ABSTRACT
In this study we evaluate both proximal and more distal transcriptional regulation of the 5' flanking region of the rat cholecystokinin gene in transfected GH3 (rat pituitary tumor) cells. Transcriptional activity was measured on the intact (-400 to +73) 5' flanking region of cholecystokinin (CCK), as well as with DNA constructs, which were deleted in both the conventional 5' to 3', as well as an unconventional 3' to 5' direction. Our in vivo studies indicate complex phorbol ester and forskolin interactions in the 10-base pair region between -130 and -140. We conclude, there are at least two transcriptional factors involved in regulation of the rat CCK transcription in this region. In vitro studies utilizing heterologous nuclear (HeLa) extract, as well as purified transcription factors AP-2 and NF-kappa B, identify overlapped AP-2- and NF-kappa B-responsive elements within the 17-base pair sequence between -149 and -134 of the distal 5' flanking region. In this region complex transcriptional regulation occurs, which indicates inhibition of AP-2 CCK promoter complexing by NF-kappa B. Six-point mutations introduced into this sequence prevent AP-2 and NF-kappa B binding to CCK promoter, as well as its transcriptional activation by phorbol ester and forskolin in GH3 cells.
Subject(s)
Cholecystokinin/genetics , DNA-Binding Proteins/metabolism , NF-kappa B/metabolism , Promoter Regions, Genetic/genetics , Response Elements/genetics , Transcription Factors/metabolism , Animals , Base Sequence , Binding, Competitive , Colforsin/antagonists & inhibitors , Colforsin/pharmacology , Consensus Sequence/genetics , DNA/genetics , DNA/metabolism , DNA Footprinting , DNA-Binding Proteins/antagonists & inhibitors , Deoxyribonuclease I/metabolism , HeLa Cells , Humans , Molecular Sequence Data , Mutation , Protein Binding , Rats , Tetradecanoylphorbol Acetate/antagonists & inhibitors , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-2 , Transcription Factors/antagonists & inhibitors , Transcriptional Activation/drug effects , Transfection , Tumor Cells, CulturedABSTRACT
Liposome mediated gene transfer has a great potential in gene therapy. In this review we discuss the physical and chemical properties of cationic liposomes that affect their abilities to mediate gene transfer into eukaryotic cells. The specific focus is on functional domains of cationic lipids. We address polar head variations, counterions, linker bonds, acyl chain variations, as well as composition of liposomes. We additionally discuss different functional groups of lipids affecting lipid bilayer packing, lipid association with DNA, fusion with the cellular membranes and the release of transferred DNA from endosomes into the cytoplasm.
Subject(s)
Gene Transfer Techniques , Lipids/chemistry , Liposomes/chemistry , Animals , Cations , DNA/chemistry , DNA/metabolism , Humans , Liposomes/metabolism , Liposomes/pharmacology , Structure-Activity RelationshipABSTRACT
BACKGROUND: Cationic liposomes may be used in gene transfer. However, different liposome configurations have varying efficiency in different tissues. AIMS: To compare multiple lipids during gene transfer into the intestinal mucosa, liver and central nervous system in the adult rat. We evaluate different lipid aliphatic and polar head domains. MATERIALS AND METHODS: Nine cationic or neutral phospholipids, combined with a cationic cholesterol derivative, have been compared to Lipofectin. Transfection was into GH3 cells and the adult rat brain, liver or intestinal mucosa. Results Optimum DNA:lipid ratio was lowest (1:2) in the intestinal mucosa and highest in GH3 cells (1:40). Lipofectin ", was most effective in brain and GH3 cells but had no activity in intestinal mucosa. Saturated cationic lipids transfect differently in GH3 cells and GI mucosa than in liver and brain. However, with saturated neutral phospholipids, GH3 cells, intestinal mucosa and liver transfect similarly. DOTAP the longest unsaturated cationic lipid (18:1) was most effective in the intestine, whereas DMEPC the shortest saturated neutral lipid (14:0) was optimal in the liver. CONCLUSIONS: In this study we propose a rational approach, based on systematic variations of lipids, to optimize liposome mediated gene transfer into the ventricular system of the brain, the liver and gastro-intestinal tract in the adult rat. Additionally, we demonstrate the feasibility of gene transfer into the mucosal cells of the gastro-intestinal tract as well as throughout the ventricular system of the rat brain. This requires liposomes which contain a cationic cholesterol derivative.
Subject(s)
Brain/metabolism , Gene Transfer Techniques , Intestinal Mucosa/metabolism , Liver/metabolism , Animals , Cell Line , Cholesterol/analogs & derivatives , DNA/metabolism , Lipid Metabolism , Liposomes , Luciferases/genetics , Luciferases/metabolism , Male , Phosphatidylethanolamines/metabolism , Phospholipids/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Brain/gut cholecystokinin (CCK) has well-established translational and post-translational differences. By contrast, CCK transcription regulation is reported to be the same in brain and gut. Accordingly, the rat CCK gene has been evaluated for differential brain/gut transcription initiation. Using the 5'-flanking region of the rat CCK gene, DNase I protection assays were performed. We evaluated reporter constructs deleted of the conventional transcription start site in cell culture. Finally, brain and gut mRNA was evaluated using both a reverse transcription serial primer polymerase chain reaction assay as well as rapid amplification of cDNA ends (RACE) analysis. TFIID protein protects against DNase I digestion between -177 and -196 bp. In tissue culture, spontaneous 5'-flanking region transcription initiation occurs until deletion proceeds upstream of -140 bp. RACE analysis performed on mRNA from the rat brain identifies heretofore undescribed transcription initiation at -43 bp 5' as well as at +1,212 (in exon II). These alternative transcription initiation sites are utilized in brain, but not gut. The rat CCK gene has alternative 5'-flanking region transcription initiation sites. These alternative sites are utilized only in the brain. These data may provide insights into how brain and gut respond to their differing physiological demands.
Subject(s)
Brain/metabolism , Cholecystokinin/biosynthesis , Intestine, Small/metabolism , Transcription, Genetic , Animals , Cholecystokinin/genetics , Cloning, Molecular , Genes, Reporter , Luciferases/biosynthesis , Mutagenesis, Site-Directed , Organ Specificity , Pituitary Neoplasms , Polymerase Chain Reaction , Promoter Regions, Genetic , Rats , Recombinant Fusion Proteins/biosynthesis , Sequence Deletion , Transfection , Tumor Cells, CulturedABSTRACT
Three components of the male yellowfin Baikal sculpin pheromonal signal have been isolated from urine by diethyl ether extraction, thinayer chromatography (TLC), and high-performance liquid chromatography (HPLC). Using mass spectrometry, we have identified two of them as testosterone (T) and 11ß-hydroxytestosterone (11HT). These steroids are synthesized in testes during full spermatogenesis, and they are excreted in male urine with milt. The third component is not a steroid. It is more likely to be a polyene alcohol (farnesol). 2Z,6E-Farnesol possesses behavioral activity.
