ABSTRACT
Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive and specific detection in real time. However, the intracellular monitoring of metabolite levels still poses great challenges due to the often complex nature of detected metabolites. Here, we provide a systematic analysis of thiamin pyrophosphate (TPP) metabolism in Escherichia coli by using a TPP-sensing riboswitch that controls the expression of the fluorescent gfp reporter. By comparing different combinations of reporter fusions and TPP-sensing riboswitches, we determine key elements that are associated with strong TPP-dependent sensing. Furthermore, by using the Keio collection as a proxy for growth conditions differing in TPP levels, we perform a high-throughput screen analysis using high-density solid agar plates. Our study reveals several genes whose deletion leads to increased or decreased TPP levels. The approach developed here could be applicable to other riboswitches and reporter genes, thus representing a framework onto which further development could lead to highly sophisticated detection platforms allowing metabolic screens and identification of orphan riboswitches.
Subject(s)
Biosensing Techniques , Escherichia coli , Metabolic Networks and Pathways , Riboswitch , Thiamine Pyrophosphate , Riboswitch/genetics , Biosensing Techniques/methods , Escherichia coli/genetics , Escherichia coli/metabolism , Thiamine Pyrophosphate/metabolism , Metabolic Networks and Pathways/genetics , Green Fluorescent Proteins/metabolism , Green Fluorescent Proteins/genetics , Genes, Reporter , Gene Expression Regulation, Bacterial , Genome, BacterialABSTRACT
The rat intermediate pituitary lobe is one of the rare tissues that is not a known glucocorticoid target and is devoid of immunoreactive glucocorticoid receptor. The intermediate lobe is poorly vascularized and receives a dopaminergic and serotonergic innervation from the hypothalamus. In previous studies we demonstrated that removal of this hypothalamic input results in the appearance of immunoreactive glucocorticoid receptor in the intermediate lobe, as demonstrated with the use of in vitro intermediate pituitary cultures and two in vivo experimental situations. We now show that this appearance of the glucocorticoid receptor is presumably due to removal of hypothalamic dopamine from the intermediate lobe cells, since in this study dopamine (or its potent agonist bromocriptine) inhibits expression of the glucocorticoid receptor in intermediate pituitary cells in primary culture, as demonstrated by [3H] dexamethasone binding and immunocytochemistry. The dopamine antagonist haloperidol blocks the inhibitory effects of the dopamine agonist. In contrast to the intermediate pituitary cells, bromocriptine does not affect glucocorticoid receptor expression in anterior pituitary cells in culture. The differential dopaminergic regulation of glucocorticoid receptor expression in the pituitary gland raises questions about possible effects of dopamine on glucocorticoid receptor levels and glucocorticoid response in other dopamine target tissues, especially in the brain.
Subject(s)
Dopamine/physiology , Pituitary Gland/metabolism , Receptors, Glucocorticoid/metabolism , Animals , Bromocriptine/pharmacology , Cells, Cultured , Dexamethasone/metabolism , Dopamine/pharmacology , Haloperidol/pharmacology , Histocytochemistry , Immunologic Tests , Male , Pituitary Gland/drug effects , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Pituitary Gland, Posterior/drug effects , Pituitary Gland, Posterior/metabolism , RatsABSTRACT
Monolayer cell cultures of embryonic chicken pancreas contain functionally active insulin, glucagon and somatostatin-containing cells as evidenced by immunohistochemical and radioimmunoassay techniques. Hormone release is in relation to the number of each cell type present and responds to known specific secretory stimuli. The relatively high numbers of D-cells and amounts of immunoreactive somatostatin released by this preparation makes this system a suitable model for studies of somatostatin function and secretion.
Subject(s)
Pancreas/analysis , Somatostatin/metabolism , Animals , Cell Count , Cells, Cultured , Chick Embryo , Glucagon/analysis , Histocytochemistry , Immunochemistry , Insulin/analysis , Pancreas/embryology , Radioimmunoassay , Somatostatin/analysisSubject(s)
Athletic Injuries , Skiing , Adolescent , Adult , Aged , Female , Humans , Male , Middle AgedABSTRACT
Addition of anti-somatostatin serum to rat islets of Langerhans which had been previously maintained in organ culture for 48h, resulted in a marked stimulation of glucagon release with no change in the rate of insulin secretion during the entire 90 min period during which the secretion of both hormones was followed. Prior saturation of the anti-somatostatin serum with somatostatin prevented the stimulatory acting on glucagon release. These data strongly suggest a physiological role for endogenous somatostatin in the control of glucagon secretion.