ABSTRACT
The present study generated a polyclonal antibody (AP86/3) that recognises a peptide sequence of the h5-HT(3B) receptor subunit. Western blot analysis of homogenates prepared from cell lines expressing either homomeric (h5-HT(3A)) or heteromeric (h5-HT(3A/3B)) receptors, as well as immunocytochemical studies with the same cell lines, indicated that AP86/3 recognised, selectively, the 5-HT(3B) subunit. Immunohistochemical labelling was also apparent in cells in the rat hippocampus that displayed the distribution and morphology of interneurones.
Subject(s)
Antibodies/metabolism , Hippocampus/cytology , Hippocampus/immunology , Receptors, Serotonin/immunology , Receptors, Serotonin/metabolism , Animals , Antigen-Antibody Reactions , Cell Line , Humans , Immune Sera/metabolism , Immunohistochemistry , Male , Rabbits , Rats , Rats, Wistar , Receptors, Serotonin, 5-HT3ABSTRACT
Suspension of human epidermal cells in methylcellulose-containing medium induces CYP1A1 by a mechanism requiring functional Ah receptor (AhR). In present work CYP1A1 mRNA was induced in a variety of cultured rat epithelial cells by suspension, but the induction was transient, with CYP1A1 mRNA reaching maximal levels by 5 h and disappearing by 12 h. Though the methylcellulose itself contained no detectable ligand, (a) suspension activated the AhR, as judged by mobility shift assays, (b) the AhR competitive inhibitor alpha-naphthoflavone inhibited suspension-mediated induction, and (c) induction was dependent upon dioxin responsive transcriptional elements in the CYP1A1 promoter. The rapid disappearance of CYP1A1 mRNA after 5 h of suspension was unaffected by the addition of TCDD but was prevented by the inclusion of the protein synthesis inhibitor cycloheximide. Thus the downregulation appears to be mediated by a novel short-lived protein induced or activated by suspension.
Subject(s)
Cytochrome P-450 CYP1A1/genetics , Animals , Base Sequence , Cells, Cultured , Culture Media , Cycloheximide/pharmacology , DNA Primers/genetics , Gene Expression/drug effects , Genes, Reporter , Keratinocytes/drug effects , Keratinocytes/metabolism , Methylcellulose , Polychlorinated Dibenzodioxins/pharmacology , Promoter Regions, Genetic , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/metabolismABSTRACT
The purpose of this study was to determine whether the level of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-hydroxy-2'-deoxyguanosine) (8-oxo-dG), a major mutagenic DNA oxidation product, is enhanced in newborn rat liver DNA as a consequence of oxidative stress incurred during the early postnatal period. 32P-postlabeling showed this adduct to increase approximately 2-fold from the 20th day of gestation (2 days before birth) to a peak level at 50-53 h after birth. Postnatal levels exceeded fetal levels at all time points investigated, i.e. 0.5-1, 8, 24, 50-53, 100, 216 and 432 h after birth. Increased formation of this mutagenic DNA lesion during the critical postnatal phase when there is rapid cell proliferation in all tissues is proposed to contribute to carcinogenesis in susceptible tissues later in life.
