Biomedical subjects

Tadashi Ogawa

Publications and source records attributed to Tadashi Ogawa.

At least 19 recordsLinked to original sources

PubMed2006

Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli.

Phosphoenolpyruvate inhibited Escherichia coli NADP-isocitrate dehydrogenase allosterically (Ki of 0.31 mM) and isocitrate lyase uncompetitively (Ki' of 0.893 mM). Phosphoenolpyruvate enhances the uncompetitive inhibition of isocitrate lyase by increasing isocitrate, which protects isocitrate dehydrogenase from the inhibition, and contributes to the control through the tricarboxylic acid cycle and glyoxylate shunt.

Escherichia coli↗

PubMed2006

Oxidative inactivation of reduced NADP-generating enzymes in E. coli: iron-dependent inactivation with affinity cleavage of NADP-isocitrate dehydrogenase.

Treatment of E. coli extract with iron/ascorbate preferentially inactivated NADP-isocitrate dehydrogenase without affecting glucose-6-phosphate dehydrogenase. NADP-Isocitrate dehydrogenase required divalent metals such as Mg(2+), Mn(2+ )or Fe(2+) ion. Iron/ascorbate-dependent inactivation of the enzyme was accompanied with the protein fragmentation as judged by SDS-PAGE. Catalase protecting the enzyme from the inactivation suggests that hydroxyl radical is responsible for the inactivation with fragmentation. TOF-MS analysis showed that molecular masses of the enzyme fragments were 36 and 12, and 33 and 14 kDa as minor components. Based on the amino acid sequence analyses of the fragments, cleavage sites of the enzyme were identified as Asp307-Tyr308 and Ala282-Asp283, which are presumed to be the metal-binding sites. Ferrous ion bound to the metal-binding sites of the E. coli NADP-isocitrate dehydrogenase may generate superoxide radical that forms hydrogen peroxide and further hydroxyl radical, causing inactivation with peptide cleavage of the enzyme. Oxidative inactivation of NADP-isocitrate dehydrogenase without affecting glucose 6-phosphate dehydrogenase shows only a little influence on the antioxidant activity supplying NADPH for glutathione regeneration, but may facilitate flux through the glyoxylate bypass as the biosynthetic pathway with the inhibition of the citric acid cycle under aerobic growth conditions of E. coli.

Binding Sites↗

PubMed2006

Alanine racemase of alfalfa seedlings (Medicago sativa L.): first evidence for the presence of an amino acid racemase in plants.

We demonstrated several kinds of D-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa (Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition of L- or D-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of L- and D-alanine. The K(m) and V(max) values of enzyme for L-alanine were 29.6 x 10(-3) M and 1.02 mol/s/kg, and those for D-alanine are 12.0 x 10(-3) M and 0.44 mol/s/kg, respectively. The K(eq) value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5'-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5'-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far.

Alanine↗

PubMed2006

Neuronal dynamics of bottom-up and top-down processes in area V4 of macaque monkeys performing a visual search.

Visual selection is thought to be guided by both bottom-up intrinsic visual saliency and top-down visual attention. We examined how the relative importance of each of these processes dynamically changes over the course of a visual search in area V4 of two macaque monkeys. The animals were trained to perform a multidimensional visual search task in which a search array that consisted of two singleton stimuli, unique in shape and color, and four nonsingleton stimuli was presented. The task was to select one of the singletons as the saccade target, depending on the instructed search dimension, which was switched between shape and color. The strengths of the neural modulations caused by the bottom-up and top-down factors were, respectively, evaluated by assessing the dependence of the visual responses on the singleton type and the behavioral significance of the receptive field stimulus. We found that the initial visual responses of V4 neurons predominantly specified the singleton type of the receptive field stimulus, while the late presaccadic response primarily specified its behavioral significance. For comparison, recordings were also made from the frontal eye field (FEF) of one monkey and we found that the predominant activity of FEF neurons signaled the behavioral significance of the receptive field stimulus over time. These findings suggest that area V4 might function as an intermediate stage where both sensory- and behavior-based signals are dynamically represented, depending on their online requirements during a visual search, whereas the FEF predominantly represents behavior-based signals for upcoming saccade responses.

Action Potentials↗

PubMed2006

Detection of low-molecular weight allergens resolved on two-dimensional electrophoresis with acid-urea polyacrylamide gel.

Two-dimensional electrophoresis with immobilized pH gradient (IPG) followed by acetic acid/urea-polyacrylamide gel electrophoresis (AU-PAGE) was developed for the detection of low-molecular weight food allergens. Wheat proteins were used to test the applicability of AU-PAGE for the analysis of food allergens. Isoelectric focusing (IEF) for first dimension was performed with IPG pH 3-10. AU-PAGE was performed as a second-dimensional electrophoresis and high resolution was obtained, especially for proteins below 15 kDa. For immunodetection, the proteins resolved on AU gel were transferred to a polyvinylidene difluoride membrane. The assembly of semidry electroblotting for AU gel was set reversed as for sodium dodecyl sulfate (SDS)-PAGE gel. The electroblotted membrane was immunolabeled with serum from a radio-allergosorbent test-positive individual for wheat to identify allergenic proteins. Protein spots strongly recognized by the patient's serum were chosen for further analysis. Mass spectrometry analysis revealed that these proteins were alpha-amylase/trypsin inhibitors and lipid transfer protein. The system developed in this study was shown to be useful as a standard protocol for the separation of low-molecular weight proteins. Moreover, the IPG strips on which IEF was performed could be used either for SDS-PAGE or AU-PAGE by only changing equilibrating conditions, allowing for a wide range of allergen analysis.

Acetic Acid↗

PubMed2006

Evaluation of available IgE-binding epitope data and its utility in bioinformatics.

This paper reviews the role played by IgE-binding epitopes in eliciting clinical symptoms, the types of IgE-binding epitopes in allergenic proteins, the methods used to identify IgE-binding epitopes, and the availability of IgE-binding epitopes in allergenic sources. Finally, bioinformatics methods to assess protein allergenicity using knowledge of IgE-binding epitopes are discussed.

Allergens↗

PubMed2006

Sodium cromoglycate inhibits absorption of the major soybean allergen, Gly m Bd 30K, in mice and human intestinal Caco-2 cells.

Our previous data showed that Gly m Bd 30K was absorbed from the gastrointestinal tract and circulated in blood in mice. This study was conducted to determine the mechanism and identify the inhibitor of such absorption. Using sandwich ELISA and immunoblotting, we found that intact Gly m Bd 30K was absorbed from apical to basolateral solutions and intracellularly accumulated by Caco-2 cells in a dose- and time-dependent manner. The absorption and intracellular accumulation of Gly m Bd 30K were significantly suppressed when Caco-2 cells were treated with sodium cromoglycate (SCG) (0-50 mmol/L) in a dose-dependent manner. In 24-d-old mice orally treated with SCG (10-1000 mg/kg body weight), plasma Gly m Bd 30K concentration decreased significantly 30-120 min after Gly m Bd 30K (2000 mg/kg body weight) administration. Moreover, inhibitors that suppress the clathrin-dependent endocytosis dansylcadaverine, the caveolae-dependent endocytosis nystatin and clathrin, and the caveolae-dependent endocytosis methyl-beta-cyclodextrin had inhibitory effects on the absorption and intracellular accumulation of Gly m Bd 30K by Caco-2 cells. These data indicate that Gly m Bd 30K is absorbed and intracellularly accumulated in Caco-2 cells via clathrin- or caveolae-dependent endocytosis. We propose that the absorption and intracellular accumulation of Gly m Bd 30K are inhibited by SCG via clathrin- or caveolae-dependent endocytosis.

Alkaline Phosphatase↗

PubMed2005

Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats.

BACKGROUND & AIMS: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. METHODS: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor beta receptor (AdTbeta-TR), which works as a dominant-negative receptor, bacterial beta-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1-21 days. RESULTS: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdTbeta-TR-, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdTbeta-TR-treated rats. CONCLUSIONS: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdTbeta-TR-treated livers indicate that transforming growth factor beta is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor beta is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells.

Animals↗

PubMed2005

Dietary fat and an exogenous emulsifier increase the gastrointestinal absorption of a major soybean allergen, Gly m Bd 30K, in mice.

The mechanisms by which food allergens are absorbed and sensitized via the gastrointestinal tract have not been well characterized. In this study, the gastrointestinal absorption of a major soybean allergen, Gly m Bd 30K, in young and older mice, and the effects of dietary fat and exogenous emulsifier were investigated. In Expt. 1, Gly m Bd 30K [0, 500 or 2000 mg/kg body weight (BW)] was administered orally to 24-d-old mice, and blood was sampled at various time points over a 120-min period. Plasma Gly m Bd 30K was measured by sandwich ELISA and immunoblotting. Its concentration peaked at 30 min and was dose dependent. Intact Gly m Bd 30K and its 20-kDa fragments were identified in plasma after absorption. In Expt. 2, 24-d-old mice administered soy milk containing 1 mg Gly m Bd 30K showed a steady increase in plasma Gly m Bd 30K from 60 to 120 min that was significantly higher than that in 10-wk-old mice. In Expt. 3, when corn oil (5 or 30%) was coadministered with Gly m Bd 30K (2000 mg/kg BW) to 24-d-old mice, the plasma concentration increased significantly and generally reached a plateau after 30 min. The absorption after the coadministration of 30% corn oil and 3% sucrose fatty acid ester was higher than after the administration of 30% corn oil alone. Intact Gly m Bd 30K and its fragments that were < 20 kDa survived digestion and were absorbed into the blood. We propose that absorption was enhanced by fat carrier-mediated transport.

Allergens↗

PubMed2005

Influence of royal jelly on mouse hepatic gene expression and safety assessment with a DNA microarray.

We used a DNA microarray to compare the gene expression profiles in liver among three groups of mice fed a diet containing 5% royal jelly (RJ), a diet containing 5% RJ stored at 40 degrees C for 7 d (40-7d RJ) or a control diet which provides the same total energy as RJ. Expression of 267 genes was increased or decreased by 1.8-fold or more in animals given the RJ diet for 14 d as compared with control diet, though serum total cholesterol, triglyceride, phospholipid, glucose, insulin and leptin levels were unaffected. Many genes involved in cell growth, signal transduction, energy metabolism and transcription regulation were responsive to the RJ diet. Among the 267 genes whose expression was altered by RJ, 60% showed no change or a reduced change in response to 40-7d RJ diet. The 40-7d RJ diet contained little 57-kDa protein, identified as a possible freshness marker of RJ. Furthermore, the RJ diet did not influence the gene expression of cytochrome P450 enzymes and detoxifying enzymes, whereas the 40-7d RJ diet increased the gene expression of glutathione S-transferase and glutathione peroxidase. Indeed, the RJ diet decreased the gene expression of cytochrome P450 4A14 (CYP4A14), which catalyzes peroxidation of endogenous lipids that is associated with nonalcoholic steatohepatitis and alcoholic liver disease, while the 40-7d RJ diet was not effective to decrease the gene expression of CYP4A14. The results indicate that the efficacy of RJ decreased and the toxicity of RJ increased during storage at high temperature. We suggest that application of DNA microarray technology to the biochemical evaluation of food safety may be effective for rapid and precise quality control.

Animals↗

PubMed2005

A novel enzyme-linked immunosorbent assay for quantification of soybean beta-conglycinin, a major soybean storage protein, in soybean and soybean food products.

Soybean (Glycine max L.) storage proteins are composed of two major components, beta-conglycinin and glycinin, corresponding to 7S and 11S globulins, respectively. Recently, soybean beta-conglycinin (7S globulin) has been reported to show beneficial functions in animals and human. To date, there is no method for the precise quantification of soybean beta-conglycinin in processed food products or soybean seeds. We report here a novel method for this purpose. At first, antibodies specifically reactive to the subunits of beta-conglycinin were prepared. And then, a direct enzyme-linked immunosorbent assay (ELISA) for the quantification of soybean beta-conglycinin in processed foods and seeds was developed. In this assay, the sample was treated with sodium dodecyl sulfate sample buffer followed by dilution with phosphate-buffered saline. The diluted samples were poured and coated onto an ELISA plate and reacted with rabbit anti-beta-conglycinin antibody and peroxidase-labeled anti-rabbit IgG. Finally, the bound peroxidase-labeled antibody was detected by colorimetric reaction. By using this system, it has been possible to measure soybean beta-conglycinin concentrations in several processed food products. In addition, this simple quantification ELISA system was demonstrated to be adaptable for the quantification of beta-conglycinin contents of various soybean cultivars.

Animals↗

PubMed2004

Target selection in area V4 during a multidimensional visual search task.

Natural scenes typically contain multiple objects that are unique in different stimulus dimensions so that an object with feature contrast to surrounding objects draws attention and pops out. Furthermore, if we have previous knowledge about the dimension in which a target object differs from the surrounding objects, we will attend to that dimension and more easily detect the target. Our aims here were to elucidate neural mechanisms underlying this type of attention by recording neuronal activities from area V4 and to investigate how visual signals encoding feature contrast between objects are modulated by attention specific to a particular dimension. To accomplish this, we trained monkeys to do a multidimensional visual search task in which two singleton stimuli, unique in the color or shape dimension, were presented with four other identical stimuli. The monkeys had to search for the singleton stimulus that was unique in the instructed dimension while the search dimension was switched between shape and color. We found that individual V4 neurons carry visual signals encoding feature contrast in either shape or color, and this signal is modulated depending on the search dimension. Population responses to the target singleton stimulus were significantly higher than to others, regardless of the search dimension. In most V4 neurons, however, significant response increases occurred only when one particular singleton stimulus was the target. These findings suggest that interaction between bottom-up signals encoding feature contrast between stimuli and top-down signals encoding search dimension occurs in V4 and facilitates adaptive selection of targets in a complex visual environment.

Animals↗

PubMed2004

Enhanced bactericidal action and masking of allergen structure of soy protein by attachment of chitosan through maillard-type protein-polysaccharide conjugation.

The soy protein-chitosan conjugate was formed by the Maillard reaction in dry state (relative humidity 65%) at 60 degrees C for 2 weeks to improve the functional properties. The antimicrobial activity of the Maillard-type soy protein-chitosan conjugates enhanced 2-3 times that of soy protein-chitosan mixture. The soy protein-chitosan conjugate showed excellent emulsifying property with the progress of Maillard-type conjugation. The allergenicity of soy protein was greatly decreased by the attachment of chitosan through Maillard reaction. The immonoblotting analysis with patient's sera revealed that soy protein-chitosan conjugate was more effective to mask the allergen structure of soy protein causing from 34 kDa-protein (Gly m Bd 30K) than soy protein-galactomannan conjugate. The Western blotting showed that allergen (34 kDa-protein) was completely masked by soy protein-chitosan conjugation, while it was not completely masked by soy protein-galactomannan conjugation.

Allergens↗

PubMed2004

Effective transfer of interleukin-12 gene to solid tumors using a novel gene delivery system, poly [D,L-2,4-diaminobutyric acid].

Delivery of the interleukin-12 (IL-12) gene to solid tumors is a promising anticancer therapy. Vectors are currently being developed to achieve safe and effective intratumoral delivery of the IL-12 gene. Poly [D,L-2,4-diaminobutyric acid] (PDBA) is a novel gene carrier that was recently described. The goal of this study was to use this gene delivery system for treatment of solid tumors. To determine the optimal conditions for transfection, established B16F10-melanomas in C57BL/6 mice were treated with intratumoral injection of the PDBA/plasmid luciferase (pLuc) complex. We determined that the optimal complex composition was 50 microg/ml pLuc and 150 microg/ml PDBA. High levels of IL-12 protein were expressed in tumors after a single injection of PDBA/murine IL-12 (pmIL-12) complex, whereas serum levels of IL-12 in treated mice were below the limits of detection. IL-12 gene therapy with the PDBA system significantly inhibited tumor growth in comparison with the controls (P<.001). Moreover, both natural killer and cytotoxic T lymphocyte activities from draining lymph nodes of PDBA/pmIL-12-treated mice were increased substantially in comparison with those of controls (P<.05). These results suggest that PDBA-mediated IL-12 gene therapy is a potential strategy for treatment of patients with solid tumors.

Aminobutyrates↗

PubMed2004

Soybean beta-conglycinin diet suppresses serum triglyceride levels in normal and genetically obese mice by induction of beta-oxidation, downregulation of fatty acid synthase, and inhibition of triglyceride absorption.

The purpose of this study was to discover the effects of soybean beta-conglycinin (7S-globulin) and glycinin (11S-globulin) on serum lipid levels and metabolism in the livers of normal and genetically obese mice. Male normal (ICR) and obese (KK-Ay) mice were fed ad libitum high fat diets for two weeks, followed by a 2-week restriction of diet (2 g diet/mouse/day) containing 20% casein, soybean beta-conglycinin, or soybean glycinin, and then sacrificed immediately. Serum triglyceride (TG), glucose, and insulin levels of beta-conglycinin-fed mice were lower than in casein- and glycinin-fed mice of both strains. In order to analyze the related events to these effects, enzyme activities and relative mRNA levels of lipid metabolism-related proteins were measured. The activities of two enzymes related to fatty acid beta-oxidation were higher while that of fatty acid synthase was lower in livers of beta-conglycinin-fed mice than of casein-fed both mice. Messenger RNA levels of acyl-CoA oxidase (fatty acid beta-oxidation related enzyme) were significantly higher in livers of beta-conglycinin-fed mice than of both casein-fed mice. On the contrary, mRNA levels of SREBP-1 and 2 tended to be lowered in livers of soy protein-fed mice than of both casein-fed mice. Fecal excretion of TG was higher in beta-conglycinin-fed mice than in casein-fed mice. Our results demonstrated that the soy beta-conglycinin diet reduced serum TG levels by acceleration of beta-oxidation, suppression of fatty acid synthase and/or increased TG fecal excretion, and also diminished serum glucose and insulin levels. Some of these events might be caused at the transcriptional levels, judged from the result that relative messenger RNA levels of lipid metabolism-related proteins were altered. These results suggest that soy beta-conglycinin could be a potentially useful dietary protein source for the prevention of hypertriglyceridemia, hyperinsulinemia, and hyperglycemia, which are recognized as risk factors for atherosclerosis.

Animals↗

PubMed2003

Low resistin levels in adipose tissues and serum in high-fat fed mice and genetically obese mice: development of an ELISA system for quantification of resistin.

Obesity is a major risk factor for insulin resistance. Resistin, an adipocyte-derived hormone-like molecule, is considered to serve as an important link between obesity and insulin resistance. However, the physiological role of resistin and the mechanism by which it neutralizes insulin action are still unclear. There are also conflicting reports that cast doubt on the cause of insulin resistance. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) system for quantification of mouse resistin levels, analyzed in relation to insulin resistance. C57BL/6J mice fed high-fat diet compared with normal diet had low resistin levels (by 70%, P<0.01) in epididymal adipose tissues. Genetically obese mice, db/db and KK-A(y), had hyperinsulinemia and hyperglycemia but low resistin levels (decreases by 83 and 90%, both P<0.01) compared with C57/BL6J mice in epididymal adipose tissues. Serum resistin levels determined by Western blotting showed a similar pattern to those in adipose tissues. Resistin levels in adipose tissues correlated with serum adiponectin concentrations positively (r=0.49). Our results indicate that the novel ELISA system is suitable for measurement of resistin levels in adipose tissues. The results do not support a role for resistin in insulin resistance.

Adiponectin↗

PubMed2003

Synthesis and antinociceptive activity of orally active opioid peptides: improvement of oral bioavailability by esterification.

To improve the oral bioavailability of a dermorphin tetrapeptide analog, N(alpha)-1-iminoethyl-Tyr-D-MetO-Phe-MebetaAla-OH (III), which has a potent analgesic activity after oral administration, various derivatives were synthesized to increase lipophilicity by esterification of the C-terminal carboxyl group and/or acylation of the phenolic hydroxyl group on Tyr1. Antinociceptive activity was evaluated after subcutaneous or oral administration using the mouse tail pressure test. As a result, increased antinociceptive activity after oral administration as well as an improved ED50(p.o.)/ED50(s.c.) ratio, which is an indicator of oral bioavailability, were found for some compounds. With regard to the improvement of bioavailability, derivatives with acylation of the phenolic hydroxyl group on Tyr1 showed better results than derivatives with esterification of the C-terminal carboxyl group. In particular, an ED50(p.o.)/ED50(s.c.) ratio equivalent to that of morphine was found for an acetylated derivative, N(alpha)-1-iminoethyl-Tyr(COMe)-D-MetO-Phe-MebetaAla-OH (7a), as well as for a methoxycarbonylated derivative, N(alpha)-1-iminoethyl-Tyr(CO2Me)-D-MetO-Phe-MebetaAla-OH (7l).

Administration, Oral↗

PubMed2003

Identification of suberization-associated anionic peroxidase as a possible allergenic protein from tomato.

A 45 kDa protein, which is recognized by IgE antibodies in sera of food-allergic patients, was purified and characterized as an allergenic protein from the tomato. The IgE-binding protein purified from tomato extract was found to be a glycoprotein with a molecular weight of approximately 45,000, an isoelectric point of 4.2, and no free N-terminal amino group. Furthermore, it was shown that the purified protein had peroxidase activity. From the amino acid sequence of a peptide fragment prepared by lysylendopeptidase digestion, the allergenic protein was identified to be the tomato suberization-associated anionic peroxidase 1 known as one of the pathogenesis-related proteins widely distributed in plants. These properties suggested the protein isolated from tomato to be a new allergenic protein in plant foodstuffs.

Adolescent↗