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Biomedical subjects

Reiko Teshima

Publications and source records attributed to Reiko Teshima.

32 records · Page 2Linked to original sources
PubMed2004

N-cadherin plays a role in the synapse-like structures between mast cells and neurites.

Communication between nerves and mast cells is a prototypic demonstration of neuro-immune interaction. Numerous studies have shown that the stimulation of nerves (or addition of neurotransmitters) can evoke activation of mast cells, and that mast cell-derived mediators can influence neuronal activity. However, the molecules involved in the membrane-membrane contacts between nerves and mast cells are still unknown. Here, we used an in vitro co-culture approach comprising interaction between immune (bone marrow-derived mast cell, BMMC) and nerve cells (superior cervical ganglia, SCG). The experiments showed clearly that the nerve-mast cell communication was supported by synapse-like structure and that N-cadherin, not E-cadherin, played an essential role in the synapse-like structure. In addition, we found that the synapse-like structure was assisted by clustering of beta-catenin to N-cadherin.

Animals↗
PubMed2003

Presence and primary sequence of a high-affinity IgG receptor on canine mastocytoma (CM-MC) cells.

Mast cells play a central role in IgE-dependent allergic responses. Although they have been reported to express only low-affinity IgG receptors and no high-affinity receptors (FcgammaRI), our recent study showed that canine mastocytoma CM-MC cells are activated by monomeric canine IgG, suggesting the presence of FcgammaRI on CM-MC cells. In the present study, we measured the affinity of canine IgG with CM-MC cells, determined the presence of the FcgammaRI protein and mRNA, and identified the cDNA sequence of it. The results showed that (7.5+/-3.1)x10(4) receptor molecules are expressed on a CM-MC cell with a Ka of (9.1+/-1.6)x10(7)M(-1) for binding to monomeric canine IgG. Canine IgG-conjugated beads precipitated an approximately 72-kDa surface protein, whose size is consistent with that of the FcgammaRI alpha subunit of humans and mouse. The expression of FcgammaRI mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR), and the cDNA encoding the FcgammaRI alpha subunit was found to be 84% and 78% similar to that of humans and the mouse, respectively. The predicted amino acid sequence was 72% and 63% identical, respectively. Canine mastocytoma CM-MC cells are therefore very useful for studying FcgammaRI-mediated signal transduction in mast cells.

Amino Acid Sequence↗
PubMed2003

The canine mast cell activation via CRP.

We report here canine mastocytoma-derived cell (CMMC) activation via two pentraxin, limulus- and human-CRP. Mast cell chemotaxis was measured by Boyden's blindwell chamber. To confirm that the cell migration was chemotactic, "checkerboard" analysis was performed. We used Fura-2 to investigate CRP-mediated cytosolic calcium elevation. To examine whether CRP-induced stimulation is mediated through G-proteins, CMMC were incubated with pertussis toxin (PTx) before use in chemotaxis assay and Ca(2+) mobilization. CMMC migration in response to CRP was both chemokinetic and chemotactic. Limulus-CRP induced a transient Ca(2+)-mobilization dose-dependently. Preincubation of the cells with PTx inhibited CRP chemotaxis and Ca(2+)-mobilization, suggesting that G-proteins of the Gi-class are involved in the chemotaxis. We suggest that CRP may participate in the migration of mast cells to inflamed tissues during an acute-phase response. CRP-mediated recruitment of mast cells might play an important role in hypersensitivity and inflammatory processes.

Animals↗
PubMed2003

Oral sensitization of W/W(v) mice with ovalbumin and possible involvement of the decrease in gammadelta-T cells.

Mast-cell-deficient WBB6F1-W/W(v) mice (W/W(v)) and congenic wild-type (+/+) mice were sensitized by oral administration of 0.1 or 1.0 mg ovalbumin (OVA) in the form of gavage every day for 9 weeks, and active systemic anaphylaxis (ASA) was induced by intraperitoneal injection of OVA. Production of OVA-specific IgG1 in response to oral sensitization of the W/W(v) mice was very high, and the production of IL-4, IL-5 and IL-10 by splenocytes re-stimulated with OVA in vitro was increased. These findings suggest that Th2-dominant helper T-cell activation had occurred. By contrast, production of OVA-specific IgG1 was low in +/+ mice, and no significant increase in production of Th2-type cytokines by the splenocytes of +/+ mice was observed. Population analysis in Peyer's patches by flow cytometry revealed that the proportion of the CD11c(+) cell in the W/W(v) mice was slightly increased after antigen stimulation. Analysis of the cell surface markers of intraepithelial lymphocytes (IELs) by flow cytometry showed that the proportion of TCRgammadelta-T cells was extremely lower in the W/W(v) mice, especially in the antigen sensitized group. The proportion of TCRgammadelta-T cells in the splenocytes of W/W(v) mice was also lower than in +/+ mice. Taken together, the above findings indicate that W/W(v) mice seems to be a good model not only for studying the induction mechanism of food allergy but for examining the role of TCRgammadelta-T cells in food-induced hypersensitivity.

Anaphylaxis↗
PubMed2003

Application of human Fc epsilon RI alpha-chain-transfected RBL-2H3 cells for estimation of active serum IgE.

We transfected the alpha-chain of human FcepsilonRI into rat basophilic leukemia cell line RBL-2H3, established several stable transfected cells, and screened them by beta-hexosaminidase release induced by sensitization with human IgE and stimulation with anti-human IgE antibody. A cloned cell line RBL-hEIa-2B12 was the strongest responder among the transfected cell clones. The concentrations of cytosolic free Ca2+ concentration in the human IgE-sensitized cells increased after stimulation with anti-human IgE antibody. Thus, it is suggested that the alpha-chain of human FcepsilonRI is associated with the beta-chain and/or gamma-chain of rat FcepsilonRI, and that they form functional high affinity IgE receptor complexes. The total IgE concentrations of the sera from allergic patients were determined by using the beta-hexosaminidase release assay, where the transfected cells were sensitized with diluted and heat-inactivated (at 56 degrees C for 30 min) serum and stimulated with anti-human IgE antibody. The IgE concentration obtained correlated with those measured by an enzyme immunoassay method. beta-Hexosaminidase release induced by stimulation with 5 times diluted serum was sometimes less than the release induced by the same serum; diluted 25 times or 125 times, suggesting that these serum contained factors that blocked IgE binding to FcepsilonRI or cross-linking by anti-human IgE antibody. The results suggested that our system will be useful for detecting FcepsilonRIalpha-bindable IgE in human serum.

Animals↗
PubMed2003

Comparative study of in vitro digestibility of food proteins and effect of preheating on the digestion.

Information on the comparative digestibility of food allergens and non-allergenic proteins is crucial when stability to digestion is to be used as a criterion to assess the allergenic potential of novel proteins. Preheating effect on in vitro digestibility has not been fully examined. In this study we investigated the preheating effect of in vitro digestibility of several proteins and their proteolytic fragments in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Five major food allergens, ovalbumin (OVA), ovomucoid (OVM), beta-lactoglobulin (BLG), bovine serum albumin (BSA), soybean trypsin inhibitor (STI), four proteins of unproven allergenicity, horseradish peroxidase (HRP), ribulose-1,5-bisphosphate carboxylase/oxidase (RBC), phosphinothricin acetyltransferase (PAT) and zein from corn, and plant lectin, concanavalin A (Con A) were preheated (at 100 degrees C for 5 min) or not preheated, and then digested in SGF or SIF. Food allergens were relatively stable in both SGF and SIF. Among the allergens, digestibility of OVA in both SGF and SIF was markedly decreased, and BLG and STI were relatively stable after preheating. Digestibility of ConA in SGF and SIF was markedly decreased by preheating. Digestibility of non-allergenic proteins in SGF was higher than the allergenic proteins. From these results, because of the marked increase of the digestibility in several proteins by preheating, systematic information concerning the effect of food treatment on protein digestion is necessary to assess the relationship between allergenic potential and the digestibility of food protein.

Allergens↗
PubMed2003

Determination of enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase by LC/MS.

A liquid chromatography-mass spectrometry (LC/MS) method for determining the enzymatic activity of 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase), an enzyme of the shikimate pathway, was developed. EPSP synthase catalyzes the formation of 5-enolpyruvylshikimate-3-phosphate (EPSP) from shikimate-3-phosphate (S-3-P) and phosphoenolpyruvate (PEP) in microorganisms and plants. The enzymatic activity of EPSP synthase was assessed by the determination of EPSP after a 30-min incubation with S-3-P and PEP using the LC/MS system. EPSP synthase activity is given in terms of the produced EPSP (pmol/min/mg protein). Glyphosate (N-phosphonomethyl glycine)-tolerant EPSP synthase from the Agrobacterium sp. strain CP4 (CP4-EPSP synthase) in genetically modified soybeans (GM-soybeans) was found to have an enzymatic activity of 736 EPSP pmol/min/mg protein in the presence of 3 nmol of S-3-P. In contrast, the enzyme activity of non-GM-soybeans was 21 EPSP pmol/min/mg protein. The EPSP synthase activity was markedly decreased in the non-GM-soybeans by the addition of glyphosate, but the enzyme activity of the GM-soybeans was only slightly decreased with this treatment. This LC/MS system could also be applicable to the measurement of EPSP synthase activity in different plant species and the detection of herbicide-tolerant EPSP synthase in GM foods.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2003

[Study on a method for delivering scFv recombinant antibody into cultured cells].

We try to develop a method for delivering antibody from blood circulation through blood brain barrier to brain. In order to achieve this goal, antibody has to cross cellular membrane of brain capillary endothelial cells twice. As a first step of our study, we examined the ability for scFv antibody to cross cellular membrane of RBL-2H3 cells once and be delivered into the inside of the cultured cells with the help of TAT peptide. TAT peptide was originally found in Tat protein from the HIV-1 virus and known as one of protein transduction domains. First, oligonucleotide encoding TAT peptide was linked to 5' terminal of gene fragment of scFv antibody by PCR technology. TAT-linked scFv gene fragment was subcloned into pET-23b vector and successfully expressed in E. coli as inclusion body. After solubilization and purification, TAT-linked scFv recombinant protein was added to the culture of RBL-2H3 cells. TAT-linked scFv delivered into RBL-2H3 cells was detected by means of immunocytochemistry using fluorescence microscopy. TAT-linked scFv crossed cellular membrane more efficiently than scFv without TAT peptide.

Animals↗
PubMed2002

Effect of dialkyl phthalates on the degranulation and Ca2+ response of RBL-2H3 mast cells.

We examined the effect of three dialkyl phthalates, di-n-butylphthalate (DBP), di-isobutylphthalate (DIBP) and di(2-ethylhexyl)phthalate (DEHP), on antigen-induced degranulation of RBL-2H3 mast cells. Exposure to 50-500 microM DBP, 50-500 microM DIBP, and 500 microM DEHP significantly potentiated antigen-induced beta-hexosaminidase release. Without antigen stimulation, the phthalates did not cause any significant increase in degranulation. Next, we examined the Ca2+ response of RBL-2H3 cells after exposure to these phthalates. The cytosolic calcium ion concentration ([Ca2+]i) of the cells clearly increased when the cells were stimulated with 50-500 microM and 50-500 microM DIBP, and increased slightly when stimulated with 50-500 microM DEHP. Digital imaging fluorescence microscope analysis showed that the addition of DBP evoked Ca2+ oscillation in individual mast cells. Finally, we investigated the relationship between the DBP-sensitive Ca2+ stores and thapsigargin (TG)-sensitive Ca2+ stores. A rise in [Ca2+](i) following challenge with DBP after TG was observed, and thus the DBP-sensitive and TG-sensitive Ca2+ stores in RBL-2H3 cells seem to be different. In conclusion, some dialkyl phthalates increase antigen-induced degranulation in RBL-2H3 cells dependent on the increase of [Ca2+]i.

Animals↗
PubMed2002

mRNA expression of multiple cytochrome p450 isozymes in four types of cultured skin cells.

BACKGROUND: Many drugs are known to induce allergic reactions in the skin. The metabolic activation of drugs resulting in the formation of protein adducts is thought to be a first step in the induction of these allergic reactions. We postulated that dermal tissue might be a site of drug activation by cytochrome P450 (CYP) isozymes. METHODS: Messenger RNA was extracted from cultured Langerhans cells, keratinocytes, fibroblasts and melanocytes from 6 individuals, and CYP mRNA expression was analyzed by RT-PCR. RESULTS: CYP1A1, 1B1 and 2E1 were found in all four cell types. CYP2A6, 2C, 2D6, 3A5, 3A7 and 4B1 mRNA was expressed in a cell-type- and/or individual-specific manner. CYP1A2, 2A7, 2B6 and 3A4 mRNA was not detectable. CONCLUSIONS: The mRNA for a variety of CYP isozymes was expressed in all four types of skin cells examined. These CYP enzymes may be involved in the pathogenesis of drug-induced allergic reactions in the skin.

Cells, Cultured↗
PubMed2002

IgG-mediated signal transduction in canine mastocytoma-derived cells.

BACKGROUND: We have reported canine cutaneous mastocytoma-derived cells named CM-MC sensitized with monomeric IgG released histamine upon anti-IgG stimulation. However, IgG or IgE-mediated signal transduction in the cells remains to be examined. METHODS: Monomeric IgG-binding to cells was measured by flow cytometry using FITC-anti-IgG. IgG-mediated protein tyrosine phosphorylation was studied by Western blotting using anti-phosphotyrosine antibody. We monitored the intracellular Ca(2+) concentration ([Ca(2+)](i)) when IgG-primed cells were activated with anti-canine IgG. Release of Ca(2+) from intracellular stores was analyzed with thapsigargin in the absence of extracellular Ca(2+). The Ca(2+) entry via store-operated Ca(2+) channel from the external environment was characterized using Ba(2+), Ni(2+) and EGTA. Cells sensitized with canine serum abundant in IgG and IgE or heat-inactivated serum were activated by anti-canine IgG or anti-canine IgE. The effect of extracellular Ca(2+) and reaction time on IgG-mediated histamine release was examined. Staurosporine and ER-27319 were used to clarify the IgG-mediated protein tyrosine phosphorylation. RESULTS: Abundant IgG-binding sites on the cell were detected by FACS analysis. Anti-IgG induced rapid protein tyrosine phosphorylation and [Ca(2+)](i) elevation. When extracellular Ca(2+) was excluded by EGTA, a mild and transient increase in [Ca(2+)](i) was observed, indicating the release of Ca(2+) from anti-IgG-sensitive intracellular Ca(2+) stores. The constant Ba(2+) entry from external environment proved the Ca(2+) influx occurred mainly via a store-operated Ca(2+) channel which was inhibited by Ni(2+) and EGTA. Canine serum-sensitized cells showed a rapid and sustained increase in [Ca(2+)](i) upon both anti-IgG and anti-IgE stimulation. The [Ca(2+)](i) elevation induced by anti-IgE was decreased in the cells sensitized with heat-inactivated serum. Histamine release from CM-MCs was absolutely dependent on extracellular Ca(2+), and reached equilibrium within 5 min. Staurosporine inhibited the tyrosine phosphorylation of 38-, 65-, 70-, 80-kD proteins. ER-27319 inhibited the tyrosine phosphorylation of 38- and 70-kD proteins. Staurosporine also inhibited IgG-mediated [Ca(2+)](i) elevation and histamine release in a dose-dependent manner. CONCLUSIONS: Canine cutaneous mastocytoma-derived (CM-MC) cells were activated by both IgG- and IgE-mediated mechanisms. IgG-mediated protein tyrosine phosphorylation and Ca(2+) influx were similar to those mediated by IgE. CM-MC cells are useful for the study of allergic inflammation caused by IgG-dependent mechanisms.

Acridines↗
PubMed2002

Effect of subchronic feeding of genetically modified corn (CBH351) on immune system in BN rats and B10A mice.

Subchronic animal feeding studies to examine the effect on the immune system of genetically modified corn CBH351, which contains the Cry9C protein derived from Bacillus thuringiensis subspecies tolworthi, were conducted in female BN rats and B10A mice. The studies were designed to compare the effect of a line of genetically modified corn CBH351 (GM corn) with that of isoline corn (non-GM corn). Heat-treated corn meal was incorporated into the diets of the rats and mice at a concentration of 50%. The study duration was 13 weeks. Growth, food intake, and organ weights of the thymus, spleen, and liver were compared between animals fed the non-GM and GM lines. The histological findings in thymus, spleen, mesenteric lymph nodes, Peyer's patches, small intestines, liver, kidney, and bone marrow, and the presence of Cry9C-specific IgE, IgG, IgG1 and IgA antibodies in serum were also compared. The results showed no significant differences in growth, feeding value, or the histological findings in immunity-related organs between the animals fed the GM and non-GM lines. Production of Cry9 C-specific IgE and IgA was not detected in the serum of either group. Production of Cry9C-specific IgG and IgG1 was slightly increased in the 50% GM groups of BN rats. No Cry9C-specific IgG or IgG1 was detected in the serum of BN rats fed the diet containing 5% GM-corn In conclusion, no immunotoxic activity was detected in the GM-corn-fed rats and mice in this subchronic dietary study.

Animals↗
PubMed2002

Increased digestibility of two products in genetically modified food (CP4-EPSPS and Cry1Ab) after preheating.

We performed experiments on in vitro digestion of newly expressed proteins by SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) to assess the allergenicity of food components derived from biotechnological modification. For newly expressed proteins, we chose CP4-EPSPS (5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4) and Cry1Ab derived from Bacillus thuringiensis subsp. kurstaki strain HD-1. The former is expressed in GM-soybeans and the latter is expressed in GM-corns. Firstly, we examined the digestibility of purified CP4-EPSPS and Cry1Ab by SGF. Both proteins were rapidly digested within 60 sec. After preheating, the digestibility by SGF was slightly increased. Secondly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SGF. The digestion time of both proteins by SGF was almost the same as that of the purified proteins. Thirdly, the digestibility of CP4-EPSPS and Cry1Ab by SIF was examined. The digestion time of these proteins was 240 min or more. However, digestibility of these proteins by SIF was dramatically increased by preheating, and the digestion time was less than 5 sec. Fourthly, CP4-EPSPS in GM-soybean extracts and Cry1Ab in GM-corn extracts were digested by SIF. Digestion time of both proteins by SIF was almost the same as that of the purified proteins. From these results, we concluded that the digestibility of both CP4-EPSPS and Cry1Ab by SGF and SIF was increased by preheating. Therefore, we suggest that the allergenicity of both proteins should be extremely low because of the easy digestibility of these proteins by SGF and also by SIF with preheating.

Allergens↗