Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded ß-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4.

Several dog allergens cause allergic reactions in humans worldwide. Seven distinct dog allergens, designated Canis familiaris allergen 1 to 7 (Can f 1-Can f 7), have been identified thus far. Can f 6 shows high sequence similarity and cross-reactivity with Fel d 4 and Equ c 1, major cat and horse allergens, respectively. This study was conducted on the allergenic epitopes of Can f 6 based on its structural characterization. We demonstrated that sera from 18 out of 38 (47%) dog-sensitized patients reacted to recombinant Can f 6 protein (rCan f 6). We then determined the crystal structure of rCan f 6 by X-ray crystallography, which exhibited a conserved tertiary structural architecture found in lipocalin family proteins. Based on the tertiary structure and sequence similarities with Fel d 4 and Equ c 1, we predicted three IgE-recognizing sites that are possibly involved in cross-reactivity. Substituting three successive amino acids in these sites to triple alanine decreased IgE reactivity to the allergen. However, the degree of reduction in IgE reactivity largely depended on the site mutated and the serum used, suggesting that Can f 6 is a polyvalent allergen containing multiple epitopes and Can f 6-reactive sera contain varied amounts of IgE recognising individual Can f 6 epitopes including those predicted in this study. We also demonstrated that the predicted epitopes are partly involved in IgE cross-reactivity to Fel d 4. Interestingly, the effect of the mutation depended on whether the protein was structured or denatured, indicating that the bona fide tertiary structure of Can f 6 is essential in determining its IgE epitopes.

Venous or arterial blood components trigger more brain swelling, tissue death after acute subdural hematoma compared to elderly atrophic brain with subdural effusion (SDE) model rats.

Acute subdural hematoma (ASDH) is a frequent complication of severe head injury, whose secondary ischemic lesions are often responsible for the severity of the disease. We focused on the differences of secondary ischemic lesions caused by the components, 0.4ml venous- or arterial-blood, or saline, infused in the subdural space, evaluating the differences in vivo model, using rats. The saline infused rats are made for elderly atrophic brain with subdural effusion (SDE) model. Our data showed that subdural blood, both venous- and arterial-blood, aggravate brain edema and lesion development more than SDE. This study is the first study, in which different fluids in rats' subdural space, ASDH or SDE are compared with the extension of early and delayed brain damage by measuring brain edema and histological lesion volume. Blood constituents started to affect the degree of ischemia underneath the subdural hemorrhage, leading to more pronounced breakdown of the blood-brain barrier and brain damage. This indicates that further strategies to treat blood-dependent effects more efficiently are in view for patients with ASDH.

Roles of Ets-1 and p70S6 kinase in chondrogenic and gliogenic specification of mouse mesencephalic neural crest cells.

Fibroblast growth factors (FGFs) have been shown to promote the chondrogenic and gliogenic specification of mouse mesencephalic neural crest cells through Notch signaling [Nakanishi, K., Chan, S.Y., Ito, K., 2007. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 124, 190-203; Ijuin, K., Nakanishi, K., Ito, K., 2008. Different downstream pathways for Notch signaling are required for gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 125, 462-474]. In the present study, we analyzed FGF signaling pathways in chondrogenic and gliogenic specification. The promotion of chondrogenesis by FGF-2 was significantly suppressed by U0126, an inhibitor of the extracellular signal-regulated protein kinase (Erk) pathway, and by Erk-1 siRNA. Chondrogenesis was also prevented by the dominant negative Ets-1 expression vector. In contrast, Ets-1 was irrelevant to gliogenesis. The promotion of gliogenesis by FGF-2 was not only inhibited by U0126 but also by LY294002 and rapamycin, inhibitors of the Akt pathway, and by Akt-1 siRNA. Furthermore, gliogenesis was dramatically prevented by blocking the expression of p70S6 kinase (p70S6k), which is activated by both the Erk and Akt pathways, with p70S6k siRNA. These results suggest that Ets-1 activated by the Erk pathway promotes chondrogenic specification and p70S6k activated by both the Erk and Akt pathways plays an important role in gliogenic specification.