ABSTRACT
ß-thalassemia is one of the most common genetic disorders worldwide. Concerted efforts are being made to prevent the disease, as the medical and economic burden of thalassemia represents a major public health problem. The molecular diagnosis of the ß-globin mutations that cause the disease currently involves a combination of classic methodologies. A microarray-based assay for parallel one-shot detection of mutations has been developed, but the assay remains too expensive for routine application. We developed a cost-effective plastic fiber-based DNA chip for the fast and reliable detection of 25 types of ß-thalassemia mutations. Assay conditions were established and genotyping was successfully performed on a genomic sample from a ß-thalassemia patient. Our data show that this ß-thalassemia genotyping chip is an advantageous platform for mass genotyping because of its low cost, rapid results, and reliability.
Subject(s)
Genotyping Techniques , Oligonucleotide Array Sequence Analysis/methods , beta-Globins/genetics , beta-Thalassemia/diagnosis , beta-Thalassemia/genetics , Genotype , Humans , Multiplex Polymerase Chain Reaction , Mutation , Oligonucleotide Array Sequence Analysis/economics , Reproducibility of ResultsABSTRACT
Barrier function of the epidermis is maintained by precise expression of keratinocyte-specific structural proteins to form the cornified cell envelope (CE). Loricrin, a major component of the CE, is expressed at the late stage of keratinocyte differentiation. In this study, we reveal the isoform-specific function of protein kinase C (PKC) in the regulation of loricrin expression. Both PKCdelta and PKCeta have been recognized as differentiation-promoting isoforms. However, loricrin expression was inversely controlled by PKCdelta and PKCeta in cultured keratinocytes and 3D skin culture; i.e. loricrin expression was decreased by PKCdelta and increased by PKCeta. To clarify the mechanisms that PKCdelta and PKCeta oppositely regulate the loricrin expression, we examined the expression of activator protein-1 (AP-1) family proteins, which modulate the transcription of loricrin and are downstream molecules of PKC. PKCdelta decreased c-Jun expression, whereas PKCeta increased JunD, which are positive regulators of loricrin transcription. These findings suggest that inverse effects of PKCdelta and PKCeta on loricrin expression attributes to the expression of c-Jun and JunD.
Subject(s)
Gene Expression Regulation , Keratinocytes/metabolism , Membrane Proteins/genetics , Protein Kinase C-delta/metabolism , Protein Kinase C/metabolism , Transcription Factor AP-1/metabolism , Cells, Cultured , Humans , Protein Kinase C/genetics , Protein Kinase C-delta/genetics , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA Interference , Transcription Factor AP-1/geneticsABSTRACT
BACKGROUND: Thymus and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically attracts T-helper (Th) 2 cells positive for the CC chemokine receptor 4 (CCR4(+)). Corticosteroids reduce airway inflammation, as reflected by reduced numbers of eosinophils and T cells and reduced expression of cytokines. We investigated TARC production and the inhibitory effects of corticosteroids on TARC expression in a murine model of allergic asthma. METHODS: BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA) with alum. Once daily for 1 week, mice received injections of dexamethasone or 0.2 ml saline (control), then 1 h later inhaled aerosolized 1% OVA for 30 min. Mice were killed 24 h after OVA challenge for bronchoalveolar lavage and lung tissue examination. RESULTS: TARC was expressed mainly in the bronchial epithelial cells. Dexamethasone attenuated OVA-induced airway eosinophilia, lymphocyte infiltration, and airway hyperresponsiveness. Dexamethasone also decreased TARC production in the bronchoalveolar lavage fluid and decreased expression of TARC mRNA and TARC protein in lung tissue. CONCLUSIONS: The corticosteroid dexamethasone inhibits TARC production in a murine model of allergic asthma in vivo. The beneficial effect of corticosteroids in bronchial asthma is due in part to their direct inhibitory effects on TARC production.
Subject(s)
Adrenal Cortex Hormones/pharmacology , Asthma/immunology , Chemokines, CC/biosynthesis , Dexamethasone/pharmacology , Animals , Asthma/drug therapy , Asthma/pathology , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Cell Count , Chemokine CCL17 , Chemokines, CC/genetics , Chemokines, CC/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Lung/immunology , Lung/pathology , Male , Methacholine Chloride/immunology , Mice , Mice, Inbred BALB C , Ovalbumin/immunology , RNA/chemistry , RNA/genetics , Receptors, CCR4 , Receptors, CXCR3 , Receptors, Chemokine/immunology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
In isolated rat pancreatic acini, protein expression of RhoA and Rho-associated kinase, ROCK-II, and the formation of immunocomplex of RhoA with ROCK-II were enhanced by CCK-8, carbachol, and the phorbol ester TPA. The ROCK-specific inhibitor, Y-27632, did not alter basal amylase secretion, whereas it potentiated CCK-stimulated pancreatic enzyme secretion in vitro. During caerulein-induced pancreatitis occurring in mice in vivo, Y-27632 enhanced serum amylase levels and the formation of interstitial edema and vacuolization at 12-18h after the first injection of caerulein. Y-27632 in turn inhibited the recovery of protein expression of ROCK-II at 18h after the first caerulein injection. These results suggest that RhoA and ROCK-II assemble normal CCK-stimulated pancreatic enzyme secretion and prevent caerulein-induced acute pancreatitis.
