14-Deoxy-11,12-Didehydroandrographolide Ameliorates Glucose Intolerance Enhancing the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 Signaling Pathway and Inducing GLUT4 Expression in Myotubes and Skeletal Muscle of Obese Mice.

14-Deoxy-11,12-didehydroandrographolide (deAND), a bioactive component of Andrographis paniculata, has antidiabetic activity. AMP-activated protein kinase (AMPK) regulates glucose transport and ameliorates insulin resistance. The aim of the present study was to investigate whether activation of AMPK is involved in the mechanism by which deAND ameliorates insulin resistance in muscles. deAND amounts up to 40 [Formula: see text]M dose-dependently activated phosphorylation of AMPK[Formula: see text] and TBC1D1 in C2C12 myotubes. In addition, deAND significantly activated phosphorylation of LKB1 at 6 h after treatment, and this activation was maintained up to 48 h. deAND increased glucose uptake at 18 h after treatment, and this increase was time dependent up to 72 h. Compound C, an inhibitor of AMPK, suppressed deAND-induced phosphorylation of AMPK[Formula: see text] and TBC1D1 and reversed the effect on glucose uptake. In addition, the expression of GLUT4 mRNA and protein in C2C12 myotubes was up-regulated by deAND in a time-dependent manner. Promotion of GLUT4 gene transcription was verified by a pGL3-GLUT4 (837 bp) reporter assay. deAND also increased the nuclear translocation of MEF-2A and PPAR[Formula: see text]. After 16 weeks of feeding, the high-fat diet (HFD) inhibited phosphorylation of AMPK[Formula: see text] and TBC1D1 in skeletal muscle of obese C57BL/6JNarl mice, and deactivation of AMPK[Formula: see text] and TBC1D1 by the HFD was abolished by deAND supplementation. Supplementation with deAND significantly promoted membrane translocation of GLUT4 compared with the HFD group. Supplementation also significantly increased GLUT4 mRNA and protein expression in skeletal muscle compared with the HFD group. The hypoglycemic effects of deAND are likely associated with activation of the LKB1/AMPK[Formula: see text]/TBC1D1/GLUT4 signaling pathway and stimulation of MEF-2A- and PPAR[Formula: see text]-dependent GLUT4 gene expression, which account for the glucose uptake into skeletal muscle and lower blood glucose levels.

Identification the Cross-Reactive or Species-Specific Allergens of Tyrophagus putrescentiae and Development Molecular Diagnostic Kits for Allergic Diseases.

Mite allergens are considerable factors in the genesis of allergic diseases. The storage mite Tyrophagus putrescentiae (Tp) appears in contaminated foods and household surroundings. The current diagnostic tools for Tp allergy are mostly based on crude extracts and still contain shortcomings. This study aimed to investigate the immunoglobulin E (IgE)- responsiveness profiles of Tp-allergic patients and develop a molecular diagnostic method using recombinant allergens. Allergenic components were characterized as cross-reacting or species-specific allergens, in which the effective combinations of recombinant allergens were developed and analyzed in terms of the prediction accuracy for clinical diagnosis. Seven recombinant allergens were cloned and generated to detect the IgE responsiveness of the Tp allergy. A survey on the prevalence of mite allergy showed there were higher sensitizations with IgE responsiveness to house dust mites (HDM) (78.9-80.9%) than to storage mites Tp (35.6%). Prevalence of sensitization to Tp was higher in elderly subjects. The principal IgE-binding components of Tp were Tyr p 1, Tyr p 2 and Tyr p 3. Prediction accuracy for Tp allergy by IgE-responsiveness combination D (Tyr p 1, Tyr p 2 & Tyr p 3) was with high precision (100%). Avoiding the cross-reactivity of Dermatophagoides pteronyssinus, the prediction accuracy of IgE-responsiveness combination H+ (Tyr p 1, Tyr p 2, Tyr p 3, Tyr p 7, Tyr p 8, Tyr p 10 & Tyr p 20) was suitable for Tp-specific diagnosis. Panels of Tp allergens were generated and developed a diagnostic kit able beneficial to identify IgE-mediated Tp hypersensitivity.

Andrographis paniculata Improves Insulin Resistance in High-Fat Diet-Induced Obese Mice and TNFα-Treated 3T3-L1 Adipocytes.

Pro-inflammatory cytokines interfere with blood glucose homeostasis, which leads to hyperglycemia. Andrographis paniculata (AP) has been shown to possess anti-inflammatory activity and to reduce blood glucose levels in diabetes. The two major bioactive diterpenoids in AP, andrographolide (AND) and 14-deoxy-11,12-didehydroandrographolide (deAND), have potent anti-inflammatory activity. We studied whether APE (an ethanolic extract of AP), AND, and deAND could improve a high-fat diet (HFD)-induced hyperglycemia in vivo and TNF[Formula: see text]-induced impairment of insulin signaling in vitro. Male C57BL/6JNarl mice were fed a normal diet (ND) or the HFD, and the fatty mice were treated with APE, deAND, or AND for 16 weeks. 3T3-L1 cells were used to study the underlying mechanisms by which APE, deAND, or AND attenuated TNF[Formula: see text]-induced insulin resistance. The HFD significantly induced obesity, hyperglycemia, insulin resistance, and inflammation, whereas APE and deAND significantly ameliorated HFD-induced obesity, hyperglycemia, insulin resistance, and TNF[Formula: see text] production. The HFD significantly impaired insulin signaling by decreasing the protein expression of p-IRS1 tyr632 and p-AKT ser473, as well as the membrane translocation of GLUT4 in response to insulin stimulation in epididymal adipose tissue. HFD-impaired the membrane translocation of GLUT4 was significantly reversed by deAND and APE. In addition, deAND and APE markedly reversed the detrimental effect of TNF[Formula: see text] on the insulin signaling pathway and glucose uptake in 3T3-L1 cells. Despite no significant positive effect on p-AS160, a trend for recovery by deAND and APE was observed. These results suggest that deAND and APE protect against HFD-induced insulin resistance by ameliorating inflammation-driven impairment of insulin sensitivity.

Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule.

We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

Detection of group 2 Dermatophagoides pteronyssinus allergen for environmental monitoring of dust mite infestation.

Aeroallergen avoidance has been promoted in order to prevent sensitization and the correlation between the level of allergen exposure and sensitization has been reported. The aims of this study were to monitor environmental mite infestation and to design an effective Der p 2 detection kit to estimate the number of mites in house dust samples. House dust samples were collected from 6 carpets and 2 mattresses monthly from April 2010 to March 2011. The total number of mites was counted under microscopes and Der p 2 concentrations were measured using Der p 2 ELISA kits. The detection kit was constituted using Der p 2 specific mouse monoclonal antibody as capture antibody, and rabbit polyclonal antibody as detection antibody. Both Der p crude extract and rDer p 2 were used as internal standards. The number of mites in the dust samples was significantly higher in the mattresses as compared with that in the carpets and the total number of dust mites was higher in the summer than any other seasons. The concentration of Der p 2 components in Der p crude extract was analyzed and the results showed that each gram of Der p crude extract contained 53.4 mg of Der p 2. When the number of mites and Der p 2 concentration were measured for the correlation analysis, the results showed that there was a good correlation between Der p 2 and number of mites with R(2) = 0.9667. Dust mites were significantly increased in the dust samples collected from mattresses especially in the summer. The good correlation between Der p 2 concentration and mite numbers indicated that the measurement of Der p 2 can be used to replace direct mite counting. Using the Der p 2 detection method to monitor environmental mite infestation may be beneficial for allergic subjects to prevent disease activation.

Identification of allergenic component Tyr p 8 from Tyrophagus putrescentiae and cross-reactivity with Der p 8.

Group 8 mite allergens exhibit sequence homology to glutathione S-transferases (GSTs), such as that from Dermatophagoides pteronyssinus (Der p 8). GSTs have been identified as important allergens in studies of allergens from house dust mites, cockroaches, and fungi. Our objective was to purify the native group 8 allergen from Tyrophagus putrescentiae (nTyr p 8) and generate recombinant Tyr p 8 (rTyr p 8) for immunological characterization. The allergenicity was determined by antibody recognition, IgE inhibition, and triggering of the basophil-sensitized release of histamine, using T. putrescentiae hypersensitivity sera. The results showed that the mRNA transcript of nTyr p 8 is 657 bp long, contains 218 amino acids with a molecular mass of 26 kDa, and exhibits 83% sequence homology to Der p 8. Serum samples from the allergic patients with an IgE-positive response to T. putrescentiae were analyzed to determine their IgE response to rTyr p 8. The results showed that the sera of 48 subjects (45.3%) had specific IgE against rTyr p 8. However, sera of only 19 subjects (17.9%) had specific IgE against rTyr p 8 after D. pteronyssinus absorption. Histamine release was observed from T. putrescentiae-allergic subjects in the presence of rTyr p 8. Both the nTyr p 8 and T. putrescentiae crude extract had been demonstrated to possess GST enzymatic activity. Although the specific binding of serum IgE to rTyr p 8 was only 17.9%, which indicates that rTyr p 8 was not a major allergen, the positive response to rTyr p 8 was due to the cross-reactivity with Der p 8. The group 8 mite allergen might be of use in the design of a suitable allergen for diagnosis and for the development of novel immunotherapies.