Purification of soybean cupins and comparison of IgE binding with peanut allergens in a population of allergic subjects.

Identification, purification and characterization of allergens is crucial to the understanding of IgE-mediated disease. Immunologic and structural studies with purified allergens is essential for understanding relative immunogenicity and cross-reactivity. In this work, the complex soybean 7S vicilins (Gly m 5) with three subunits and 11S legumins (Gly m 6) with five subunits were purified and characterized along with purified peanut allergens (Ara h 1, 2, 3, and 6) by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Individual subjects plasma IgE binding was tested from subjects allergic to soybeans and or peanuts by immunoblotting, ImmunoCAP™ and ISAC™ ImmunoCAP chip, comparing these soybean proteins with those of purified peanut allergens; vicilin (Ara h 1), 2S albumin (Ara h 2 and Ara h 6) and 11S globulin (Ara h 3). Results show differences between methods and subjects demonstrating the complexity of finding answers to questions of cross-reactivity.

Evaluating the potential allergenicity of dietary proteins using model strong to non-allergenic proteins in germ-free mice.

Currently no validated animal model is predictive of human responses in ranking purified dietary proteins in the prevalence or potency of food allergy in humans. Since the gastrointestinal microbiota is thought to influence oral tolerance, we hypothesize that a germ-free mouse model will more accurately predict atopic human responses than conventional mice. Germ-free C3H/HeN mice were immunized with 60 µg Ara h 2, BLG, or LOX by three weekly intraperitoneal (IP) injections with alum adjuvant. One week following the final immunization an IP challenge of 500 µg of Ara h 2, BLG, or LOX was administered. Thirty minutes post-challenge clinical scores were graded and body temperatures recorded. The presence of protein-specific IgE and mast cell protease concentrations in mouse sera were determined using ELISA. Upon challenge germ-free mice sensitized with Ara h 2 and BLG exhibited significantly more severe clinical scores compared to germ-free mice immunized with LOX. Hypothermic responses in challenged mice differed between the three proteins post-challenge. Results indicate that this model can differentiate between potent and non-allergens based on temperature drop, clinical scores, and biomarkers. Additional proteins with known human exposure and allergenicity are needed to confirm the predictive accuracy.

Conserved miRNAs and their targets identified in lettuce (Lactuca) by EST analysis.

MicroRNAs (miRNAs) are a newly identified class of endogenous, non-coding, short ( approximately 21nt) RNAs that play important roles in regulating gene expression at post-transcriptional level by targeting mRNA cleavage or translational inhibition in plants and animals. Though there are lots of differences between plant miRNAs and animal miRNAs, most of these tiny RNAs are highly conserved in each kingdom. Here, we show the conserved miRNAs in lettuce (Lactuca) identified using EST (expressed sequence tag) analysis. Namely, all previously known miRNAs in other plant species were blasted against lettuce EST sequences to select novel miRNAs in lettuce by a series of filtering criteria. By this strategy, we found a total of 21 conserved miRNAs belonging to 12 miRNA families. After analyzing the conservation and evolution of lettuce miRNAs and their counterparts in other plant species, we revealed that though miRNAs are highly conserved, some specific sites are more likely to mutate. To confirm the expression of identified miRNAs in lettuce, an RT-PCR approach was employed. Moreover, all identified lettuce miRNAs were used to search their potential target genes by miRU web-server from TIGR database available at http://www.tigr.org and a total of 63 potential targets for 10 identified miRNA families in lettuce were found. Similar to previous works, some miRNA targets are transcription factors involved in lettuce growth and development, metabolism, and stress responses.

Computational identification of microRNAs and their targets in wheat (Triticum aestivum L.).

microRNAs (miRNAs) are a class of endogenous, non-coding, short (approximately 21 nt) RNAs directly involved in regulating gene expression at the post-transcriptional level. Previous reports have noted that plant miRNAs in the plant kingdom are highly conserved, which provides the foundation for identification of conserved miRNAs in other plant species through homology alignment. Conserved miRNAs in wheat are identified using EST (Expressed Sequence Tags) and GSS analysis. All previously known miRNAs in other plant species were blasted against wheat EST and GSS sequences to select novel miRNAs in wheat by a series of filtering criteria. From a total of 37 conserved miRNAs belonging to 18 miRNA families 10 conserved miRNAs comprising 4 families were reported in wheat. MiR395 is found to be a special family, because three members belonging to the same miR395 family are clustered together, similar to animal miRNAs. MiRNA targets are transcription factors involved in wheat growth and development, metabolism,and stress responses.