Dietary pulverized konjac glucomannan suppresses scratching behavior and skin inflammatory immune responses in NC/Nga mice.

BACKGROUND: Feeding with pulverized konjac glucomannan (PKGM) suppresses the development of eczema and hyper-IgE production in NC/Nga mice, a model of atopic dermatitis. This study aimed to examine the effects of PKGM on scratching behavior and skin inflammatory immune responses in NC/Nga mice. METHODS: Four-week-old NC/Nga mice were maintained for 8 or 9 weeks on diet containing PKGM. Scratching behavior and clinical symptoms were evaluated every 2 weeks. Effects of PKGM on cutaneous inflammation were evaluated by histopathological analysis. Local expression levels of substance P and proinflammatory cytokines were measured by ELISA. RESULTS: An increase in scratching behavior was evident from 6 weeks of age in control mice, but this symptom was dose-dependently inhibited in PKGM-fed mice. Continuous PKGM feeding then significantly inhibited eczematous skin lesions including hyperkeratosis, dermal mastocytosis and eosinophilia. Concomitantly, cutaneous overproductions of substance P, IL-10, IL-4, and TNF-alpha were all suppressed in PKGM-fed mice. CONCLUSIONS: PKGM feeding markedly suppressed development of scratching behavior, substance P expression with mastocytosis, and skin inflammatory immune responses in NC/Nga mice.

Decrease in the allergenicity of Japanese cedar pollen allergen by treatment with positive and negative cluster ions.

BACKGROUND: Japanese cedar pollinosis is a severe allergic disease in Japan. The most effective means of decreasing allergic inflammation reactions is still avoidance of the aeroallergen. Recently, a novel air purification system using positively and negatively charged cluster ions was developed to create comfortable living environments. We aimed to assess the ability of existing technology to lower allergenicity of Japanese cedar pollen. METHODS: A Japanese cedar pollen extract was nebulized from the top of a cylindrical container with 2 or 4 ion-generating devices. The extract in a mist was passed through the space filled with or without plasma cluster ions for 90 s, and the ion-treated or nontreated extract was then collected in a Petri dish at the bottom of the container. RESULTS: The ion-exposed extract was significantly diminished in its reactivities to anti-Cry j 1 or anti-Cry j 2 antiserum and to human allergic sera IgE on ELISA. SDS-PAGE analysis revealed that ion exposure induced protein degradation in the pollen extract. Similarly, the ion treatment impaired about 80% of the binding to pooled sera IgE from patients allergic to Japanese cedar pollen on ELISA inhibition. Furthermore, intracutaneous and conjunctival reaction tests showed a remarkable diminution in the allergenicity of the ion-irradiated extract. CONCLUSION: Ion irradiation resulted in a remarkable decrease in in vitro and in vivo allergenicities of atomized Japanese cedar pollen extracts.

A new immunomodulatory function of low-viscous konjac glucomannan with a small particle size: its oral intake suppresses spontaneously occurring dermatitis in NC/Nga mice.

BACKGROUND: Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions: the higher viscosity displayed the stronger potency. However, the high-viscous GM powders, ordinary konjac powder and highly purified GM were mostly unsuitable for the application to various food industries. Our aims are to develop new physiological functions of low-viscous GM powder, pulverized GM or re-granulated fine GM, using a murine model of atopic dermatitis. METHODS: Male 4-week-old NC/Nga mice were fed for 8 weeks on diets containing 5% of two high-viscous and two low-viscous GM powders, respectively. RESULTS: Striking suppression against the aggravation of dermatitis, the increase in scratching behaviors, and the rise in IgE levels was recognized only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. Eczema prevention in the fine GM-fed mice was accompanied by a significant decrease in their plasma IFN-gamma levels, a positive regulatory cytokine for atopic skin inflammation. CONCLUSION: Only the pulverized GM possessed the ability to suppress the development of dermatitis in NC/Nga mice. This is the new immunomodulatory function of low-viscous GM with a small particle size.

Liver transplantation-induced antihistone H1 autoantibodies suppress mixed lymphocyte reaction.

BACKGROUND: In a rat model of orthotopic liver transplantation (OLT), recipient serum after OLT (post-OLT serum) has been reported to prevent allograft rejection. However, the molecular identities of immunosuppressive factors, which are in the early stage of post-OLT, remain elusive. This study was aimed to identify immunodominant suppressive factors present in early post-OLT serum. METHODS: The immunosuppressive activities of post-OLT serum, immunoglobulin (Ig) G-depleted serum, and purified IgG were evaluated in vitro by inhibition of the mixed lymphocyte reaction (MLR). Autoantigens recognized by the MLR-inhibitory IgG in early post-OLT serum were identified by the internal protein sequencing. RESULTS: Recipient post-OLT serum inhibited MLR, and its immunosuppressive activity vanished by means of the elimination of OLT-inducible IgG. IgG from post-OLT sera (2-3 weeks) specifically reacted to 31-, 34-, and 73-kDa autoantigens on splenic cells. The internal sequences of the doublet 31- and 34-kDa antigens coincided completely with those of histone H1 molecules. The levels of histone H1-specific antibodies were transiently increased to a plateau around 2 to 3 weeks after OLT but decreased in the later tolerogenic phase. Immunodepletion of antihistone H1 autoantibodies from early post-OLT serum abolished the MLR-inhibitory activity. Furthermore, rabbit polyclonal antibody-directed histone H1 not only suppressed MLR but also prolonged allograft survival. CONCLUSIONS: In this article, the authors provide evidence that autoreactive antibodies against histone H1, which are transiently induced at the early stage by liver transplantation, are a major OLT-induced graft survival factor.

Two-dimensional IgE-binding spectrum of Japanese cedar (Cryptomeria japonica) pollen allergens.

BACKGROUND: Japanese cedar (Cryptomeria japonica) pollen is one of the most prevalent sources of the allergens that elicit rhinitis and conjunctivitis. Only Cry j 1 and Cry j 2 have been well characterized as the major allergens of this pollen. OBJECTIVES: The aims of this study were to complete the repertoire of C. japonica pollen allergens, to investigate their variability with respect to IgE-reactive patterns and to identify the isoforms of Cry j 1 and Cry j 2 by proteome analysis. METHODS: Proteins in C. japonica pollen separated on two-dimensional (2-D) polyacrylamide gel electrophoresis were immunodetected with IgE in sera of 40 subjects allergic to C. japonica pollen. Mass fingerprinting was used to elucidate the diversity of the major allergens. RESULTS: 2-D immunolabeling with individual patients' sera showed the distinguishable IgE-binding patterns inlaid with 4-87 spots from a total of 131 IgE-binding protein spots. At least 12 Cry j 1 (27.5-75% of IgE-binding frequency) and 3 Cry j 2 (32.5-40%) isoforms were localized. In total, 31 spots were found to be more reactive than the highest IgE-reactive isoform of Cry j 2. CONCLUSIONS: The proteomics approaches showed great interindividual variation of IgE-binding patterns to C. japonica proteins and contributed to the repertoire of numerous C. japonica allergens other than Cry j 1 and Cry j 2. Protein microsequencing demonstrated more complicated multiplicity in Cry j 1 than previously known and new isoforms in Cry j 2.

The ability of konjac-glucomannan to suppress spontaneously occurring dermatitis in NC/Nga mice depends upon the particle size.

Konjac glucomannan (GM) is a well-known dietary fiber with various beneficial functions, which the higher viscous GM is proposed to have the stronger potencies. Application of highly viscous GM to various food industries is limited for the sake of long elapsed time required to solubilize the GM. Thus, the exploration into any physiological function of low-viscous GM remained undone for a long time. Male 4-week-old NC/Nga mice, a model of atopic dermatitis (AD), were fed for 8 weeks on diets containing 5% each of a high-viscous GM powder, ordinary konjac or highly purified GM, and a low-viscous GM powder, pulverized GM or re-granulated fine GM. Striking suppression to the aggravation of dermatitis and the increases in scratching behaviors and IgE levels was significantly observed only in mice fed on the pulverized GM diet, but not in mice fed on the other GM diets or a control diet. We concluded that the low-viscous GM possesses a novel immunoregulatory function and the intensity of the function depends upon the particle size rather than the viscosity.

Evidence of isozymes for delta6 fatty acid desaturase in rat hepatocytes.

The expression of delta6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, alpha-linolenic acid, and tetracosapentaenoic acid into the respective delta6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two delta6 desaturase isozymes in rat hepatocytes.

Production and properties of phytase and acid phosphatase from a sake koji mold, Aspergillus oryzae.

We identified three types of acid phosphatase (ACP-I, ACP-II, and ACP-III) produced by Aspergillus oryzae in a submerged culture using only phytic acid as the phosphorous substrate. The optimum pH for the activities of the three enzymes was in the range of 4.5 to 5.5. Analysis of the substrate specificities of these enzymes revealed that ACP-I and ACP-III were acid phosphatases, and ACP-II was a phytase. These enzymes were produced during different periods of mycelial growth: ACP-II was produced during the early phase of cultivation (around 24 h), and ACP-I was produced between 24 to 72 h. ACP-III was detected after the production of ACP-I and ACP-II had ceased. The release of phosphate from phytic acid was expected to be due to the cooperative hydrolysis of these enzymes.

Extracellular secretion of free fatty acids by disruption of a fatty acyl-CoA synthetase gene in Saccharomyces cerevisiae.

To elucidate the molecular mechanism governing fatty acid transport across the cell membrane, we first isolated a Saccharomyces cerevisiae mutant, B-1, that exhibits a reduced acyl-CoA oxidase activity and an increase in free fatty acid accumulation. Following mutagenesis of B-1, a mutant, YTS51, which secretes free fatty acids, was isolated. The concentration of free fatty acids in the YTS51 culture medium was about 17 times higher than that in B-1. The mutation that causes the fatty acid secretion phenotype occurred at a single allele, and this phenotype was suppressed by the introduction of a single copy of FAA1, a gene for acyl-CoA Synthetase, to the mutant. Although the mutation expressing this phenotype was not within FAA1 in YTS51, the disruption of FAA1 in the wild-type strain resulted in fatty acid secretion even though the level of fatty acid secretion was less than that in YTS51. We consider that YTS51 is a suitable model to elucidate the molecular basis of the fatty acid transport process.

Production of two types of phytase from Aspergillus oryzae during industrial koji making.

In our previous study, it was determined that phytase produced by Aspergillus oryzae plays an important role in supplying phosphate to yeast in the process of making sake. During koji making, two types of phytase (Phy-I and Phy-II) are produced. The purified phytases have high thermal and pH stability, in comparison to phytase purified from a submerged culture (ACP-II). In the present study, Phy-I and Phy-II retained their activities for 45 h. The NH2-terminal sequence of Phy-1, which is eight amino acids in length, was identical to that of ACP-II, but the molecular weights of these two forms, as estimated by SDS-PAGE, were quite different from each other (Phy-I, 120 kDa; ACP-II, 58 kDa). From the NH2-terminal amino acid sequence analysis of the predominant phytase (Phy-II), a molecular weight of 116 kDa was expected to reflect a new type of phytase produced only in koji culture. The substrate specificity of Phy-II was sufficiently broad that it hydrolyzed not only phytic acid and p-nitro phenyl phosphate, but also glucose 6-phosphate and glycerol 1-phosphate. In the process of making koji, Phy-I was produced at an early stage, followed by Phy-II; with both phytases being thought to function to hydrolyze phytic acid cooperatively.

Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes.

Seven strains of marine microbes producing a significant amount of docosahexaenoic acid (DHA; C22:6, n-3) were screened from seawater collected in coastal areas of Japan and Fiji. They accumulate their respective intermediate fatty acids in addition to DHA. There are 5 kinds of polyunsaturated fatty acid (PUFA) profiles which can be described as (1) DHA/docosapentaenoic acid (DPA; C22:5, n-6), (2) DHA/DPA/eicosapentaenoic acid (EPA; C20:5, n-3), (3) DHA/EPA, (4) DHA/DPA/EPA/arachidonic acid (AA; C20:4, n-6), and (5) DHA/DPA/EPA/AA/docosatetraenoic acid (C22:4, n-6). These isolates are proved to be new thraustochytrids by their specific insertion sequences in the 18S rRNA genes. The phylogenetic tree constructed by molecular analysis of 18S rRNA genes from the isolates and typical thraustochytrids shows that strains with the same PUFA profile form each monophyletic cluster. These results suggest that the C20-22 PUFA profile may be applicable as an effective characteristic for grouping thraustochytrids.

A high-molecular-weight mite antigen (HM1) fraction aggravates airway hyperresponsiveness of allergic mice to house dusts and whole mite cultures.

BACKGROUND: The house dust mite Dermatophagoides farinae is the most common aeroallergen causing human allergic asthma. Previously, we demonstrated that a high-molecular-weight allergenic fraction (HM1), which was abundant in D. farinae extracts, induced a proliferative response of T cells from healthy donors. The induction was mediated through the activation of macrophages without MHC class II restriction. In this study, we investigate whether HM1 influences the development of airway inflammation in murine models of asthma. METHODS: BALB/c mice were injected twice intraperitoneally with D. farinae fecal extract (Dff) at an interval of 5 days. They were exposed daily to aerosolized antigen (group 1: Dff, group 2: HM1, group 3: HM1-depleted Dff and group 4: PBS) for 10 days. The effect of HM1 on their airway inflammation was evaluated by measuring acetylcholine-induced airway hyperresponsiveness and inflammatory cell infiltration in lung tissue. RESULTS: The inhalation of the whole fecal extract or the HM1 fraction induced airway hyperresponsiveness which was detectable after 24 h and was maintained for as long as 120 h. The inhalation of extract depleted of the HM1 fraction induced hyperresponsiveness measured at 24 h but this was not maintained for 120 h. Macrophage infiltration was significantly prolonged in mice inhaling the whole extract and the HM1 fraction compared to the HM1-depleted extract. CONCLUSION: The inhalation of the high-molecular-weight HM1 fraction of D. farinae prolonged airway hyperresponsiveness and macrophage inflammation in a mouse model of hypersensitivity. The results indicate that the HM1 fraction which can induce T cell proliferation through macrophage activation may play a role in the duration of airway responsiveness.

Identification and expression of a rat fatty acid elongase involved in the biosynthesis of C18 fatty acids.

A major part of the palmitic acid (C16:0) generated by fatty acid synthase is converted into stearic acid (C18:0) via carbon chain elongation. Here, we describe the cloning and expression of a rat hepatic enzyme, rELO2, responsible for the elongation of C16:0, presumably at the condensing reaction. Heterologous expression experiments in a yeast, Saccharomyces cerevisiae, demonstrated the elongation activity of rELO2 on C16:0 and to a lesser extent, C18:0 and fatty acids with low desaturation degree. This was distinct from that rELO1, a rat homolog of HELO1, which preferably catalyzed the elongation of mono- and polyunsaturated fatty acids of C16-C20. The Northern analysis showed that the expression of rELO2, but not rELO1, in hepatocytes was activated by the cycles of fasting and refeeding rats on a fat-free diet. Under these conditions, the rELO1 was expressed constitutively in various tissues but the rELO2 transcripts were detected predominantly in liver.

Induction of a proliferative response of T cells by a high-molecular antigen in Dermatophagoides farinae feces.

BACKGROUND: We have previously demonstrated that high-molecular mite antigen (HM1) from Dermatophagoides farinae feces is an allergen which binds to mite-allergic patients IgE. HM1 also induced a proliferative response in lymph node cells from mite-immunized mice as well as nonimmunized mice. In the present study, we demonstrated that HM1 induced T cell proliferation and investigated the HM1-stimulated T cell proliferative pathways using nonallergic human peripheral blood mononuclear cells (PMBC). METHODS: Blood samples were obtained from 10 healthy donors. Using primary culture, T cell response stimulated with HM1 was performed on purified T cells, CD19+ cell-depleted PBMC and CD11b+ cell-depleted PBMC. In addition, interleukin (IL)-5 and interferon (IFN)-gamma produced by mite-allergic and healthy donors stimulated with HM1 were estimated by enzyme immunoassay. RESULTS: T cell proliferation was detected only in CD19+ cell-depleted PBMC. When T cells were cocultured with CD11b+ cells they recovered their proliferative response to HM1. In addition, the pathway of HM1-stimulated T cell proliferation did not involve HLA class II restriction. Both activated CD11b+ cells and their conditioned media were needed to induce HM1-stimulated T cell proliferation. Furthermore, HM1 induced IFN-gamma production in both healthy and allergic donors. CONCLUSION: The high-molecular mite antigen, HM1, induced a proliferative response of T cells in healthy as well as allergic donors, without HLA class II restriction. Our results suggest that further investigation of HM1 could constitute a valuable avenue of research into complex allergic diseases.

Der f 16: a novel gelsolin-related molecule identified as an allergen from the house dust mite, Dermatophagoides farinae.

Allergen from the house dust mite (Dermatophagoides sp.) is a major trigger factor of allergic disorders, and its characterization is crucial for the development of specific diagnosis or immunotherapy. Here we report the identification of a novel dust mite (Dermatophagoides farinae) antigen whose primary structure belongs to the gelsolin family, a group of actin cytoskeleton-regulatory proteins. Isolated mite cDNA, termed Der f 16, encodes 480 amino acids comprising a four-repeated gelsolin-like segmental structure, which is not seen in conventional gelsolin family members. Enzyme immunoassay indicated that recombinant Der f 16 protein, prepared using an Escherichia coli expression system, bound IgE from mite-allergic patients at 47% (8/17) frequency. This is the first evidence that the gelsolin family represents a new class of allergen recognizable by atopic patient IgE.

Toward elucidating the full spectrum of mite allergens--state of the art.

Our research has focused on the molecular design of immunotherapeutic vaccines and the advancement of mite-allergy diagnosis. Here, we describe the research history of the major group 1 and group 2 allergens, immunoelectrophoretic analyses covering the complete spectrum of mite allergens, our results on allergens with distinctive characteristics (a conjunctival congestion-eliciting antigen [LM2], an immunotherapeutic antigen [HM2] with high efficacy and without definite adverse reactions, and a potent T-cell stimulatory antigen [HM1] with secretion of IFN-gamma), the full spectrum and immunochemical properties of the major and other important mite allergens (including our newly described allergens: a pan-allergen [tropomyosin, group 10], a potent T-cell stimulatory allergen [M-177, apolipophorin, group 14] and its peptide fragments Mag1 and Mag3, a moderate IgE-binding allergen [gelsolin/villin, group 16], an EF-hand Ca2+-binding allergen [group 17], and a less IgE-binding allergen [heat shock protein 70]), and prospects for the development of immunotherapeutic and diagnostic agents.

Purification and characterization of a novel fungal alpha-glucosidase from Mortierella alliacea with high starch-hydrolytic activity.

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable alpha-amylase activity. Here, a alpha-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal alpha-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55 degrees C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-alpha-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50 degrees C.