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Summary Background Previous studies have shown that rat peritoneal mast cells and mast cell model rat basophilic leukaemia (RBL-2H3) cells generate intracellular reactive oxygen species (ROS) in response to antigen challenge. However, the physiological significance of the burst of ROS is poorly understood. Objective The present study was undertaken to investigate the role of superoxide anion in mediator release in rat and human cell systems. Methods RBL-2H3 cells were directly stimulated with anti-rat FceRI α-subunit monoclonal antibody (mAb). For the analysis of human cell system, leucocytes were isolated by dextran sedimentation from healthy volunteers or from patients, and challenged either with anti-human FceRI mAb or with the relevant antigens. Superoxide generation was determined by chemiluminescence-based methods. The releases of histamine and leukotrienes (LT)s were determined by enzyme-linked immunosorben assay (ELISA). Results Cross-linking of FceRI on RBL-2H3 cells or on human leucocytes from healthy donors by the anti-FceRI mAb resulted in a rapid generation of superoxide anion, as determined by chemiluminescence using superoxide-specific probes. Similarly, leucocytes from patients generated superoxide anion in response to the challenge with the relevant allergen but not with the irrelevant allergen. Furthermore, diphenyleneiodonium (DPI), a well-known inhibitor of flavoenzymes suppressed the superoxide generation and the release of histamine and LTC4 induced by the anti-FceRI mAb or by allergen in parallel. Conclusion These results indicate that both RBL-2H3 cells and human basophils generate superoxide anion upon FceRI cross-linking either by antibody or by allergen challenge and that blockade of the generation prevents the release of allergic mediators. The findings strongly support the role of superoxide generation in the activation of mast cells and basophils under both physiological and pathological conditions. The findings suggest that drugs regulating the superoxide generation have potential therapeutic use for allergic disorders.

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.

Mast cells play a central role in immediate allergic reactions mediated by immunoglobulin E. It has recently been reported that mast cells generate intracellular reactive oxygen species (ROS) in response to stimulation with divergent physiologically relevant stimulants. However, the physiological role of ROS is poorly understood. Here we demonstrate that mast cell model rat basophilic leukemia (RBL-2H3) cells generate ROS in response to antigen and the calcium-ionophore A23187 via activation of diphenyleneiodonuim (DPI)-sensitive enzyme and that blockade of ROS generation by DPI suppresses histamine release induced by either stimulant. Increased tyrosine phosphorylation of pp125(FAK) and a 77-kDa protein coprecipitating specifically with the kinase occurred in parallel with the secretion, and blockade of ROS generation by DPI also suppressed the tyrosine phosphorylation of both proteins. These findings suggest that ROS generated by a flavoenzyme-dependent mechanism may be involved in histamine release through the pp125(FAK) pathway.

Background: Urinary iodine is a good biochemical marker for control of iodine deficiency disorders. Our aim was to develop and validate a simple, rapid, and quantitative method based on the Sandell–Kolthoff reaction, incorporating both the reaction and the digestion process into a microplate format. Methods: Using a specially designed sealing cassette to prevent loss of vapor and cross-contamination among wells, ammonium persulfate digestion was performed in a microplate in an oven at 110 °C for 60 min. After the digestion mixture was transferred to a transparent microplate and the Sandell–Kolthoff reaction was performed at 25 °C for 30 min, urinary iodine was measured by a microplate reader at 405 nm. Results: The mean recovery of iodine added to urine was 98% (range, 89–109%). The theoretical detection limit, defined as 2 SD from the zero calibrator, was 0.11 μmol/L (14 μg/L iodine). The mean intra- and interassay CVs for samples with iodine concentrations of 0.30–3.15 μmol/L were ≤10%. The new method agreed well with the conventional chloric acid digestion method (n = 70; r = 0.991; y = 0.944x + 0.04; Sy|x = 0.10) and with the inductively coupled plasma mass spectrometry method (n = 61; r = 0.979; y = 0.962x + 0.03; Sy|x = 0.20). The agreement was confirmed by difference plots. The distributions of iodine concentrations for samples from endemic areas of iodine deficiency diseases showed similar patterns among the above three methods. Conclusions: Our new method, incorporating the whole process into a microplate format, is readily applicable and allows rapid monitoring of urinary iodine.

A rice gene encoding a novel isoform of translation elongation factor-1beta subunit (termed EF-1beta2) was isolated and characterized. The gene comprises of eight exons, and encodes a 226-amino-acid protein. Expression of EF-1beta2 mRNA is abundant in seeds and cultured cells, but is considerably low in the tissues of the rice seedling. Antiserum raised against an EF-1beta2 synthetic peptide detected a protein with a relative molecular mass of about 32 kDa, indicating the EF-1beta2 gene is actually expressed in rice tissues. EF-1beta2 showed a close similarity to the cognate subunits from plant (beta and beta').

Aldolase A (AldB) gene is one of the liver-specific genes, which is activated in the fetal stage. As a first step to investigate the functional relationship between transcription and DNA replication, we intended to determine the initiation zone of replication nearest to the AldB gene region. BrdU-labeled nascent DNA was obtained from G1/S arrested hepatoma cells at various times after entering S phase. Hybridization of the newly synthesized, BrdU-labeled DNA with probes corresponding to regions spanning about 26 Kb, revealed that replication zone locates within the AldB gene region. This result, together with the result of hybridization of nascent DNA obtained by alkaline sucrose density-gradient centrifugation, suggested that the initiation zone is located within a more defined region (about 1.0 Kb) containing AldB promoter. In the predicted initiation zone, a purine-rich element which shows high homology to known mammalian origin sequences and other replication components are found. Further, autonomously replicating activity of this initiation zone was examined by DNA transfection. The results showed that the predicted initiation zone confers replication initiation in Cos-1 cells.

Extracts of Chilean medicinal plants were evaluated in vitro for their activities against influenza virus proliferation in MDCK cells. The most potent extract obtained was from Muehlenbeckia hastulata (Polygonaceae), known as Quilo in Chile, from which three active principles were isolated and identified as pheophorbide a (1), hypericin (2) and protohypericin (3). Minimum inhibitory concentration (MIC) values of 42 ng/ml for compound 1, 2.1 ng/ml for compound 2 and 1.5 ng/ml for the authentic hypericin were determined by using an endpoint assay which comprises pre-incubation of serially diluted specimens with a given amount of the influenza virus, incubation of the pre-incubated virus/specimen with MDCK cells and determination of the hemagglutination (HA) titer of the culture supernatant. Compound 3 was easily converted to 2 on exposure to visible light and, in due course, showed an anti-influenza virus activity (3.1 ng/ml) similar to 2. Although compounds 1–3 were previously isolated from other plants, this is the first report of their isolation from M. hastulata. The high content of 1 (0.06% dry weight of whole plant) is noteworthy. In addition, this is the first report on the isolation of compounds 2 and 3 from a plant other than the genus Hypericum.

A highly sensitive sandwich-type chemiluminescence enzyme immunoassay for plasma endothelin-I (ET-1), involving no extraction steps, has been developed. Two populations of polyclonal antibodies were used in the present study: One is specific to the C-terminus of the endothelin family of peptides; the other, a Fab' fragment against the N-terminal core region of ET-1, is coupled with horseradish peroxidase (HRP). Labeled HRP activity was measured by using an enhanced chemiluminescence reaction of luminol/hydrogen peroxide. The assay was sensitive enough to detect 0.5 ng/L (0.05 pg/well) of plasma ET-1 and had no significant cross-reactivities with other related peptides, including endothelin-3 and the endothelin precursor peptide, big ET-1. Reliability of the assay was confirmed by comparison with an extraction-based procedure and a commercial kit.

To analyze the differentiation-related glycolipids, we have recently developed human monoclonal anti-i antibodies (mAbs) using a combination of EBV-transformation and bovine i-active glycolipid (NeuAc alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4Glc beta 1-->1Cer)-containing liposome immune lysis assay (LILA). Using complement cytolysis with these mAbs, we found the occurrence of a surface antigen (Ag) in the EBV-negative but not in the EBV-positive cell lines. The fresh EBV infection reveals that the suppressed expression of the antigen was a result of the EBV infection. The Ag recognized with these mAbs appeared to have a very low density on the B cell lines. Unexpectedly, thin layer chromatogram (TLC)-immunostaining using the mAbs revealed that the major immunoreactive substance in the EBV-negative B cell lines was an extremely minor glycolipid that was distinct from the i-active glycolipid however, this was not the case in the EBV-positive B cell lines.

A stable and highly sensitive chemiluminescent detection system for horseradish peroxidase (HRP)/luminol/hydrogen peroxide using a newly designed thiazole compound enhancer has been established. Some additives for the chemiluminescent reaction were explored to overcome some defects of the reaction such as rapid decay and high background of light emission. Recrystallization of luminol and the addition of several detergents into the reacting solution were effective to increase specific light emissions. The addition of skim milk into the reacting solution reduced the background. Consequently, skim milk combined with a detergent increased the signal to noise ratio about 20 times compared with the reactions in the absence of both additives. The optimal concentration of enhancer and the addition of egg albumin stabilized the emission. In the new method, 6x 10(-18) mol of HRP was detectable. This would be the most sensitive enhanced chemiluminescent detection system for HRP. Furthermore, we could detect picogram per milliliter (10(-17) mol) concentrations of a trace component in biological materials such as endothelin-1 by employing this reaction.

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).

The hepatitis B virus (HBV) core antigen was purified by mild procedures, including hydroxyapatite column chromatography, with care taken to avoid the degradation of the particles. Circular dichroism (CD) of the HBV core particles in saline showed low intensities of negative ellipticities in the region dominated by amide bond absorption. Acid treatment of the particles induced a remarkable change in the CD spectrum, with the appearance of a positive extremum at about 208 nm. The amino acid composition and the COOH-terminal residue of the isolated core polypeptide( M r 21000–21500) were shown to be essentially the same as those of the polypeptide deduced from the nucleotide sequences which had been proposed for the HBV core antigen by other laboratories. We failed to detect any NH 2 -terminal dansyl-derivatives from the core polypeptide by the dansyl-Edman method. We also showed by the method of fluorescein polarization that the core polypeptide conjugated with fluorescein isothiocyanate has an affinity for serum alubumin. This may indicate a state of disassembled or non-assembled core polypeptide in sera.

The ultrastructure of the nucleocapsid component of human hepatitis B virus (core particle) was studied by negative staining-carbon film technique. Using this method an improved image of core particles was obtained in respect of resolution and contrast. Two-dimensional crystalline arrays of core particles were formed in vitro. Under these arrays the distance between the particle centers was 28.3 nm, corresponding to the capsid diameter, when analyzed through optical diffraction patterns. Positively stained images of these arrays revealed that core particles contain an electron-dense center of nucleoid-like area about 21 nm in diameter. The capsid surface rarely exhibited small capsomeres, ie, small spheres or ring-like structures measuring 4.0-4.2 nm. From the dimension of these structures and the analysis by Markham's rotational technique, it was suggested that each of these capsomeres is an individual subunit (monomer) and 180 of these subunits build up the core particle capsid according to the icosahedral symmetry (T = 3), but not clustering into distinct morphological features.

Highly purified and homogenous hepatitis B core particles were obtained from an autopsy liver. The core particles consisted mainly of an electrophoretically single major polypeptide with a molecular weight of 20,000 daltons. When antigenic conversion from hepatitis B core antigen (HBeAg) to hepatitis B e antigen was achieved by treating these core particles by sonication or by passing them through an anti-HBc IgG-conjugated Sepharose 4B column, no appreciable changes were found in the above protein composition. The same antigenic conversion was also achieved by centrifugation of core particles in CsCl, which revealed the process of morphological disintegration accompanied by antigenic conversion. These observations may support the hypothesis that HBeAg resides on a protein conformation which consists of a polypeptide sharing HBe antigenicity.

Japanese cedar pollinosis is a type I allergic disease mediated by immunoglobulin E (IgE) antibodies to Japanese cedar (Cryptomeria japonica) pollen antigen (CPAg). By using 22 dogs consisting of 20 dogs aged 3 months and 2 dogs aged 3 years, immunization was performed by subcutaneous injections of CPAg with aluminum hydroxide gel. Variable levels of CPAg-specific IgE antibody response were detected in 21 of the 22 immunized dogs two weeks after the second immunization. This study provided an experimental sensitization system with CPAg in dogs, which will be useful for further immunological studies on Japanese cedar pollinosis.