Cloning, expression, and characterization of Der f 7, an allergen of Dermatophagoides farinae from China.

A full-length cDNA encoding house dust mite allergen Der f 7 from Dermatophagoides farina (Acari: Pyroglyphidae) from China was cloned, sequenced, and successfully expressed. A reference sequence (GenBank accession AY283292) was used to design polymerase chain reaction primers. Analysis revealed eight mismatched nucleotides in five Der f 7 cDNA clones, and the projected amino acid sequence contained six incompatible residues. These results suggest that the sequence of Der f 7 may be polymorphic. Further bioinformatic analysis revealed that the mature Der f 7 allergen had a molecular mass of approximately 21.88 kDa and a theoretical isoelectric point of 4.90. Der f 7 protein secondary structure was composed of a helix (56.63%), extended strand (5.10%), and random coil (38.27%). Group 7 allergens are present in Pyroglyphidae, Acaridae, and Glycyphagidae families, and homology analysis revealed a 86% similarity between Der f 7 and Der p 7. Furthermore, a phylogenetic tree constructed of group 7 allergens from different mite species revealed that Der f 7 and Der p 7 clustered with 100% bootstrap support. Bioinformatics-driven characterization of Der f 7 allergen as conducted in this study may contribute to diagnostic and therapeutic applications for dust mite allergies.

[Preliminary investigation on phylogenetic relationship among three common species of house dust mites].

Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei were used for the investigation. The cDNA fragment coding for Der f1 and Der f2 were amplified by PCR, cloned and sequenced. By bioinformatics softwares, the amino acid sequences for Der f1 and Der f2 were deduced and compared with those for the groups 1 and 2 allergens of D. pteronyssinus and E. maynei available in GenBank. Amino acid sequence similarity analysis showed that Der p1 shared 84% identical residues with Eur m1 and 83% with Der f1. Similarly, Der p2 shared 87% identical residues with Eur m2 and 68% with Der f2. In the two phylogenetic trees constructed with group 1 and 2 allergens, D. pteronyssinus was clustered with E. maynei but not with D. farinae, although D. pteronyssinus and D. farinae belong to the same genus. D. pteronyssinus should be more similar to E. maynei than to D. farinae at evolutional level, which was not consistent with the conventional taxonomical relationship based on their morphological characteristics.

Blocking beta 2-adrenergic receptor inhibits dendrite ramification in a mouse model of Alzheimer's disease.

Dendrite ramification affects synaptic strength and plays a crucial role in memory. Previous studies revealed a correlation between beta 2-adrenergic receptor dysfunction and Alzheimer's disease (AD), although the mechanism involved is still poorly understood. The current study investigated the potential effect of the selective ß2-adrenergic receptor antagonist, ICI 118551 (ICI), on Aß deposits and AD-related cognitive impairment. Morris water maze test results demonstrated that the performance of AD-transgenic (TG) mice treated with ICI (AD-TG/ICI) was significantly poorer compared with NaCl-treated AD-TG mice (AD-TG/NaCl), suggesting that ß2-adrenergic receptor blockage by ICI might reduce the learning and memory abilities of mice. Golgi staining and immunohistochemical staining revealed that blockage of the ß2-adrenergic receptor by ICI treatment decreased the number of dendritic branches, and ICI treatment in AD-TG mice decreased the expression of hippocampal synaptophysin and synapsin 1. Western blot assay results showed that the blockage of ß2-adrenergic receptor increased amyloid-ß accumulation by downregulating hippocampal α-secretase activity and increasing the phosphorylation of amyloid precursor protein. These findings suggest that blocking the ß2-adrenergic receptor inhibits dendrite ramification of hippocampal neurons in a mouse model of AD.

Cloning, sequence analysis and expression in E. coli of the group 3 allergen of Dermatophagoides farinae.

BACKGROUND: The dust mites, which are mostly represented by Dermatophagoides spp. (Acari: Pyroglyphidae), are the major sources of indoor allergens. Identification and characterization of these mite allergen molecules are an important step in the development of new effective diagnostic procedures and possible therapeutic strategies for allergic disorders associated with dust mites. METHODS: Total RNA was extracted from Dermatophagoides farinae. The gene coding for Der f 3 was amplified by RT-PCR with the primers designed based on previous sequence published in GenBank. The target gene was cloned intermediately into pMD19-T plasmid and finally into plasmid pET28a (+), expressed in E. coli BL21 at the aid of the inducer isopropyl-D-thiogalactopyranoside (IPTG). The physicochemical properties, spatial structure of the allergen were analyzed with bioinformatics software. RESULTS: The cDNA coding for group 3 allergen of Dermatophagoides farinae from China was cloned and expressed successfully. Sequencing analysis showed that there were nineteen mismatched nucleotides in five Der f 3 cDNA clones in comparison with the reference (GenBank Accession No. AY283291), which resulted in deduced amino acid sequence incompatibility in eleven residues. Bioinformatics analysis revealed that the Der f 3 pro-protein was an extracellular hydrophobic protein, consisting of 259 amino acids with a 16 amino acid signal peptide. The protein was deduced to have three chymotrypsin active sites (53-68 AA, 108-122 AA and 205-217 AA), one N-glycosylation site, one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, and five N-myristoylation sites. CONCLUSIONS: Der f 3 is an extracellular hydrophobic protein which possesses multiple activation and phosphorylation sites. Polymorphism may exist in the Der f 3 gene but this needs to be further confirmed in the future.

[Molecular chaperone GRP75 reprove cells from injury caused by glucose deprivation].

Glucose-regulated proteins 75(grp75) is a member of hsp70 family. The expression of grp75 is upregulated during glucose starvation (such as ischemia). To evaluate grp75 function, CHL cells were cultured with glucose-free media for 20 h (A) and glucose-free media for 12 h + glucose-containing media for 8 h (ischemia reperfusion) (B). A constructed rat grp75 cDNA expression vector (pcDNA/grp75) was transfected into CHL cells and a cell strain that stably overexpressed grp75 was obtained. The transfected cells and untransfected cells(control group) were cultured with A or B. By MTT, LDH leakage measurement and flow cytometry analysis, growth rate of untransfected cells in B is significantly lower than that in glucose-containing media for 20 h (C) (p < 0.05) and A (p < 0.05). Growth rate of transfected cells is apparently higher than that of control group in B (p < 0.01). LDH liberation percentage of untransfected cells in B is obviously higher than that in C(p < 0.01) and it is not different from A(p > 0.05). LDH liberation percentage of transfected cells is apparently lower than that of control group in B(p < 0.01). Apoptosis of transfected cells is obviously lower by flow cytometry analysis. These results provide evidence for the cytoprotective function of grp75 during glucose starving and ischemia reperfusion.