Core epitope analysis of 16 kDa allergen from tartary buckwheat.

Tartary buckwheat is widely accepted as its nutritionalvalue. Some allergic reactions hinder its utilization. This research focused on evaluating the core epitope of 16 kDa allergen (Fag t 2) in tartary buckwheat. Six B- and seven T cell epitopes of Fag t 2 were predicted, and six B cell epitope-mutants were expressed in Pichia pastoris. Bioinformatics analysis and SDS-PAGE demonstrated that the molecular weight, isoelectric point and spatial structures of six mutant allergens were similar with Fag t 2, with the same signal peptide sequences and α-amylase inhibitor domain. There was no significant change in mutants' spatial conformation confirmed by Circular Dichroism. The position K132N and peptides at 108-117 and 132-141 were the core B- and T cell epitopes of Fag t 2 confirmed by competitive inhibition ELISA and dot blot. This result was of great significance on the study of allergen epitopes in prevention and treatment of hypersensitivity.

Zinc homeostasis plays an important role in the prevention of obesity-induced cardiac inflammation, remodeling and dysfunction.

Obesity often leads to cardiovascular diseases, such as obesity-related cardiac hypertrophy (ORCH), due to chronic cardiac inflammation. Zinc is structurally and functionally essential for many transcription factors, therefore it not only has anti-inflammatory and anti-oxidative stress functions, but also has insulin-like function, however, its role in the development of obesity-associated cardiac pathogenesis and the potentially underlying mechanism(s) remains unclear. This review aims to summarize the available evidence on the role of zinc homeostasis in the prevention of ORCH. It was recently reported that when four-week old mice were fed either high fat diet (HFD) or normal diet containing deficient, adequate or supplemented zinc, HFD induced obesity and ORCH along with increased phosphorylation of p38 MAPK and increased expression of B-cell lymphoma/ leukemia 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These effects were further aggravated by zinc deficiency and significantly alleviated by zinc supplementation. Mechanistically administration of a p38 MAPK specific inhibitor in HFD-fed mice for 3 months did not affect HFD-induced obesity and increased expression of BCL10 and CARD9, but completely abolished HFD/obesity-induced cardiac hypertrophy and inflammation. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate-induced increased p38 MAPK activation and atrial natriuretic peptide expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38 MAPK. Taken together with other recent studies, we concluded that HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signaling. Zinc supplementation ameliorates ORCH through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation.

Characterization of 16-kDa major allergen with α-amylase inhibitor domain in tartary buckwheat seeds.

Tartary buckwheat (Fagopyrum tataricum, TB) is an important functional food containing proteins with balanced amino acid composition and more flavonoids than common buckwheat (Fagopyrum esculentum, CB). Buckwheat contains highly potent allergens that trigger an allergic reaction via an IgE mediated response. In this work, the full-length cDNA encoding Fag t 2 from tartary buckwheat seeds was cloned by screening the cDNA library of seed-filling period. The recombinant Fag t 2 (rFag t 2) expressed in Pichia pastoris SMD1168H was purified by purified by immobilized metal affinity chromatography. It demonstrated that Fag t 2 was a major allergen in tartary buckwheat with the activity of IgE binding and pepsin resistance, along with the thermal stability. The identification of natural Fag t 2 (n Fag t 2) confirmed that the Fag t 2 protein belongs to the 2S albumin family, only existing in embryo. Most interesting, we discovered that Fag t 2 had a α-amylase inhibitor domain near the end of C-terminal. The possible activity of α-amylase inhibitor of Fag t 2 will be detected in subsequent studies.

Direct detection of rutin-degrading isozymes with polyacrylamide gel electrophoresis.

Rutin-degrading enzymes (RDEs) specifically hydrolyze the glycosidic linkages of rutin, producing quercetin and rutinose. Here we report a reliable and sensitive polyacrylamide gel electrophoresis and staining method for the detection of RDE isozymes, which is based on the aqueous solubility difference between rutin and quercetin, as well as the ultraviolet absorbance of quercetin. With this novel method, we achieved a detection limit of 12 ng with 107 U of RDE activity, enabling us to detect at least five RDE isozymes in tartary buckwheat seeds.