The 1st step initiation essential for allergen-specific IgE antibody production upon the 2nd step: Induction of non-specific IgE+ small B cells containing secondly-sensitized allergen-specific ones in mice firstly-sensitized with an allergen.

There was a significant amount of non-specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)-specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain-, but not mite feces- or pollen-specific IgE+ cells and an i.n. injection of papain induced papain-specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces-specific IgE+ cells in the lymph nodes nor mite feces-specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen-specific IgE+ small B cells in the lymph nodes on Day 10, when non-specific IgE Ab titers reached a peak in the serum, implying induction of related allergen-specific IgE+ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen-specific IgE Abs in the serum. These results indicate that when firstly-sensitized non-specific IgE+ small B cells in mouse lymph nodes include some secondly-sensitized allergen-specific ones, mice produce IgE Abs specific for the secondly-injected allergen.

Biofortified indica rice attains iron and zinc nutrition dietary targets in the field.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 µg g(-1) iron (Fe) and 16 µg g(-1) zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 µg g(-1) Fe and 28 µg g(-1) Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 µg g(-1) in glasshouse settings; in contrast, under field conditions, 4 µg g(-1) was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 µg g(-1) Fe and 45.7 µg g(-1) Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Ion-beam irradiation, gene identification, and marker-assisted breeding in the development of low-cadmium rice.

Rice (Oryza sativa L.) grain is a major dietary source of cadmium (Cd), which is toxic to humans, but no practical technique exists to substantially reduce Cd contamination. Carbon ion-beam irradiation produced three rice mutants with <0.05 mg Cd⋅kg(-1) in the grain compared with a mean of 1.73 mg Cd⋅kg(-1) in the parent, Koshihikari. We identified the gene responsible for reduced Cd uptake and developed a strategy for marker-assisted selection of low-Cd cultivars. Sequence analysis revealed that these mutants have different mutations of the same gene (OsNRAMP5), which encodes a natural resistance-associated macrophage protein. Functional analysis revealed that the defective transporter protein encoded by the mutant osnramp5 greatly decreases Cd uptake by roots, resulting in decreased Cd in the straw and grain. In addition, we developed DNA markers to facilitate marker-assisted selection of cultivars carrying osnramp5. When grown in Cd-contaminated paddy fields, the mutants have nearly undetectable Cd in their grains and exhibit no agriculturally or economically adverse traits. Because mutants produced by ion-beam radiation are not transgenic plants, they are likely to be accepted by consumers and thus represent a practical choice for rice production worldwide.

Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition.

To address the problem of iron-deficiency anemia, one of the most prevalent human micronutrient deficiencies globally, iron-biofortified rice was produced using three transgenic approaches: by enhancing iron storage in grains via expression of the iron storage protein ferritin using endosperm-specific promoters, enhancing iron translocation through overproduction of the natural metal chelator nicotianamine, and enhancing iron flux into the endosperm by means of iron(II)-nicotianamine transporter OsYSL2 expression under the control of an endosperm-specific promoter and sucrose transporter promoter. Our results indicate that the iron concentration in greenhouse-grown T(2) polished seeds was sixfold higher and that in paddy field-grown T(3) polished seeds was 4.4-fold higher than that in non-transgenic seeds, with no defect in yield. Moreover, the transgenic seeds accumulated zinc up to 1.6-times in the field. Our results demonstrate that introduction of multiple iron homeostasis genes is more effective for iron biofortification than the single introduction of individual genes.

Essential role of macrophages in the initiation of allergic rhinitis in mice sensitized intranasally once with cedar pollen: regulation of class switching of immunoglobulin in B cells by controlling interleukin-4 production in T cells of submandibular lymph nodes.

The production of allergen-specific IgE antibodies (Abs) in allergen-sensitized patients or animals has a mutual relationship with the immunologic response leading to allergic rhinitis. We recently reported that, after an intranasal injection of cedar pollen into mice, an interleukin-4 (IL-4)-dependent increase in serum nonspecific IgE Abs was a prerequisite for the production of serum allergen-specific IgE Abs. Here, we explored which lymphoid organs were responsive to the intranasally injected allergen and how IL-4 and IgE Abs were produced in the lymphocytes. Time-dependent changes in the total cell numbers and in in vitro IgE Ab production in various lymphoid organs revealed that the submandibular lymph nodes were the main responsible organ. After treatment with allergen (for IgE production) or allergen and complete Freund's adjuvant (for IgG production), we separated submandibular lymph node cells into macrophage-, lymphocyte-, and granulocyte-rich populations by discontinuous Percoll density-gradient centrifugation. Unexpectedly, bulk cells, but not the lymphocyte- or macrophage-rich populations, produced significant amounts of IL-4, IgE, and IgG; whereas production was restored by addition of Mac-1(+) cells from the macrophage-rich to the lymphocyte-rich fraction. Furthermore, a combination of the lymphocyte-rich population (for IgG [or IgE]) production) and the macrophage-rich population (for IgE [or IgG]) production) produced a large amount of IgE (or IgG). These results indicate that, in the initiation of allergic rhinitis, macrophages in the submandibular lymph nodes are essential not only for IL-4 or immunoglobulin production, but also for class switching of immunoglobulin in lymphocytes.

Absence of nasal blockage in a Japanese cedar pollen-induced allergic rhinitis model mouse.

BACKGROUND: Japanese cedar pollen-induced allergic rhinitis in a guinea pig model clearly induced not only sneezing but also biphasic nasal blockage. To date, there have only been a few reports on models of murine allergic rhinitis which clearly show nasal blockage. Therefore, in order to try and develop such a model, we administered multiple dosages of intranasal pollen or purified antigen protein Cry j 1. METHODS: B10.S mice were sensitized by intranasal instillations of either pollen extract or Cry j 1 twice a day for 7 days, which was adsorbed on Al(OH)(3). Subsequently, once a week, the mice were given multiple intranasal instillation challenges of either the pollen suspension or Cry j 1 and the frequency of sneezing was observed after respective challenges were made. Specific airway resistance (sRaw) was measured as an indicator for nasal blockage. Cry j 1-specific IgE levels were measured using an enzyme-linked immunosorbent assay. RESULTS: The serum Cry j 1-specific IgE level showed clear elevation only in the group sensitized by Cry j 1 + Al(OH)(3) and then challenged by Cry j 1. No elevations were seen in the groups sensitized by pollen extract + Al(OH)(3) followed by a pollen suspension challenge. There was an immediate increase in sneezing after challenges in all of the sensitized-challenged groups. Nevertheless, no increases in sRaw in any of the groups were detected at any of the time points during the 8 hours following the challenges. CONCLUSIONS: Cry j 1 may be more effective than crude antigens for efficient sensitization/challenge in mice. No increase in sRaw occurred, even in mice that possessed high amounts of Cry j 1-specific IgE and that exhibited sneezing.

IL-4-dependent induction of IgE basophils in peripheral blood and IgE B cells in spleen as respective indicators of allergen sensitization and a precursor of cells secreting allergen-specific IgE antibody.

It was recently reported by us that either primary i.n. or i.p. injection of cedar pollen extract into BALB/c mice, or a second s.c. injection of the allergen into i.v. or s.c. sensitized mice, causes an IL-4-dependent increase in total IgE serum antibody to produce allergen-specific IgE antibody upon further s.c. sensitization. To determine the biology of total IgE antibody, in the present study IgE+ cells in peripheral blood or lymphoid tissues of allergen-sensitized BALB/c mice have been characterized. In peripheral blood, mice sensitized one to three times with the allergen produced a 2.5- to 4-fold increase in the number of IgE+ cells, with a time-course similar to that of the concentration of total IgE antibody in serum. These IgE+ cells were basophils. On the other hand, the number of IgE+ cells in the lymphoid tissues did not change significantly after an i.n., i.p., i.v. or s.c. injection of allergen into the mice, whereas a second s.c. injection of the allergen into the i.v.-, but not into the i.n.-, i.p.- or s.c.-, sensitized mice induced a small number of IgE+/IgM+/B220+ B cells in the spleen. In contrast, IgE+ cells were not seen in the blood or spleen of IL-4 -/- mice after sensitization with the allergen. These results suggest that IgE+ basophils in the peripheral blood, and IgE+ B cells in the spleen, might be IL-4-dependently induced as an indicator of sensitization with allergen, and a precursor of cells secreting allergen-specific IgE antibody, respectively.

The transcription factor IDEF1 regulates the response to and tolerance of iron deficiency in plants.

Iron is essential for most living organisms and is often the major limiting nutrient for normal growth. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified the rice transcription factor IDEF1, which specifically binds the iron deficiency-responsive cis-acting element IDE1. IDEF1 belongs to an uncharacterized branch of the plant-specific transcription factor family ABI3/VP1 and exhibits the sequence recognition property of efficiently binding to the CATGC sequence within IDE1. IDEF1 transcripts are constitutively present in rice roots and leaves. Transgenic tobacco plants expressing IDEF1 under the control of the constitutive cauliflower mosaic virus 35S promoter transactivate IDE1-mediated expression only in iron-deficient roots. Transgenic rice plants expressing an introduced IDEF1 exhibit substantial tolerance to iron deficiency in both hydroponic culture and calcareous soil. IDEF1 overexpression leads to the enhanced expression of the iron deficiency-induced transcription factor gene OsIRO2, suggesting the presence of a sequential gene regulatory network. These findings reveal cis element/trans factor interactions that are functionally linked to the iron deficiency response. Manipulation of IDEF1 also provides another approach for producing crops tolerant of iron deficiency to enhance food and biomass production in calcareous soils.

Expression and enzyme activity of glutathione reductase is upregulated by Fe-deficiency in graminaceous plants.

Glutathione reductase (GR) plays an important role in the response to biotic and abiotic stresses in plants. We studied the expression patterns and enzyme activities of GR in graminaceous plants under Fe-sufficient and Fe-deficient conditions by isolating cDNA clones for chloroplastic GR (HvGR1) and cytosolic GR (HvGR2) from barley. We found that the sequences of GR1 and GR2 were highly conserved in graminaceous plants. Based on their nucleotide sequences, HvGR1 and HvGR2 were predicted to encode polypeptides of 550 and 497 amino acids, respectively. Both proteins showed in vitro GR activity, and the specific activity for HvGR1 was 3-fold that of HvGR2. Northern blot analyses were performed to examine the expression patterns of GR1 and GR2 in rice (Os), wheat (Ta), barley (Hv), and maize (Zm). HvGR1, HvGR2, and TaGR2 were upregulated in response to Fe-deficiency. Moreover, HvGR1 and TaGR1 were mainly expressed in shoot tissues, whereas HvGR2 and TaGR2 were primarily observed in root tissues. The GR activity increased in roots of barley, wheat, and maize and shoot tissues of rice, barley, and maize in response to Fe-deficiency. Furthermore, it appeared that GR was not post-transcriptionally regulated, at least in rice, wheat, and barley. These results suggest that GR may play a role in the Fe-deficiency response in graminaceous plants.

OsZIP4, a novel zinc-regulated zinc transporter in rice.

Zinc (Zn) is an essential element for the normal growth of plants but information is scarce on the mechanisms whereby Zn is transported in rice (Oryza sativa L.) plants. Four distinct genes, OsZIP4, OsZIP5, OsZIP6, and OsZIP7 that exhibit sequence similarity to the rice ferrous ion transporter, OsIRT1, were isolated. Microarray and northern blot analysis revealed that OsZIP4 was highly expressed under conditions of Zn deficiency in roots and shoots. Real-time-PCR revealed that the OsZIP4 transcripts were more abundant than those of OsZIP1 or OsZIP3 in Zn-deficient roots and shoots. OsZIP4 complemented a Zn-uptake-deficient yeast (Saccharomyces cerevisiae) mutant, Deltazrt1,Deltazrt2, indicating that OsZIP4 is a functional transporter of Zn. OsZIP4-synthetic green fluorescent protein (sGFP) fusion protein was transiently expressed in onion epidermal cells localized to the plasma membrane. In situ hybridization analysis revealed that OsZIP4 in Zn-deficient rice was expressed in shoots and roots, especially in phloem cells. Furthermore, OsZIP4 transcripts were detected in the meristem of Zn-deficient roots and shoots. These results suggested that OsZIP4 is a Zn transporter that may be responsible for the translocation of Zn within rice plants.

Three nicotianamine synthase genes isolated from maize are differentially regulated by iron nutritional status.

Nicotianamine synthase (NAS) is an enzyme that is critical for the biosynthesis of the mugineic acid family of phytosiderophores in graminaceous plants, and for the homeostasis of metal ions in nongraminaceous plants. We isolated one genomic NAS clone, ZmNAS3, and two cDNA NAS clones, ZmNAS1 and ZmNAS2, from maize (Zea mays cv Alice). In agreement with the increased secretion of phytosiderophores with Fe deficiency, ZmNAS1 and ZmNAS2 were positively expressed only in Fe-deficient roots. In contrast, ZmNAS3 was expressed under Fe-sufficient conditions, and was negatively regulated by Fe deficiency. This is the first report describing down-regulation of NAS gene expression in response to Fe deficiency in plants, shedding light on the role of nicotianamine in graminaceous plants, other than as a precursor in phytosiderophore production. ZmNAS1-green fluorescent protein (sGFP) and ZmNAS2-sGFP were localized at spots in the cytoplasm of onion (Allium cepa) epidermal cells, whereas ZmNAS3-sGFP was distributed throughout the cytoplasm of these cells. ZmNAS1 and ZmNAS3 showed NAS activity in vitro, whereas ZmNAS2 showed none. Due to its duplicated structure, ZmNAS2 was much larger (65.8 kD) than ZmNAS1, ZmNAS3, and previously characterized NAS proteins (30-38 kD) from other plant species. We reveal that maize has two types of NAS proteins based on their expression pattern and subcellular localization.

Cloning an iron-regulated metal transporter from rice.

Rice cDNA and genomic libraries were screened in order to clone an Fe(II) transporter gene. A cDNA clone highly homologous to the Arabidopsis Fe(II) transporter gene IRT1 was isolated from Fe-deficient rice roots. The cDNA clone was named OsIRT1. A genomic clone corresponding to the cDNA was also obtained, sequenced and analysed. When expressed in yeast cells, OsIRT1 cDNA reversed the growth defects of the yeast iron-uptake mutant. Northern blot analysis revealed that OsIRT1 mRNA was predominantly expressed in roots and was induced by Fe- and Cu-deficiency. This suggests that OsIRT1 is a functional metal transporter for iron, and is actively engaged in Fe uptake from soils, especially under limiting conditions.

cDNA microarray analysis of gene expression during Fe-deficiency stress in barley suggests that polar transport of vesicles is implicated in phytosiderophore secretion in Fe-deficient barley roots.

To acquire Fe from soil, graminaceous plants secrete mugineic acid family phytosiderophores (MAs) from their roots. The secretion of MAs increases in response to Fe deficiency, and shows a distinct diurnal rhythm. We used a microarray that included 8987 cDNAs of rice EST clones to examine gene expression profiles in barley roots during Fe-deficiency stress. Approximately 200 clones were identified as Fe-deficiency-inducible genes, of which seven had been identified previously. In order to meet the increased demand for methionine to produce MAs, Fe-deficiency enhances the expression of genes that participate in methionine synthesis, as well as recycling methionine through the Yang cycle. Of these 200 genes, approximately 50 exhibited different transcription levels in Fe-deficient roots at noon and at night. Northern blot analysis of time course experiments confirmed that five of these genes exhibited a diurnal change in their level of expression. The diurnal changes in the expression of these genes suggest that polar vesicle transport is involved in the diurnal secretion of MAs.

IDI7, a new iron-regulated ABC transporter from barley roots, localizes to the tonoplast.

A new Fe-deficiency-induced cDNA, IDI7, was isolated from the roots of Fe-deficient barley (Hordeum vulgare L. cv. Ehimehadaka no. 1). The transcript levels of IDI7 in roots strongly correlated with iron nutritional status, and induction by Fe-deficiency was restricted to roots. Excess treatment with heavy metal ions, such as copper, manganese, and zinc, did not cause obvious IDI7 induction in either leaves or roots. IDI7 encodes a 644 amino acid protein, and has features typical of ATP-binding cassette (ABC) transporters. Phylogenetic analysis revealed that IDI7 is closely related to the half-type ABC protein subfamily, which includes mammalian transporters associated with antigen processing (TAPs). A transiently expressed fusion protein of IDI7 to green fluorescent protein (GFP) was localized to tonoplasts in suspension-cultured tobacco (Nicotiana tabacum L.) cells. IDI7 and its orthologues are thought to comprise a new class of ABC transporters, located in the tonoplasts of higher plants. A possible Fe-deficiency adaptation role for IDI7 in barley root cells, involving transport across the tonoplast, is proposed.