Capillary electrophoresis for analysis of microheterogeneous glutelin subunits in rice (Oryza sativa L.).

Glutelin, the major storage protein of rice seed, consists of microheterogenous subunits and partially exists in a macromolecular form that is polymerized by intersubunit disulfide bonds. In order to analyze the glutelin subunits using high-throughput CE, we first identified a sample preparation procedure suitable for CE. The polymerized glutelin treated with a reductant could not dissociate into its constituent monomer subunits when it was dissolved in an acidic solution. However, the glutelin dissociated into its subunits and component α and ß polypeptides when it was denatured and reduced by an appropriate amount of urea and 2-mercaptoethanol at a specific incubation time and temperature. The molecular species of the completely dissociated α and ß polypeptides were identified and quantitatively analyzed by CE using glutelin mutants. The CE analysis also demonstrated that the actual subunit variation in terms of the charge and/or size of the ß polypeptides is much smaller than predicted when compared with that of α polypeptides, even under denaturing and reducing condition. Thus, the combined analytical system described here will be useful for basic and applied research, such as the kinetic characterization of higher-order structure and the quantitative evaluation of glutelin in a large number of diverse rice varieties.

Identification and variation of glutelin alpha polypeptides in the genus Oryza assessed by two-dimensional electrophoresis and step-by-step immunodetection.

To obtain fundamental information for nutritional improvement of rice (Oryza sativa) seed proteins, the alpha polypeptides of the major storage protein glutelin varied over the genus Oryza were qualitatively and quantitatively characterized with unique methods. The polypeptides were maximally separated by two-dimensional electrophoresis (2D-PAGE) composed of nonequilibrium pH gradient gel electrophoresis (NEPHGE) and higher temperature SDS-PAGE. Then the subunit for each polypeptide spot was identified with the sequential immunodetection called a step-by-step detection method, making use of highly subunit-specific antibodies. The comparative analysis showed considerable variation in the accumulation level of A-type and B-type glutelin subunits and found unknown glutelin subunits that were unable to be identified with the antibodies used. Wild species accumulating a high amount of lysine-rich B-type glutelin subunits and unknown unique subunits were identified as they might play a crucial role in nutritional quality improvement of the cultivated rice.

Diversity of rice glutelin polypeptides in wild species assessed by the higher-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subunit-specific antibodies.

In efforts to find genetic resources with high nutritional value of rice seed, we assessed the diversity of the major storage protein glutelin in 13 wild and 2 cultivated rice species by a unique SDS-PAGE method and subunit-specific antibodies. Maximum separation of microheterogeneous glutelin alpha-polypeptides, which is a prerequisite for the diversity evaluation, could be attained by SDS-PAGE performed at higher temperature (45 degrees C) than the generally employed temperatures (4-25 degrees C). Seven antipeptide antibodies were raised against subunit-specific epitope sequences designed at five sites from four variable regions spanning the glutelin alpha-polypeptides. High specificity of each antibody was confirmed using rice glutelin mutants, and demonstrated considerable variation in amino acid sequence and accumulation level of glutelin subunit in wild species, in combination with the higher-temperature SDS-PAGE. The degree of the variation was, however, changed according to the site of variable regions and the type of subunit. Some wild species accumulated nutritious GluB subunits more than cultivated rice. The wild species Oryza longiglumis and O. brachyantha had glutelin with low reactivity against most antibodies examined in this study, reflecting the significant divergence. Such wild species may hopefully serve as important genetic resources for nutritional improvement of cultivated rice.

Effect of procaterol, a beta(2) selective adrenergic receptor agonist, on airway inflammation and hyperresponsiveness.

BACKGROUND: beta-agonists are frequently used as bronchodilators for asthma as not only a reliever but also a controller, and their utility has increased with the development of long-acting beta(2) selective drugs. Although anti-inflammatory effects of beta(2) selective-agonists have been reported in vitro, side effects on augmentation of airway hyperresponsiveness by chronic use of beta(2) selective-agonists have been described in several reports. In this study, we investigated the effects of procaterol, a second-generation beta(2)-agonist, on airway inflammation in vivo using an antigen-specific murine model of asthma. METHODS: Mice immunized with ovalbumin (OVA) + alum and challenged with inhaled ovalbumin were orally administered procaterol during the challenge. After inhalation, the mice were tracheostomized and placed in a body box under controlled ventilation to measure airway resistance before and after acetylcholine inhalation. RESULTS: Administration of procaterol at a clinical dose equivalent did not augment airway hyperresponsiveness, inflammation of the airway wall, or subsequent airway wall thickening induced by OVA inhalation. BALF cell analysis revealed that the eosinophil number in the BALF was significantly reduced in procaterol-treated mice compared to untreated mice. CONCLUSIONS: Oral administration of procaterol at a clinical dose did not augment airway responsiveness, but did reduce eosinophil inflammation.

The two subfamilies of rice glutelin differ in both primary and higher-order structures.

Rice glutelin, which accounts for 70-80% of the total proteins of the seeds, consists of two nutritionally different subfamilies (A and B types). Although the similarity in primary sequences between the two subfamilies is as high as 60%, we established conditions to discriminate the two subfamilies when low amounts of antigen are analyzed by immunoblot methods. The glutelin alpha polypeptides can be resolved into six bands labeled alpha1 to alpha6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that glutelin exists as a polymerized and a smaller molecular weight form. Immunoblot analysis of SDS-PAGE resolved polypeptides showed that alpha2, alpha3, and alpha4 are an A type and that these A types as well as alpha1, a B type, are polymerized. The polymerization tendency clearly differed between the two subfamilies except for alpha1, which may be derived from GluB-4 as suggested by analysis using Escherichia coli expression systems of glutelin cDNA regions corresponding to alpha polypeptides. GluB-4 and all the A type subunits have an extra Cys residue in the hypervariable regions, corresponding to the C-terminal region of alpha polypeptide. Accordingly, the extra Cys residue is hypothesized to be responsible for the polymerization of glutelin.

Mutation screening of the muscarinic m2 and m3 receptor genes in asthmatics, outgrow subjects, and normal controls.

Muscarinic receptors are important in the development of airway hyperresponsiveness. In some patients with asthma and in animal models of hyperreactivity, functional abnormalities in these receptors are suggested to contribute to disease. Here, we have screened for single nucleotide polymorphisms in the coding region of human muscarinic m2 and m3 receptor genes using direct fluorescence sequencing. DNA samples from 102 current asthmatics and 58 who had outgrown asthma ("outgrow" patients) were compared with 70 random non-asthmatic controls. A mutation characterized by a single base substitution (A1050G, Ser350Ser) was identified in the muscarinic m2 receptor gene. This polymorphism was common and was represented in all three groups studied. In contrast, in the m3 receptor coding region examined, we found a very rare nucleotide variant (C261T, Ile87Ile), identified in only one of the 230 samples genotyped. Therefore, neither A1050G in the m2 receptor nor C261T in the m3 receptor is likely to be functionally significant for airway hyperresponsiveness in asthma. Our data suggest that both the m2 and m3 receptor genes are highly conserved, and no significant genetic mutations are related to their possible functional changes in human asthma.

Destructive pulmonary embolism in a patient with community-acquired staphylococcal bacteremia.

We report a 17-year-old man with destructive pulmonary embolism caused by Staphylococcus aureus bacteremia. The patient was not immunocompromised and had neither underlying diseases nor risk factors, such as concomitant influenza viral infection, which exacerbate staphylococcal infections. The rapid and extensive progression of pulmonary involvement in all lung fields make this a rare case; there have been few reports in the literature describing a similar radiographic appearance in patients with community-acquired staphylococcal bacteremia. In-vitro studies did not demonstrate the production of enterotoxins or toxic shock syndrome toxin 1 (TSST-1) by the isolated strain, but genetic analysis detected Panton-Valentine leukocidine gene from the strain. Subsequent empyema with bilateral pneumothorax was prolonged because of superinfection with both methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Optional surgical treatments, including thoracostomy and thoracopneumoplasty, finally improved his condition.

Attenuation of airway hyperresponsiveness in a murine asthma model by neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF).

Asthma is recognized as an inflammatory disease in which various cytokines are involved. Among these, granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to play a critical role in the survival of eosinophils and in the activation of antigen-presenting cells (APC). We studied the effects of neutralization of GM-CSF in a murine model of asthma, to elucidate its role in enhanced airway responsiveness and in airway inflammation. A/J mice, which are genetically predisposed to acetylcholine hyperresponsiveness, were immunized with ovalbumin (OA) and alum. Thereafter, the mice were subjected to a two-week regimen of OA inhalation, during which either goat anti-mouse polyclonal GM-CSF antibody or isotype control goat IgG was administered intranasally. Pulmonary function was then analyzed using whole body plethysmography before and after acetylcholine (Ach) inhalation. Here we show that OA inhalation following OA immunization increased airway responsiveness to acetylcholine and induced GM-CSF as well as IL-4 and IL-5 mRNA expression in the lung. The administration of GM-CSF-neutralizing antibody during OA inhalation significantly reduced this increased airway hyperresponsiveness and also inhibited airway inflammation. Thus, endogenous GM-CSF plays an important role in the process of airway inflammation and airway hyperresponsiveness after antigen-specific immunity has been established.

[Usefulness of patient education and quality of life (QOL) evaluation in asthma patients].

Achieving successful treatment of bronchial asthma depends on its control by the patient. We implemented a program of educating asthma patients and conducted a QOL survey to objectively evaluate the patients'conditions. Thirty-nine asthma patients who were receiving treatment with an inhaled corticosteroid [beclomethasone dipropionate (BDP) ] on an outpatient basis at our hospital, received instructions on proper drug administration in cooperation with the Pharmacy department of our hospital. The QOL survey (SF-36 and Marks et al. AQLQ) was conducted at the initial education session and again two months later. Thirty-two patients (82.0%) responded that they would like to continue to receive instructions on the administration of drugs. Significant improvements in Social, Concerns, Marks, and Scales were observed after the education. In addition, even those patients who stated that they did not want to receive instructions showed significant improvements in their QOL scores. The usefulness of patient education can be assessed by the change in the patients' QOL scores.