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H Ushio

Publications and source records attributed to H Ushio.

At least 37 records · Page 2Linked to original sources
PubMed1999

Na(+)-dependent and Na(+)-independent transport of L-arginine and L-alanine across dog intestinal brush border membrane vesicles.

We prepared intestinal brush border membrane vesicles (BBMVs) from beagle dogs fed a commercial diet (protein content: 24-26%), and investigated the characteristics of transport for basic and neutral amino acids across the intestinal BBMVs. To determined the kinetic parameters for L-arginine and L-alanine uptake, their total uptake was resolved into three routes: (1) Na(+)-dependent carrier-mediated transport; (2) Na(+)-independent carrier-mediated transport; and (3) simple diffusion. We could observe subtle, but clear-cut, Na(+)-dependent basic amino acid transport for the first time among studies with intestinal BBMVs prepared from mammals fed a normal diet. The Na(+)-dependent system for L-arginine transport can be best characterized as 'low affinity, low capacity', in contrast to that for L-alanine transport, which is 'low affinity, high capacity'. Maximal velocities of the Na(+)-dependent carrier-mediated transport are estimated to be higher for both L-arginine and L-alanine in dog intestinal BBMVs than in rabbit intestinal BBMVs reported previously. These results suggest that food habit of mammals is an important factor to decide the characteristic of system B0,+, a Na(+)-dependent carrier-mediated transport system common to basic and neutral amino acids across intestinal brush border membranes, as is protein content of the diet.

Alanine↗
PubMed1999

Transport of N(G)-nitro-L-arginine across intestinal brush border membranes by Na+ -dependent and Na+-independent amino acid transporters.

PURPOSE: To clarify the transport mechanism of NG-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). METHODS: Dog intestinal BBM vesicles were used. RESULTS: The time course of L-NNA uptake showed a Na+ -dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na+, indicating participation of Na+ -dependent and Na+ -independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na+. N(G)-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. CONCLUSIONS: The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na+ differently from that across plasma membranes of nonepithelial cells, because B0,+ and b0,+ amino acid transporters function partly in L-NNA transport across intestinal BBM.

Amino Acid Transport Systems↗
PubMed1998

IL-12 reverses established antigen-specific tolerance of contact sensitivity by affecting costimulatory molecules B7-1 (CD80) and B7-2 (CD86).

Cutaneous painting with reactive haptens induces contact sensitivity (CS) responses that are in vivo examples of T cell immunity. In contrast, high dose i.v. administration of the hapten can induce tolerance. We investigated the effect of IL-12 on reversal of this tolerance and attempted to determine in vitro the mechanism of this reversing effect by measuring proliferation and IFN-gamma production by CS effector T cells stimulated with hapten-conjugated APC, and we also measured CS ear swelling in vivo. The in vitro responses of T cells to hapten-APC became absent in tolerized mice, paralleling impaired in vivo CS responses. Addition of IL-12 to cultures manifesting this fully established in vitro tolerance completely restored impaired responses of tolerized T cells. The reversing effects of IL-12 were not blocked by anti-IFN-gamma mAb, but were blocked by mAbs against B7-1, more strongly by anti-B7-2, and by both Abs together. Additional in vivo ear-swelling response experiments confirmed the reversing effects of IL-12 on established tolerance. To examine whether the IL-12 effect depended on stimulation of IFN-gamma, we directly injected IFN-gamma into tolerized mice. This partially mimicked but did not fully reconstitute the effects of IL-12. In summary, IL-12 abrogation of established tolerance of CS may have been partially due to endogenous production of IFN-gamma, but appeared mainly due to direct activation of the tolerized T cells by affecting signaling through costimulatory molecules B7-1 and B7-2.

Animals↗
PubMed1998

Role of nerve growth factor in cutaneous wound healing: accelerating effects in normal and healing-impaired diabetic mice.

Four full-thickness skin wounds made in normal mice led to the significant increase in levels of nerve growth factor (NGF) in sera and in wounded skin tissues. Since sialoadenectomy before the wounds inhibited the rise in serum levels of NGF, the NGF may be released from the salivary gland into the blood stream after the wounds. In contrast, the fact that messenger RNA and protein of NGF were detected in newly formed epithelial cells at the edge of the wound and fibroblasts consistent with the granulation tissue produced in the wound space, suggests that NGF was also produced at the wounded skin site. Topical application of NGF into the wounds accelerated the rate of wound healing in normal mice and in healing-impaired diabetic KK/Ta mice. This clinical effect of NGF was evaluated by histological examination; the increases in the degree of reepithelialization, the thickness of the granulation tissue, and the density of extracellular matrix were observed. NGF also increased the breaking strength of healing linear wounds in normal and diabetic mice. These findings suggested that NGF immediately and constitutively released in response to cutaneous injury may contribute to wound healing through broader biological activities, and NGF improved the diabetic impaired response of wound healing.

Animals↗
PubMed1998

Antioxidizing potentials of BHA, BHT, TBHQ, tocopherol, and oxygen absorber incorporated in a Ghanaian fermented fish product.

Raw whole fishes (Horse mackerel, Trachurus japonicus) were degutted and separately treated with antioxidants BHA, BHT, TBHQ and tocopherol before fermentation, by completely immersing the samples in 0.1% antioxidant solutions. Fish samples were then salted, fermented and dried to mark the end of processing. A portion of the control samples were packed in an oxygen absorber during storage for 2 months at room temperature to study the effect of oxygen scavenging on lipid oxidation. The moisture content and total lipid decreased with processing and storage. An inductive effect of the fish oil was observed with 2-thiobarbituric acid values and the formation of lysophos-phatidylcholine (LPC) for TBHQ and BHA treated fish during the fermentation process. Free fatty acid formation was detected in all fish samples throughout processing and storage. For samples treated with tocopherol, only *-tocopherol was detected at the end of 2 months storage. Although TBHQ showed the best antioxidative effect during processing, it was the fastest synthetic antioxidant to be depleted. However, relatively high levels of BHA were present in the sample after 2 months storage. Red color patches, suspected to be antioxidant degradative products, were observed around the operculum of TBHQ treated samples after processing.

Absorption↗
PubMed1998

Quantitative determination of butylated hydroxyanisole, butylated hydroxytoluene, and tert-butyl hydroquinone in oils, foods, and biological fluids by high-performance liquid chromatography with fluorometric detection.

Concentrations of synthetic antioxidants butylated hydroxyanisole, butylated hydroxytoluene, and tert-butyl hydroquinone were quantified using a high-performance liquid chromatograph with spectrofluorometric detector. The antioxidants were separated and eluted on a reversed-phase column by gradient of a mixture of H2O/acetonitrile/acetic acid (66.5: 28.5:5, by vol) and a mixture of acetonitrile/acetic acid (95:5, vol/vol). The eluants were monitored at emission and excitation wavelengths of 310 and 280 nm, respectively. Calibration curves obtained using peak areas against concentration showed high coefficients of multiple determination (R2 > 0.99) for all antioxidants. Known concentrations of added antioxidant standards were recoverable within 98-99% from oils and over 93% from mouse blood. This method requires minimum sample extraction and purification before analysis and provides a relatively high percentage recovery. The method has been applied successfully for the measurement of antioxidant concentrations in oils, dried foods, and biological fluids.

Antioxidants↗
PubMed1997

Human platelets can initiate T cell-dependent contact sensitivity through local serotonin release mediated by IgE antibodies.

To investigate the role of human platelets in initiating Ag-specific contact sensitivity (CS), a mouse model employing human platelets was used. Intravenous injection of immune lymphoid cells containing Ag-specific CS-effector Thy1+ B220- T cells that were depleted of CS-initiating Thy1+, B220+ cells together with human platelets presensitized in vitro with Ag-specific IgE mAb led to elicitation of CS responses in recipient mice. The fact that this response was blocked by preincubation of platelets with an irrelevant IgE or with a mixture of anti-Fc epsilonRI alpha mAb and anti-Fc epsilonRII mAb suggested that IgE Fc epsilonR on platelets were involved. When platelets that were presensitized with Ag-specific IgE mAb were incubated in vitro with specific Ag in the presence of fresh mouse serum, a significant net release of [3H]serotonin (5-hydroxytryptamine, 5-HT) was observed. Furthermore, in vitro depletion of 5-HT from platelets or in vivo pretreatment of recipients with a 5-HT2A receptor antagonist (ketanserin) abolished the IgE-dependent CS initiation mediated by platelets. These results show that human platelets can initiate T cell-dependent CS responses through IgE mAb, and this CS initiation is mediated by 5-HT released from the platelets in an Ag-specific manner.

Animals↗
PubMed1997

The stability and degradation pathway of recombinant human parathyroid hormone: deamidation of asparaginyl residue and peptide bond cleavage at aspartyl and asparaginyl residues.

PURPOSE: The stability of recombinant human parathyroid hormone (rhPTH) was examined under acidic to alkaline conditions; its degradation pathways were elucidated from resultant products. METHODS: Degradation assay was performed in the pH range 2 to 10 at 40, 50 and 60 degrees C. The approximate molecular mass and pI values of the degradation products were estimated by electrophoresis. FAB-MS peptide mapping and amino acid composition analysis were used to determine these structures. The amount of each respective product was determined by HPLC. RESULTS: At pH2, eight degradation products were found: 1-30rhPTH, 1-74rhPTH, 1-71rhPTH, 1-56rhPTH, 1-45rhPTH, 46-84rhPTH, 31-84rhPTH and Asp76-rhPTH; these were mainly as a consequence of peptide bond cleavage of the amide bond of Asp. At pH9, five products were found: isoAsp16-rhPTH, Asp16-rhPTH, Asp57-rhPTH, Asp76-rhPTH, 17-84rhPTH; the main degradation pathway was deamidation of Asn via a cyclic imide intermediate. Degradation products resulting from cleavage at Asp were increased in proportion to the extent that pH was lowered below 5. As pH was increased above 5, so were products resulting from deamidation of Asn. Correspondingly, levels of intact rhPTH were at a peak at pH5. CONCLUSIONS: Degradation of rhPTH under acidic conditions predominantly occurs by cleavage at Asp, whereas, above pH5, deamidation of Asn is the more prominent, rhPTH is most stable at pH5.

Amino Acid Sequence↗
PubMed1997

Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.

Atopic dermatitis (AD) usually develops in patients with an individual or family history of allergic diseases, and is characterized by chronic relapsing inflammation seen especially in childhood, association with IgE hyperproduction and precipitation by environmental factors. However, the exact etiology of AD has been unclear. To further explore the pathogenesis and treatment of AD, a suitable animal model is required. We found that skin lesions, which were clinically and histologically very similar to human AD, spontaneously appeared on the face, neck, ears and dorsal skin of inbred NC/Nga mice when they were raised in non-sterile (conventional) circumstances, but not under specific pathogen-free conditions. Plasma levels of total IgE in conventional NC/Nga mice were markedly elevated from 8 weeks of age, correlating with clinical skin severity of dermatitis. Immunohistochemical examination of the skin lesion showed increased numbers of mast cells and CD4+ T cells containing IL-4 necessary for IgE synthesis. Thus, NC/Nga mice suffered from dermatitis very similar to human AD with IgE hyperproduction, which may be triggered by some environmental factor(s).

Animals↗
PubMed1996

Gamma delta T cells from tolerized alpha beta T cell receptor (TCR)-deficient mice inhibit contact sensitivity-effector T cells in vivo, and their interferon-gamma production in vitro.

Contact sensitivity (CS) responses to reactive hapten Ag, such as picryl chloride (PCl) or oxazolone (OX), are classical examples of T cell-mediated immune responses in vivo that are clearly subject to multifaceted regulation. There is abundant evidence that downregulation of CS may be mediated by T cells exposed to high doses of Ag. This is termed high dose Ag tolerance. To clarify the T cell types that effect CS responses and mediate their downregulation, we have undertaken studies of CS in mice congenitally deficient in specific subsets of lymphocytes. The first such studies, using alpha beta T cell-deficient (TCR alpha -/-) mice, are presented here. The results clearly show that TCR alpha -/- mice cannot mount CS, implicating alpha beta T cells as the critical CS-effector cells. However, TCR alpha -/- mice can, after high dose tolerance, downregulate alpha +/+ CS-effector T cells adoptively transferred into them. By mixing ex vivo and then adoptive cell transfers in vivo, the active downregulatory cells in tolerized alpha -/- mice are shown to include gamma delta TCR+ cells that also can downregulate interferon-gamma production by the targeted CS-effector cells in vitro. Downregulation by gamma delta cells showed specificity for hapten, but was not restricted by the MHC. Together, these findings establish that gamma delta T cells cannot fulfill CS-effector functions performed by alpha beta T cells, but may fulfill an Ag-specific downregulatory role that may be directly comparable to reports of Ag-specific downregulation of IgE antibody responses by gamma delta T cells. Comparisons are likewise considered with downregulation by gamma delta T cells occurring in immune responses to pathogens, tumors, and allografts, and in systemic autoimmunity.

Adoptive Transfer↗
PubMed1995

Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor.

We investigated the inhibitory activity of nerve growth factor (NGF) on apoptosis of rat peritoneal mast cells (PMCs) and compared it with that of recombinant stem cell factor (rSCF), which is a mast cell growth factor. When PMCs were incubated up to 72 hours in the presence of control medium, internucleosomal fragmentation of DNA indicating apoptosis was detected by agarose gel electrophoresis and flow cytometry. The aged PMCs showed morphological changes typical for apoptosis, such as chromatin condensation and loss of microvilli of the cell membrane. Addition of NGF or rSCF prevented development of the characteristic DNA fragmentation and decreased the proportion of apoptotic cells with low DNA content values in a dose-dependent manner. Polyclonal antibody to NGF completely abolished the inhibitory activity of NGF but not of rSCF. NGF-induced PMCs were in the G0/G1 phase of the cell cycle, but rSCF transited them from the G0/G1 phase to the S/G2M phase, suggesting that NGF, unlike rSCF, may have no proliferation activity to PMCs. By flow cytometric analysis with antibodies to NGF receptors p75LNGFR and p140trk, we defined that PMCs expressed p140trk but not p75LNGFR. Addition of herbimycin A or K-252a, tyrosine kinase inhibitors, to NGF resulted in blockage of the NGF-induced p140trk phosphorylation and restriction of the inhibitory activity of NGF on apoptosis of PMCs. These results indicated that NGF suppressed apoptosis of rat PMCs through the p140trk tyrosine phosphorylation and possessed no proliferative activity. Thus, NGF may act as a key factor to promote survival of connective tissue-type mast cells.

Animals↗
PubMed1995

Adoptive cell transfer of contact sensitivity-initiation mediated by nonimmune cells sensitized with monoclonal IgE antibodies. Dependence on host skin mast cells.

A role for mast cell release of serotonin (5-HT), via Ag-specific factors derived from Thy-1+ B220+ lymphoid cells in the initiation of murine contact sensitivity (CS) has been suggested. However, because CS in mast cell-deficient mice was intact, a role for mast cells in CS initiation was unclear. Therefore, we examined whether CS could be initiated by i.v. injection of nonimmune mixed lymphoid cells that were sensitized in vitro with IgE. When naive mice received IgE-sensitized nonimmune spleen or lymph node cells, or IgE-sensitized purified mast cells, together with immune CS-effector B220- T cells, which therefore were depleted of CS-initiating, Thy-1+, B220+ cells, which could not transfer CS, then reconstitution of CS occurred. Mast cell-deficient W/Wv mice could not elicit this IgE-dependent CS ear swelling, but when mast cell deficiency was reversed by ear injection of normal bone marrow-derived cultured mast cells, then CS was restored. In vitro pretreatment with irrelevant monoclonal anti-OVA IgE prevented CS initiation mediated by Ag-specific, IgE mAb-sensitized cells, presumably by blocking sensitization with IgE. Thus Fc epsilon R on the normal lymphoid cells were involved. When ketanserin, a 5-HT2 receptor antagonist, was injected i.v. before cell transfer, CS initiation via IgE-sensitized cells and CS were no longer elicited. Thus, in this system, IgE Abs bound to circulating IgE Fc epsilon R bearing lymphoid cells sensitized in vitro (most likely basophils), probably mediated early activation of these circulating basophils to release mediators, causing 5-HT release from cutaneous mast cells, to mediate CS initiation.

Animals↗
PubMed1995

Peptide mapping of recombinant human parathyroid hormone by enzymatic digestion and subsequent fast-atom bombardment mass spectrometry.

Peptide maps of recombinant human parathyroid hormone (rhPTH) were determined by both trypsin and V-8 protease digestion with subsequent fast-atom bombardment mass spectrometry (FAB-MS). Coverage of the sequence was 85% when using trypsin and 90% when using V-8 protease. Five rhPTH variants that were recombinantly produced as models of Asn deamidated type degradation products were measured, and molecular weight differences between their respective deamidated peptide fragments were completely detected. In the V-8 protease digests of some variants, characteristic peptide ions caused by the deamidation were observed and this greatly facilitated the assignment and recognition of the deamidated position. Our data suggest that FAB-mapping of rhPTH via the protease digestion methods used, appears to have great potential for structural investigations of the peptide.

Amino Acid Sequence↗
PubMed1995

Oxidation of recombinant human parathyroid hormone: effect of oxidized position on the biological activity.

PURPOSE: To determine the oxidation products of recombinant human parathyroid hormone (rhPTH) treated with H2O2, the amino acid residue oxidized, and the biological activity of the oxidation products. METHODS: Oxidized residues were determined by CNBr cleavage, trypsin digestion and subsequent fast atom bombardment mass spectrometry. The biological activity of each oxidized rhPTH was examined in rat osteosarcoma cell adenylate cyclase assay. RESULTS: Three oxidized products were isolated, namely, Met at position 8 (Met8) sulfoxide, Met at position 18 (Met18) sulfoxide and both positions Met sulfoxide. It appears that the Met8 and Met18 oxidized forms are intermediates in the generation of the Met doubly oxidized form. All oxidized forms possessed reduced biological activity, more so for oxidation at Met8 than at Met18. CONCLUSIONS: The region around Met8 is important for the activity of the parathyroid hormone.

Chromatography, High Pressure Liquid↗
PubMed1995

Mechanisms of eosinophilia in mice infested with larval Haemaphysalis longicornis ticks.

Infestation of larval Haemaphysalis longicornis ticks induced a threefold increase of eosinophils in the peripheral blood of normal WBB6F1- +/+ mice 2 days after tick infestation. In genetically mast cell-deficient WBB6F1- W/Wv mice, a threefold increase of blood eosinophils was observed 6 days after the tick infestation. However, marked infiltration of eosinophils was detected in the tick infestation sites of the WBB6F1- +/+ mice but not the WBB6F1- W/Wv mice. When the mast cell deficiency of WBB6F1- W/Wv mice had been rescued locally by intradermal injections of WBB6F1- +/+ mouse-derived cultured mast cells, a rapid increase of blood eosinophils and tissue infiltration of eosinophils were revealed following tick infestation. The intravenous (i.v.) injection of immune spleen or lymph node cells obtained from WBB6F1- +/+ mice 10 days after tick infestation led to significant eosinophilia in naive recipient mice. Treatment with anti-Thy-1.2 or anti-CD4 monoclonal antibody (mAb) and complement (C) completely abolished the eosinophilia; the early response (2 days after tick challenge) is dependent on mast cells at the feeding site, and the late response (6 days after tick challenge) is dependent on T lymphocytes. Since amplified interleukin-5 (IL-5) cDNA was detectable in the spleen cells 4 days after tick infestation, the late response might be mediated by IL-5. The infiltration of eosinophils at the feeding site of skin appeared to be dependent on mast cells.

Animals↗
PubMed1994

Poor response of cultured mast cells derived from mi/mi mutant mice to nerve growth factor.

Decreased numbers of mast cells and abnormalities in the phenotype of mast cells are observed in the skin of mi/mi mutant mice. Recently, the mi locus was identified to encode a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors. Since nerve growth factor (NGF) has been reported to influence the proliferation and the phenotype of cultured mast cells (CMCs), we compared the effect of NGF between mi/mi and control normal (+/+) CMCs. Addition of NGF to the suboptimal dose of recombinant murine interleukin-3 (rmIL-3) increased the plating efficiency of +/+ CMCs, but not of mi/mi CMCs. Although +/+ CMCs were berberine sulfate-negative when cultured with rmIL-3 alone, +/+ CMCs became berberine sulfate-positive when cultured in the presence of both rmIL-3 and NGF, which suggests increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. +/+ CMCs significantly bound 125I-NGF, but mi/mi CMCs did not, which suggests a defect of NGF receptors in mi/mi CMCs. Both p75 and p140 molecules are known to be involved in the formation of NGF receptors. Although the expression of p140 messenger (m)RNA was comparable between +/+ and mi/mi CMCs, the expression of p75 mRNA was significantly lower in mi/mi CMCs than in +/+ CMCs. Taken together, the poor response of mi/mi CMCs to NGF appeared to be attributable to the impaired transcription of the p75 gene.

Animals↗
PubMed1994

Carp parvalbumin binds to and directly interacts with the sarcoplasmic reticulum for Ca2+ translocation.

Interaction between two relaxing factors, the sarcoplasmic reticulum and parvalbumin, in carp fast skeletal muscle was investigated. Immunoblotting using an anti-parvalbumin antibody revealed that parvalbumin bound to the light sarcoplasmic reticulum isolated from carp fast skeletal muscle in the presence of Ca2+. Parvalbumin enhanced Ca2+ uptake activity of the light sarcoplasmic reticulum. Furthermore, using a photoreactive cross-linker, we detected a protein in the light sarcoplasmic reticulum which bound to parvalbumin in a Ca(2+)-dependent manner. These results suggest that parvalbumin may directly interact with the sarcoplasmic reticulum in contraction-relaxation cycle of carp fast skeletal muscle.

Animals↗