, and Glu showed lesser interaction in that order. The generality of the rule was confirmed by the test with urine-derived human L chain instead of a particular V(L). The results demonstrate that H95 plays a central role in deciding the V(H)/V(L) interaction of human Fvs that have most commonly found frameworks.

Amino Acid Sequence↗
PubMed2002

Nonopioid and neuropathy-specific analgesic action of the nootropic drug nefiracetam in mice.

Nootropic drug nefiracetam and related compounds are used in diseases with learning and memory deficits. Recent studies have implicated relationships between learning, memory, and chronic pain. Thus, in the present report, we have studied the effects of nootropic drug nefiracetam on the thermal and mechanical hyperalgesia induced by partial sciatic nerve ligation or streptozotocin treatment in mice. In the thermal paw withdrawal test, p.o., s.c., i.t., and i.c.v. administration of nefiracetam dose dependently reversed the thermal hyperalgesia observed in nerve-injured mice. Nefiracetam (p.o. and i.t.) also significantly reversed the thermal hyperalgesia observed in streptozotocin-induced diabetic mice. In the paw pressure test, p.o. and i.t. administration of nefiracetam dose dependently reversed the mechanical hyperalgesia observed in both nerve-injured and diabetic mice. In contrast, nefiracetam had no effect in sham-operated or control nondiabetic mice in all paradigms. Among other pyrrolidine nootropics (p.o.), aniracetam produced significant analgesic effects. Other analogs also had some, but not significant, analgesic effects. Finally, nefiracetam (p.o.)-induced analgesia in injured mice was not affected by opioid antagonist naloxone (s.c., i.t., and i.c.v.) but was dose dependently inhibited by nicotinic antagonist mecamylamine (i.t. and i.c.v.). The analgesic effect of i.t. nefiracetam was also blocked by i.t. mecamylamine pretreatment. Together, these findings suggest that nefiracetam, a new member of the piracetam group of cognition enhancers, could be a good therapeutic tool against neuropathic pain. We also demonstrate that nefiracetam-induced analgesic action was nonopioid in nature and was due to stimulation of nicotinic cholinergic system at spinal and supraspinal levels.

Analgesia↗
PubMed2002

Luteolin as an anti-inflammatory and anti-allergic constituent of Perilla frutescens.

Oral administration of the perilla leaf extract (PLE) to mice inhibits inflammation, allergic response, and tumor necrosis factor-alpha production. We also found that PLE suppressed the tumor necrosis factor-alpha (TNF-alpha) production in vitro. Using the inhibitory activity of TNF-alpha production in vitro as the index for isolation, we searched the active constituents from PLE and isolated luteolin, rosmarinic acid and caffeic acid as active components. Among the isolated compounds, only luteolin showed in vivo activity: inhibition of serum tumor necrosis factor-alpha production, inhibition of arachidonic acid-induced ear edema, inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced ear edema and inhibition of oxazolone-induced allergic edema. These results suggest that luteolin is a genuinely active constituent which is accountable for the oral effects of perilla.

Animals↗
PubMed2002

[Neuronal death mode switch and neurogenesis as in vivo neuroprotection].

The brain has various in vivo neuroprotective mechanisms that allow it to survive for an entire lifetime. As well as neurotrophic factor-mediated inhibition of in vivo apoptotic mechanisms through various protein kinases including Akt and MAP kinase, we propose adding the neuronal death mode switch mechanism observed under the brain ischemic stress to the list of neuroprotective mechanisms. Necrosis occurs when energy or ATP levels are markedly reduced. Lowered ATP levels cause a Na(+)-K(+)-ATPase failure, leading to an osmolysis. On the other hand, sufficient ATP is required for the apoptosome activation. Under the serum-free condition, cortical neurons rapidly die in necrosis. High-glucose treatment converted the cell death mode to apoptosis through an elevation of cellular ATP levels. This treatment also rescued the cell from death due to retinal ischemic injury. These findings suggest the possibility that ischemia-induced neuronal death could be inhibited by some drugs to elevate cellular ATP levels. Neurogenesis in the adult brain is now an important topic in neuroscience. As brain injury is reported to enhance the neurogenesis, this might be also included in the ways of in vivo neuroprotection. As lysophosphatidic acid has various activities to drive neurogenesis, the neurogenesis could also be managed by other drugs to compensate for functions lost by neuronal death.

Adenosine Triphosphate↗
PubMed2002

Detection of protein-protein interaction by bioluminescence resonance energy transfer from firefly luciferase to red fluorescent protein.

We describe a novel homogeneous protein-protein interaction assay based on bioluminescence resonance energy transfer (BRET) from mutant firefly luciferase to red fluorescent protein (DsRed). The association of glutathione S-transferase (GST)-luciferase and protein G-DsRed, which was driven by the addition of anti-GST antibody, was successfully monitored by BRET.

Journal Article↗
PubMed2002

Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase.

A sensitive homogeneous immunoassay is needed in the field of clinical diagnostics. Here we propose a rapid and potentially sensitive homogeneous immunoassay of peptide epitope based on the bioluminescence resonance energy transfer (BRET). A GST peptide tag fused to the N-terminus of firefly luciferase, and Cy3 or Cy3.5-labeled anti-peptide antibody were prepared to detect BRET from the hybrid luciferase to the fluorolabeled antibody. By measuring the spectral change due to BRET, the amount of c-myc peptide was successfully determined in a short period.

Journal Article↗
PubMed2002

Open sandwich immunoassay: a novel immunoassay approach based on the interchain interaction of an antibody variable region.

Despite their general applicability, conventional sandwich immunoassays suffer from the requirements of two antibodies recognizing different epitopes, and multiple long incubation/washing steps. In addition, the assay is not applicable to the measurement of low-molecular-weight haptens due to their size. Recently, a novel sandwich-type immunoassay that can overcome the limitations of conventional sandwich immunoassays was proposed. The assay, which utilizes antigen-dependent stabilization of an antibody variable region, requires less time and handling, and is particularly suitable for hapten measurement. The principle could be applied to several homogeneous assay formats, and the method has been shown to have superior sensitivity, as well as a lower time requirement compared to conventional assays.

Journal Article↗
PubMedDate not supplied

Detection of protein tyrosine phosphorylation by open sandwich fluoroimmunoassay.

In the era of proteomics, high-throughput screening of posttranslational modification states of proteins, especially protein phosphorylation, is considered of utmost importance. However, current protein phosphorylation detection methods depend on either the combination of proteolysis and mass spectrometry, or time-consuming immunoassay that requires inevitable washing processes. As a way to rapidly assay protein phosphorylation events, here we propose the use of Open Sandwich immunoassay that detects antigen-dependent stabilization of antibody variable region (Fv). As a model system, the heavy and light chain variable regions (V(H)/V(L)) of anti-phosphotyrosine antibody PY20 were used to evaluate its performance. When V(H)/V(L) interaction was first estimated by phage ELISA, wild-type Fv showed a modest phosphotyrosine (PY)-dependent increase in signal. However, after screening of mutants at an interface residue, one with weak V(H)/V(L) interaction (HQ39R) showed markedly improved (>200%) antigen-dependent signals. Using this mutant, two fusion proteins in which each variable region fragment was tethered to a GFP color variant were made (V(H)-eYFP/V(L)-eCFP) to monitor PY-induced fluorescence resonance energy transfer (FRET) between them. The results showed significant PY- or tyrosine phosphorylated peptide-induced enhancement in FRET in homogeneous solutions, indicating applicability of the method for rapid screening of tyrosine phosphorylation in vitro or in situ and possibly in vivo.

Antigen-Antibody Reactions↗