ABSTRACT
Epithelial-mesenchymal transformation is now recognised as an important feature of tissue remodelling. The present report concerns the role of adenovirus infection in inducing this transformation in an animal model of chronic obstructive pulmonary disease. Guinea pig primary peripheral lung epithelial cells (PLECs) transfected with adenovirus E1A (E1A-PLECs) were compared to guinea pig normal lung fibroblasts (NLFs) transfected with E1A (E1A-NLFs). These cells were characterised by PCR, immunocytochemistry, electron microscopy, and Western and Northern blot analyses. Electrophoretic mobility shift assays were performed in order to examine nuclear factor (NF)-kappaB and activator protein (AP)-1 binding activities. E1A-PLECs and E1A-NLFs positive for E1A DNA, mRNA and protein expressed cytokeratin and vimentin but not smooth muscle alpha-actin. Both exhibited cuboidal morphology and junctional complexes, but did not contain lamellar bodies or express surfactant protein A, B or C mRNAs. These two cell types differed, however, in their NF-kappaB and AP-1 binding after lipopolysaccharide stimulation, possibly due to differences in the expression of the subunits that comprise these transcriptional complexes. E1A transfection results in the transformation of peripheral lung epithelial cells and normal lung fibroblasts to a phenotype intermediate between that of the two primary cells. It is postulated that this intermediate phenotype may play a major role in the remodelling of the airways in chronic obstructive pulmonary disease associated with persistence of adenovirus E1A DNA.
Subject(s)
Adenovirus E1A Proteins/physiology , Fibroblasts/virology , Lung/virology , Mesoderm/metabolism , Actins/metabolism , Animals , Binding Sites , Blotting, Northern , Blotting, Western , Cell Transformation, Viral , Cells, Cultured , DNA, Viral , Electrophoretic Mobility Shift Assay , Epithelial Cells/metabolism , Epithelial Cells/virology , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Guinea Pigs , Immunoenzyme Techniques , Keratins/metabolism , Lung/cytology , Lung/metabolism , Mesoderm/cytology , Mesoderm/virology , Microscopy, Electron , Muscle, Smooth/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Phenotype , Polymerase Chain Reaction , Pulmonary Surfactant-Associated Protein A , RNA, Messenger , RNA, Viral , Transcription Factor AP-1 , Transfection , Vimentin/metabolismABSTRACT
The stability of housekeeping genes is critical when performing gene expression studies. To date, there have been no studies that look at the stability of commonly used housekeeping genes in alveolar macrophages. Expression levels may be affected by culture, stimulation or disease severity. The present study investigated the expression level of 10 housekeeping genes and analysed the stability of their expression in alveolar macrophages from chronic obstructive pulmonary disease patients (n = 22) who were classified according to disease severity. Guanine nucleotide-binding protein, beta polypeptide 2-like 1 (GNB2L1), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and ribosomal protein L32 (RPL32) were the most stably expressed in alveolar macrophages, irrespective of disease severity. There was no difference in the expression levels of 10 housekeeping genes between mild and moderate/severe patients. GNB2L1, HPRT1 and RPL32 were also stably expressed in alveolar macrophages cultured with no stimulation, or with interleukin-1beta, lipopolysaccharide or tumour necrosis factor-alpha stimulation. In conclusion, as fluctuations in the expression of some housekeeping genes were observed, including glyceraldehyde-3-phosphate dehydrogenase, it is recommended that guanine nucleotide binding protein, beta polypeptide 2-like 1 be used as a reference gene for alveolar macrophages in similar study designs, or that the stability of housekeeping genes be validated in alveolar macrophages prior to expression studies.
Subject(s)
GTP-Binding Proteins/genetics , Gene Expression , Hypoxanthine Phosphoribosyltransferase/genetics , Macrophages, Alveolar/metabolism , Neoplasm Proteins/genetics , Pulmonary Disease, Chronic Obstructive/genetics , Receptors, Cell Surface/genetics , Ribosomal Proteins/genetics , Aged , Bronchoalveolar Lavage , Female , Humans , Interleukin-1/pharmacology , Lipopolysaccharides/pharmacology , Male , Middle Aged , Pulmonary Disease, Chronic Obstructive/metabolism , Receptors for Activated C Kinase , Respiratory Function Tests , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The inhibition of growth of cotton seedlings (Gossypium hirsutum, var. Stardel) varied diurnally to applications of three herbicides [1,1-dimethyl-3-(alpha,alpha,alpha-trifluoro-m-tolyl) urea, 3',4'-dichloro-2-methacrylamide, and ethyl-N,N-dipropylthiocarbamate], but not to a fourth [3-(3,4-dichlorophenyl)-1, 1-dimethylurea]. Inhibition was strongest when the plants were treated at about daybreak. The rhythmic response was apparently not endogenously controlled, since most of the diurnal effect was lost under conditions of constant light and temperature.
