Safety and efficacy of short-term oral immunotherapy with Cry j 1-galactomannan conjugate for Japanese cedar pollinosis: a randomized controlled trial.

Current allergen-specific immunotherapy (AIT) for pollinosis requires long-term treatment with potentially severe side effects. Therefore, development of an AIT that is safe and more convenient with a shorter regimen is needed. This prospective, double-blind, placebo-controlled trial randomized 55 participants with Japanese cedar pollinosis (JCP) to active or placebo groups to test the safety and efficacy of short-term oral immunotherapy (OIT) with Cry j 1-galactomannan conjugate for JCP. Mean symptom-medication score as the primary outcome in the active group improved 27.8% relative to the placebo group during the entire pollen season. As the secondary outcomes, mean medication score in active group improved significantly, by 56.2%, compared with placebo during the entire pollen season. Mean total symptom score was similar between active and placebo groups during the entire pollen season. There were no severe treatment-emergent adverse events in the active and placebo groups. Therefore short-term OIT with Cry j 1-galactomannan conjugate is safe, and effective for reducing the amount of medication use for JCP.

Effect of short-term oral immunotherapy with Cry j1-galactomannan conjugate on quality of life in Japanese cedar pollinosis patients: A prospective, randomized, open-label study.

OBJECTIVE: We have recently reported that a new regimen of short-term oral immunotherapy (OIT) with the Cry j1-galactomannan conjugate for Japanese cedar pollinosis (JCP) is effective to the improvement in the symptoms and medication use during the pollen season and relatively safe. The effect of OIT on quality of life (QOL) of JCP patients has not been assessed. Therefore, we evaluated for the first time the effect of OIT on QOL during the Japanese cedar/cypress pollen season. METHODS: A prospective, randomized, open-label trial was conducted over a period of 4 months. Participants were randomly divided into two groups. The OIT and control groups comprised 23 and 24 subjects, respectively. The build-up phase was initiated 1 month before the expected pollen season. The maintenance phase was continued for 51 days during the peak of the cedar pollen season. The QOL score in the Japan Rhinoconjunctivitis Quality of Life Questionnaire (JRQLQ) No. 1 and visual analog scale (VAS) throughout the pollen season were evaluated. RESULTS: Participants receiving OIT showed significant improvements in the total QOL score and VAS throughout the pollen season compared with the control group. In addition, the mean total QOL score and VAS correlated in both groups during the pollen season. CONCLUSION: The new regimen of short-term OIT using the Cry j1-galactomannan conjugate results in meaningful improvements in QOL of JCP patients. Our findings suggest that short-term OIT using allergen-galactomannan conjugates, as well as sublingual and subcutaneous immunotherapy, improves QOL of patients with pollinosis. The study was registered in UMIN-CTR (UMIN000013408) as the name of "a prospective, randomized, open study of oral Cry j1-galactomannan conjugate immunotherapy for Japanese cedar pollen allergy".

Safety and efficacy of a new regimen of short-term oral immunotherapy with Cry j 1-galactomannan conjugate for Japanese cedar pollinosis: a prospective, randomized, open-label study.

BACKGROUND: Short-term oral immunotherapy (OIT) using the Cry j1-galactomannan conjugate for Japanese cedar pollinosis may be effective and relatively safe. However, a treatment regimen has not been established. In the present study, we examined a new OIT regimen with a build-up phase and extended the maintenance phase of OIT to the peak period of the pollen season to enhance the therapeutic effect and safety of OIT. METHODS: A prospective, randomized, open-label trial was conducted over a period of 4 months. Participants were randomly divided into two groups. The OIT group comprised 23 subjects. The build-up phase was initiated 1 month before the expected pollen season. The maintenance phase was continued for 51 days during the peak pollen season. The control group comprised 24 subjects. The symptoms and medication score, levels of allergen-specific serum antibodies throughout the pollen season, and adverse effects with OIT were evaluated. RESULTS: Participants receiving OIT showed significant improvements in total symptom scores, medication score, and total symptom-medication scores throughout the pollen season compared with the control group. The levels of allergen-specific serum IgG4 were significantly increased in the OIT group but not in the control group throughout the cedar pollen season. Importantly, no severe adverse effects were observed with OIT. CONCLUSIONS: The new regimen of short-term OIT using the Cry j1-galactomannan conjugate for Japanese cedar pollinosis is effective, relatively safe and induces immune tolerance. Thus, OIT using allergen-galactomannan conjugates may provide a rapid, effective, and thus convenient immunotherapy for pollinosis instead of SLIT or SCIT.

(-)-epigallocatechin-3-gallate inhibits fibrillogenesis of chicken cystatin.

Previous studies have reported that (-)-epigallocatechin-3-gallate (EGCG), the most abundant flavonoid in green tea, can bind to unfolded native polypeptides and prevent conversion to amyloid fibrils. To elucidate whether this antifibril activity is specific to disease-related target proteins or is more generic, we investigated the ability of EGCG to inhibit amyloid fibril formation of amyloidogenic mutant chicken cystatin I66Q, a generic amyloid-forming model protein that undergoes fibril formation through a domain swapping mechanism. We demonstrated that EGCG was a potent inhibitor of amyloidogenic cystatin I66Q amyloid fibril formation in vitro. Computational analysis suggested that EGCG prevented amyloidogenic cystatin fibril formation by stabilizing the molecule in its native-like state as opposed to redirecting aggregation toward disordered and amorphous aggregates. Therefore, although EGCG appears to be a generic inhibitor of amyloid-fibril formation, the mechanism by which it achieves such inhibition may be specific to the target fibril-forming polypeptide.

Unstable mutant lysozymes are degraded through the interaction with calnexin homolog Cne1p in Saccharomyces cerevisiae.

Cne1p is a yeast homolog of calnexin, which is a constituent of endoplasmic reticulum (ER)-associated protein quality control system in mammals. Cne1p may be involved in the degradation of misfolded lysozymes in Saccharomyces cerevisiae. To test this, c-Myc-tagged lysozymes were expressed in CNE1-deficient S. cerevisiae. The expression and secretion of an unstable lysozyme mutant G49N/D66H were enhanced and its intracellular localization was changed in the CNE1-deficient strain. Furthermore, when Cne1p was co-expressed with unstable lysozyme mutants (G49N/D66H, G49N/C76A, and K13D/G49N), its affinity to the misfolded mutant proteins was revealed by co-immunoprecipitation. The interaction with Cne1p was abrogated by the addition of tunicamycin, an inhibitor of N-glycosylation, indicating that N-linked carbohydrates might be necessary for protein binding to Cne1p. These results suggest that in yeasts, Cne1p interacts with misfolded lysozyme proteins possibly causing their retention in the ER and subsequent elimination via ER-associated degradation.

Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33.

A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of culture medium. The chitinase was purified by column chromatography after Endoglycosidase H treatment and then characterized. It showed properties similar to the original chitinase E purified from the yam tuber reported by Arakane et al. (2000). This Pichia-produced chitinase also showed strong lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability, optimum activity at higher temperature such as 70 °C, and high substrate affinity, indicating that one can use this Pichia-produced yam chitinase as a bio-control agent.

Phase I/II study of oral immunotherapy with Cry j1-galactomannan conjugate for Japanese cedar pollinosis.

OBJECTIVE: Among many immunotherapeutic approaches, oral immunotherapy (OIT) is thought to be an effective route for desensitization against a variety of allergens. However, there is little evidence that OIT is effective for airway allergic diseases such as pollen allergy. Thus, in the present study, we assessed the safety, efficacy and immune response of OIT using the Cry j1-galactomannan conjugate for Japanese cedar pollen allergy. METHODS: An open trial was conducted over a period of 4 months. The OIT group comprised of 23 subjects. Treatment was initiated 1 month before the estimated pollen season and continued for 1 month. The control group (the pharmacological treatment group without OIT) comprised of 11 subjects. The symptoms and medication score, levels of allergen-specific serum antibodies, cellular components of lymphocytes and cytokine production from peripheral blood mononuclear cells (PBMCs) were evaluated throughout the pollen season. RESULTS: The participants receiving OIT treatment showed significant improvements in total symptom scores and symptom-medication scores during the pollen season compared with the control group. The levels of allergen-specific serum IgG4 and IL-10 production in PBMCs were significantly increased in the OIT group compared with that in the control group. Importantly, no severe adverse effects were observed in the participants receiving OIT treatment. CONCLUSION: Short-term OIT using the Cry j1-galactomannan conjugate is effective, relatively safe and induces tolerant immune responses such as increased allergen-specific serum IgG4 and IL-10 production in PBMCs. These results suggest that OIT using allergen-galactomannan conjugates may provide a rapid, effective, and safe immunotherapy regimen for cedar pollen allergy.

Haploid plants carrying a sodium azide-induced mutation (fdr1) produce fertile pollen grains due to first division restitution (FDR) in maize (Zea mays L.).

KEY MESSAGE: We induced a fdr1 mutation in maize which makes haploid plants male fertile due to first division restitution; the optimum sodium azide treatment on maize kernels has been identified. Sodium azide mutagenesis experiments were performed on haploid and diploid maize plants. Kernels with haploid embryos of maize inbred line B55 were induced by pollinating with RWS pollen. These kernels were treated with 0.2, 0.5, or 1.0 mM sodium azide solution for 2 h. The 0.5 mM solution was optimal for inducing numerous albino sectors on the treated plants without significant damage. Kernels of a maize hybrid, Oh43 × B55, were treated with sodium azide solutions at concentrations of 1.5, 2.0, 2.5, and 3.0 mM. Haploids were generated by pollinating RWS pollen. The highest rate of chlorophyll mutations in seedlings (15.3 % [13/85]) was recorded with the 2.5 mM concentration. A mutated haploid plant (PP1-50) with higher pollen fertility was isolated during the experiments. This haploid plant produced four kernels on the ear after selfing. These kernels were germinated and produced ears with full seed set after selfing. The haploid plants induced from PP1-50 diploids also exhibited high pollen fertility. In situ hybridization studies showed that meiocytes in PP1-50 haploid anthers underwent first division restitution at a rate of 48 % and produced equally divided dyads. We designated the genetic factor responsible for this high pollen fertility as fdr1. PP1-50 haploid ears exhibited high levels of sterility, as seen for regular haploids. Diploid PP1-50 meiocytes in the anther underwent normal meiosis, and all selfed progenies were normal diploids. We concluded that the fdr1 phenotype is only expressed in the anthers of haploid plants and not in the anthers of diploid plants.

The periodontopathogenic bacterium Eikenella corrodens produces an autoinducer-2-inactivating enzyme.

Eikenella corrodens produces autoinducer-2 (AI-2) in the mid log phase, and AI-2 activity decreases dramatically during the stationary phase. We investigated the mechanism underlying this decrease in AI-2 activity. To analyze the mechanism, we extracted and purified AI-2 from the supernatant of mid-log-phase culture. Simultaneously, the stationary-phase culture supernatant was fractionated by ammonium sulfate precipitation. On incubating purified AI-2 and 4-hydroxy-5-methyl-3(2H)-furanone (MHF) with each fraction, the 30% fraction decreased both AI-2 and MHF activities. The data suggest that AI-2 and MHF were rendered inactive in the same manner. Heat and/or trypsin treatment of the 30% fraction did not completely arrest AI-2 inactivation, suggesting that partially heat-stable proteins are involved in AI-2 inactivation. We observed that an enzyme converted MHF to another form. This suggests that E. corrodens produces an AI-2 inactivating enzyme, and that AI-2 can be degraded or modified by it.

Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure.

Wheat (Triticum aestivum L.) is an allohexaploid, in which each of the three genomes has a high 1C content. This indicates the presence of multiple tandemly repeated sequences, which should be detectable using in situ hybridization. Some repeats have already been described, but others remain to be recognized. To discover others, 2000 plasmid wheat clones were examined for signal presence after fluorescence in situ hybridization and microscopic signal observation. Among them, 47 clones produced strong discrete signals on wheat chromosomes. Two of the newly identified clones (pTa-535 and pTa-713) were determined to have especially valuable sequences for chromosome identification. In combination with pTa-86 (the pSc119 homologous sequence), these probes enable unambiguous discrimination of all wheat chromosomes including orientation. Four newly identified sequences (pTa-465, pTa-k566, pTa-s120, and pTa-s126) were useful in that they produced discrete signals on various wheat chromosome arms. Two other clones (pTa-k288 and pTa-k229) produced GISH-like (genomic in situ hybridization) signals because they allowed the A, B, and D genomes to be distinguished simultaneously. In addition, centromere, centromere-related, and ribosomal DNA clones were identified. Also described are improvements on slide preparation and reprobing procedures. To enhance discrete signal detection, a new direct fluorescent-labeling procedure, namely the VentR (exo-) terminal extension method, was employed.

LuxS affects biofilm maturation and detachment of the periodontopathogenic bacterium Eikenella corrodens.

Previously, we reported that biofilm formation of Eikenella corrodens is regulated by autoinducer-2 (AI-2), based on observations that biofilm-forming efficiency of ΔluxS mutant was greater than that of the wild type (Azakami et al., J. Biosci. Bioeng., 102, 110-117, 2006). To determine whether the AI-2 molecule affects biofilm formation directly, we added purified AI-2 to luxS mutant and wild-type E. corrodens and compared biofilm formations by using a static assay. Results indicated that biofilm formation in E. corrodens was enhanced by the addition of AI-2. We also compared the biofilms formed by flow cell system for the luxS mutant and the wild type by using scanning electron microscopy and confocal laser scanning microscopy. The number of viable bacteria in the luxS mutant biofilm was dramatically reduced and more sparsely distributed than that of the wild type, which suggested that AI-2 might enhance the mature biofilm. Conversely, further analysis by modified confocal reflection microscopy indicated that the wild-type biofilm was matured earlier than that of the luxS mutant, and became thinner and more sparsely distributed with time. These data suggest that LuxS may facilitate the maturation and detachment of biofilm in E. corrodens.

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant.

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71-Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71-Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71-Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95-Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α-ß interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23.

Genomic recombination through plasmid-encoded recombinase enhances hemolytic activity and adherence to epithelial cells in the periodontopathogenic bacterium Eikenella corrodens.

The periodontopathogenic bacterium Eikenella corrodens has an N-acetyl-D-galactosamine (GalNAc)-specific lectin, that contributes significantly to the pathogenicity of the bacterium. Recently, we reported that plasmid-mediated genomic recombination enhances the activity of this lectin. In this study, we investigated the effects of genomic recombination on certain virulence factors. Introduction of the recombinase gene resulted in hemolysis and significantly increased bacterial adhesion to epithelial cells. It was suggested that the enhanced adhesion was attributable to increased lectin activity due to genomic recombination, because it was inhibited by the addition of GalNAc. In contrast, invasion of the epithelial cells was remarkably reduced by genomic recombination. Although we assumed that this decrease in invasion resulted from a loss of type-IV pili, the phase variant did not show any decrease in invasion activity. This suggests that type-IV pili do not contribute to the invasive ability of E. corrodens. Our results suggest that genomic recombination enhances the pathogenicity of E. corrodens.

High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures.

The barley (Hordeum vulgare L.) genome was screened to identify sequences that could be used for fluorescence in situ hybridization (FISH). From 2000 transformed bacterium colonies carrying barley clones, 56 colonies were selected on the basis of the patterns that their PCR products produced when subjected to agarose gel electrophoresis. Among them, 42 (75%) exhibited fluorescent signals on barley chromosomes after in situ hybridization using the directly labeled PCR products. Sequencing revealed seven clones, pHv-365, pHv-177, pHv-1112, pHv-689, pHv-1476, pHv-1889, and pHv-1972, to be newly identified FISH-positive sequences. The remainder possess previously described sequences such as 5S, GAA microsatellite, centromere repeats, HVT01, and pHvMWG2315 (324 bp repeat). It is shown here that a combination of five probes, which produce strong signals on barley chromosomes, pHv-38 (5S), pHv-365, pHv-961 (HVT01), GAA, and TAG microsatellites, offer unequivocal recognition of each chromosome. The combination of three probes, i.e., pHv-1123 (barley 324 bp repeat), GAA, and TAG, decorated entire chromosomes with fine dotted signals and was useful for detecting the break points of aberrant chromosomes. The signals' distributions of pHv-177, pHv-1112, and TAG were highly polymorphic. An improved reprobing procedure and its usefulness are also discussed.

Phenotypic and gene expression analyses of a ploidy series of maize inbred Oh43.

Polyploidization has repeatedly occurred during plant evolution. Although autopolyploidy is the best model to characterize the polyploidization effects in a highly controlled manner, there are limited studies on autopolyploids compared to allopolyploids. To improve our understanding of autopolyploidy effects in maize, we developed an inbred Oh43 ploidy series consisting of the diploid (2X), tetraploid (4X) and hexaploid (6X) lines and compared their phenotypes and gene expression in the mature adult leaf tissue. Our phenotypic study showed that plants of higher ploidy exhibit increased cell size but slower growth rate, later flowering, fewer tassel branches, reduced stature and fertility. Two-dimensional difference gel electrophoresis (2D DIGE) and gel electrophoresis followed by liquid chromatography and mass spectrometry (GeLC-MS) assays of the leaf proteomes revealed ~40 and 26% quantitative differentially expressed (DE) proteins, respectively, at the per genome level. A small number of qualitative DE proteins were also identified in the GeLC-MS assay. The majority of the quantitative DE proteins found in the 2D DIGE assay were present in either the 4X versus 6X or the 2X versus 6X comparison but not the 2X versus 4X comparison. Aneuploidy in some 6X plants might contribute to the more extensive changes of gene expression per genome in the 6X. Most changes of the protein expression per genome are less than twofold. Less than 5% of the DE genes exhibit a positive or negative continuous correlation through the ploidy series between their protein expression per genome, and the genome copy number. Hence, in the Oh43 ploidy series, expression for most proteins in a cell increases linearly with ploidy.

Chromosome painting for plant biotechnology.

Fluorescence in situ hybridization (FISH) is an invaluable tool for chromosome analysis and engineering. The ability to visually localize endogenous genes, transposable elements, transgenes, naturally occurring organellar DNA insertions - essentially any unique sequence larger than 2 kb - greatly facilitates progress. This chapter details the labeling procedures and chromosome preparation techniques used to produce high-quality FISH signals on somatic metaphase and meiotic pachytene spreads.