ABSTRACT
Vacuolar-type H(+)-ATPase (V-ATPase) is a multi-subunit proton pump. The proton pump is essential for the regulation of pH in various eukaryotic cellular processes. Among the 14 subunits that constitute V-ATPase, d subunit mediates coupling between cytosolic and membrane domains. Whereas d1 is expressed ubiquitously in various types of cells, its isoform d2 is only expressed in specific cells or tissues. To characterize these isoforms, we expressed and purified the isoforms of human V-ATPase d subunits using Escherichia coli over-expression system. Subunit d1 and d2 were purified as homogeneous monomers as demonstrated by dynamic light scattering (DLS) analysis. Secondary structures of d subunits were estimated to be composed of 73% α-helix and 2% ß-sheet, as analyzed using circular dichroism (CD) analysis. Although sequence identity and secondary structures of d subunits were highly similar, the relative stability against thermal stress was higher for d1 than d2. Efficient expression and purification of d subunits, together with biophysical and biochemical characterization, presented in this study is expected to facilitate further structural analysis to clarify specific inter-molecular interactions involved in multi-subunit assembly and regulation of H(+) transporters.
Subject(s)
Escherichia coli/genetics , Vacuolar Proton-Translocating ATPases/chemistry , Vacuolar Proton-Translocating ATPases/isolation & purification , Amino Acid Sequence , Circular Dichroism , Escherichia coli/metabolism , Gene Expression , Humans , Molecular Sequence Data , Protein Stability , Protein Structure, Secondary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Sequence Alignment , Vacuolar Proton-Translocating ATPases/genetics , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
The present study provides evidence of the anti-asthmatic signaling activity of an aqueous fraction of green tea using specific in vitro and in vivo assays in an ovalbumin-induced asthmatic model. Mice sensitized to ovalbumin were orally administered an aqueous extract of Camellia sinensis. The lungs of these mice were then examined by hematoxylin and eosin staining and ELISA analysis to measure cytokine expression. The aqueous extract of Camellia sinensis exhibited potent anti-asthmatic activity by increasing the expression level of tumor necrosis factor-beta and interferon-gamma and decreasing the expression of anti-asthmatic cytokines in the lung. Together, these results indicate that the aqueous fraction of Camellia sinensis is effective in alleviating asthmatic symptoms by increasing the expression of Th1 cell-specific anti-asthmatic biomarkers.
Subject(s)
Asthma/metabolism , Camellia sinensis/chemistry , Gene Expression/drug effects , Plant Extracts/pharmacology , Th1 Cells/metabolism , Animals , Asthma/drug therapy , Asthma/immunology , Biomarkers/metabolism , CD4 Antigens/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Immunohistochemistry , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-10/genetics , Interleukin-10/metabolism , Lung/pathology , Lymphotoxin-alpha/genetics , Lymphotoxin-alpha/metabolism , Male , Mice , Mice, Inbred BALB C , Ovalbumin , PhytotherapyABSTRACT
Molecular inflammation is a pivotal process in various degenerative immune diseases, including asthma and atopic dermatitis. In this study, we examined the effects of Helianthus annuus seed (HAS) aqueous extract on an in vivo anti-asthmatic model. Ovalbumin-induced mice were orally administered the aqueous extract of Helianthus annuus seeds, and their lungs were assessed by hematoxylin and eosin staining. Moreover, the expression levels of IL-4/IL-13 cytokines and IgE were determined. HAS extract induced a decrease in CD4+ cell number, IL-4/IL-13 expression, and IgE secretion levels in the lungs. Our findings collectively suggest that the HAS extract has considerable potential in reducing the asthma-like symptoms induced by a mouse ovalbumin challenge model. However, further isolation and purification of the extract is required to determine the specific factor(s) responsible for its anti-asthmatic activity.
Subject(s)
Asthma/drug therapy , Helianthus/metabolism , Phytotherapy , Plant Extracts/therapeutic use , Seeds/metabolism , Animals , Asthma/immunology , Cell Line, Tumor , Disease Models, Animal , Humans , Hydrogen Peroxide/pharmacology , Mice , Ovalbumin , Promoter Regions, Genetic/genetics , T-Box Domain Proteins/geneticsABSTRACT
Dykellic acid, a novel factor initially identified from the culture broth of Westerdykella multispora F50733, has been shown to inhibit matrix metalloprotease 9 activity, caspase-3 activity, B cell proliferation and LPS-induced IgM production, suggesting that this factor may have anti-cancer effects. In an effort to further address the possible anti-tumoral effects of dykellic acid, we used wound healing, invasion and RhoA-GTP assays to examine the effects of dykellic acid on cell migration, invasion and angiogenesis. Our results revealed that dykellic acid dose-dependently inhibits B16 cell migration and motility, and inhibits HUVEC tube formation. Western blot analysis of the active form of RhoA (RhoA-GTP) showed that dykellic acid treatment decreased the levels of RhoA-GTP. These findings collectively suggest that dykellic acid may have both anti-metastatic and anti-angiogenic acitivites, and provides the first evidence for the involvement of RhoA in dykellic acid-induced effects.
