ABSTRACT
BACKGROUND: Corticosteroid insensitivity in asthmatics is associated with an increased expression of glucocorticoid receptor-beta (GR-beta) in many cell types. T-helper type 17 (Th17) cytokine (IL-17A and F) expressions increase in mild and in difficult-to-treat asthma. We hypothesize that IL-17A and F cytokines alone or in combination, induce the expression of GR-beta in bronchial epithelial cells. OBJECTIVES: To confirm the expression of the GR-beta and IL-17 cytokines in the airways of normal subjects and mild asthmatics and to examine the effect of cytokines IL-17A and F on the expression of GR-beta in bronchial epithelial cells obtained from normal subjects and asthmatic patients. METHODS: The expression of IL-17A and F, GR-alpha and GR-beta was analysed in bronchial biopsies from mild asthmatics and normal subjects by Q-RT-PCR. Immunohistochemistry for IL-17 and GR-beta was performed in bronchial biopsies from normal and asthmatic subjects. The expression of IL-6 in response to IL-17A and F and dexamethasone was determined by Q-RT-PCR using primary airway epithelial cells from normal and asthmatic subjects. RESULTS: We detected significantly higher levels of IL-17A mRNA expression in the bronchial biopsies from mild asthmatics, compared with normal. GR-alpha expression was significantly lower in the biopsies from asthmatics compared with controls. The expression of IL-17F and GR-beta in biopsies from asthmatics was not significantly different from that of controls. Using primary epithelial cells isolated from normal subjects and asthmatics, we found an increased expression of GR-beta in response to IL-17A and F in the cells from asthmatics (P< or =0.05). This effect was only partially significant in the normal cells. Dexamethasone significantly decreased the IL-17-induced IL-6 expression in cells from normal individuals but not in those from asthmatics (P< or =0.05). CONCLUSION: Evidence of an increased GR-beta expression in epithelial cells following IL-17 stimulation suggests a possible role for Th17-associated cytokines in the mechanism of steroid hypo-responsiveness in asthmatic subjects.
Subject(s)
Asthma/immunology , Bronchi/immunology , Epithelial Cells/immunology , Interleukin-17/metabolism , Receptors, Glucocorticoid/metabolism , Adult , Cells, Cultured , Dexamethasone/pharmacology , Female , Humans , Interleukin-17/immunology , Male , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Helper-Inducer/immunology , Up-Regulation/drug effectsABSTRACT
BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.
Subject(s)
Diabetic Retinopathy/enzymology , Ischemia/enzymology , Isoenzymes/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Receptors, Immunologic , Vitreoretinopathy, Proliferative/enzymology , Adult , Aged , Animals , Astrocytes/drug effects , Astrocytes/enzymology , Astrocytes/pathology , CD36 Antigens/metabolism , Cell Division/drug effects , Cells, Cultured , Cyclooxygenase 2 , Diabetic Retinopathy/complications , Diabetic Retinopathy/drug therapy , Diabetic Retinopathy/pathology , Dinoprostone/metabolism , Disease Models, Animal , Endothelial Growth Factors/metabolism , Enzyme Inhibitors/pharmacology , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Ischemia/complications , Ischemia/pathology , Isoenzymes/antagonists & inhibitors , Lymphokines/metabolism , Male , Membrane Proteins , Mice , Mice, Inbred C57BL , Middle Aged , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/pathology , Rats , Rats, Sprague-Dawley , Receptors, Lipoprotein/metabolism , Receptors, Prostaglandin E/drug effects , Receptors, Prostaglandin E/metabolism , Receptors, Prostaglandin E, EP3 Subtype , Receptors, Prostaglandin E, EP4 Subtype , Receptors, Scavenger , Retina/drug effects , Retina/enzymology , Retina/pathology , Retinal Vessels/drug effects , Retinal Vessels/pathology , Thrombospondin 1/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factors , Vitreoretinopathy, Proliferative/complications , Vitreoretinopathy, Proliferative/drug therapyABSTRACT
Prostanoids exert significant effects on circulatory beds. They play a role in the response of the vasculature to adjustments in perfusion pressure and oxygen and carbon dioxide tension, and they mediate the actions of numerous factors. The role of prostanoids in governing circulation of the perinate is suggested to surpass that in the adult. Prostanoids are abundantly generated in the perinate. They have been implicated in autoregulation of blood flow as studied in brain and eyes. Prostaglandins are also dominant regulators of ductus arteriosus tone. The effects of these autacoids are mediated through specific G protein-coupled receptors. In addition to the pharmacological characterization of the prostanoid receptors, important advances in understanding the biology of these receptors have been made in the last decade. Their cloning and the development of animals with disrupted genes of these receptors have been very informative. The involvement of prostanoid receptors in the developing subject, especially on brain and ocular vasculature and on ductus arteriosus, has also begun to be investigated; the expression of these receptors changes with development. Some but not all of the ontogenic changes in these receptors are attributed to homologous regulation. Interestingly, in the process of elucidating their effects, functional perinuclear prostaglandin E2 receptors have been uncovered. This article reviews prostanoid receptors and addresses implications on the developing subject with attention to vascular physiology.
Subject(s)
Blood Vessels/metabolism , Prostaglandins/metabolism , Receptors, Prostaglandin/physiology , Animals , Animals, Newborn , Cerebrovascular Circulation/physiology , Ductus Arteriosus/physiology , Echocardiography , Eye/anatomy & histology , Eye/blood supply , Eye/metabolism , Humans , Models, Biological , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Regional Blood Flow , Signal Transduction/physiologyABSTRACT
This work reports the discovery and functional characterization of catalytically active hammerhead motifs within satellite DNA of the pDo500 family from several DOLICHOPODA: cave cricket species. We show that in vitro transcribed RNA of some members of this satellite DNA family do self-cleave in vitro. This self-cleavage activity is correlated with the efficient in vivo processing of long primary transcripts into monomer-sized RNA. The high sequence conservation of the satellite pDo500 DNA family among genetically isolated DOLICHOPODA: schiavazzii populations, as well as other DOLICHOPODA: species, along with the fact that satellite members are actively transcribed in vivo suggests that the hammerhead-encoding satellite transcripts are under selective pressure, perhaps because they fulfil an important physiological role or function. Remarkably, this is the third example of hammerhead ribozyme structures associated with transcribed repetitive DNA sequences from animals. The possibility that such an association may not be purely coincidental is discussed.
Subject(s)
DNA, Satellite/genetics , Gryllidae/genetics , Multigene Family/genetics , RNA Processing, Post-Transcriptional , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , Animals , Base Sequence , Catalysis/drug effects , Conserved Sequence/genetics , Databases, Factual , Gene Expression , Kinetics , Magnesium Chloride/pharmacology , Models, Genetic , Nucleic Acid Conformation , Point Mutation/genetics , RNA Processing, Post-Transcriptional/drug effects , RNA, Catalytic/biosynthesis , RNA, Catalytic/chemistry , Software , Temperature , Transcription, GeneticABSTRACT
Four immunologically related proteins that belong to the annexin family were identified in cold acclimated wheat (Triticum aestivum). Two soluble forms with molecular masses of 34 and 36 kDa were found to bind phospholipid membranes in a calcium-dependent manner. These two forms are similar to the previously reported doublet in several plant species. The other two forms, with molecular masses of 39 and 22.5 kDa, were found associated with the microsomal fraction. Biochemical analysis showed that both forms are intrinsic membrane proteins and their association with the membrane is calcium independent. This is, to our knowledge, the first report of the presence of these annexin forms in plants. Membrane purification by two phase partitioning demonstrated that the p39 form is localized to the plasma membrane. Immunoblot analysis showed that the protein level of both p39 and p22.5 increases gradually reaching a maximum level after one day of low temperature exposure. The protein accumulation was similar in both hardy and less hardy cultivars, suggesting that the accumulation is not correlated with freezing tolerance. The results are discussed with respect to the possible role of these new intrinsic membrane annexins in low temperature signal transduction pathway.
Subject(s)
Annexins/metabolism , Membrane Proteins/metabolism , Plant Proteins/metabolism , Triticum/metabolism , Annexins/genetics , Base Sequence , Cold Temperature , DNA, Plant , Membrane Proteins/genetics , Molecular Sequence Data , Plant Proteins/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Triticum/geneticsABSTRACT
The Wcs120 gene encodes a highly abundant protein which appears to play an important role during cold acclimation of wheat. To understand the regulatory mechanism controlling its expression at low temperature, the promoter region has been characterized. Electrophoretic mobility shift assays using short promoter fragments revealed the presence in nuclear extracts from non-acclimated (NA) plants of multiple DNA-binding proteins which interact with several elements. In contrast, no DNA-binding activity was observed in the nuclear extracts from cold-acclimated (CA) plants. In vitro dephosphorylation of these CA nuclear extracts with alkaline phosphatase restored the binding activity. Moreover, okadaic acid (a potent phosphatase inhibitor) markedly stimulated the in vivo accumulation of the WCS120 family of proteins. This suggests that protein phosphatases PP1 and/or PP2A negatively regulate the expression of the Wcs120 gene. In addition, both Ca(2+)-dependent and Ca(2+)-independent kinase activities were found to be significantly higher in the CA nuclear extracts. Western analysis using antibodies directed against protein kinase C (PKC) isoforms showed that a PKCgamma homolog (84 kDa) is selectively translocated into the nucleus in response to low temperature. Taken together, our results suggest that, in vivo, the expression of the Wcs120 gene may be regulated by nuclear factors whose binding activity is modulated by a phosphorylation/dephosphorylation mechanism.
Subject(s)
Cold Temperature , DNA, Plant/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Plant , Genes, Plant , Plant Proteins/genetics , Promoter Regions, Genetic , Protein Processing, Post-Translational , Triticum/genetics , Acclimatization/genetics , Base Sequence , Cell Nucleus/metabolism , DNA, Plant/genetics , DNA-Binding Proteins/physiology , Isoenzymes/biosynthesis , Isoenzymes/genetics , Molecular Sequence Data , Phosphorylation , Plant Proteins/biosynthesis , Plant Proteins/physiology , Protein Binding , Protein Kinase C/biosynthesis , Protein Kinase C/genetics , Signal Transduction , Triticum/physiologyABSTRACT
The wcs120 gene is specifically induced by low temperature (LT) and encodes a protein that is thought to play an important role in the cold acclimation process in wheat. To identify the regulatory elements involved in its LT responsiveness, the transient expression activity of different promoter regions was determined using the luciferase reporter gene. The data indicate the involvement of putative enhancer elements, negative and positive regulatory regions in the transcriptional regulation of this gene. The promoter was found to be cold-inducible in different freezing-tolerant and -sensitive monocot and dicot species, suggesting that universal transcription factors responsive to LT may be present in all plants. This promoter could be used to drive the genes needed for LT tolerance in sensitive species.
Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , Promoter Regions, Genetic/genetics , Triticum/genetics , Acclimatization/physiology , Genes, Reporter/genetics , Plant Proteins/genetics , Sequence Deletion/genetics , Temperature , Transcription Factors/genetics , Transfection/geneticsABSTRACT
The biological effects of irradiation with(12)C(+5) ion beam on plant cells have been analyzed. Protoplasts and cell suspensions ofLavatera thuringiaca, and a somatic hybrid callus (Hibiscus rosa-sinensis +Lavatera thuringiaca), were irradiated with doses from 0.05 to 50 Gy, and the effects on cell growth, cell division, cell viability and embryogenesis rates were analyzed. Irradiation with(12)C(+5) ion beam at relatively very low doses (5.0 Gy) significantly inhibited cell division, yet the survival rate and regeneration capability of the cells through somatic embryogenesis were conserved in more than 70 and 50 %, respectively. These results indicate that cell division is the most sensitive parameter to irradiation, accounting for the inhibition of colony formation and callus growth. The potential use of the(12)C(+5) ion beam in asymmetric protoplast fusion experiments is discussed.
