ABSTRACT
Akt (or PKB) is an oncogene involved in the regulation of cell survival. Akt is regulated by phosphatidylinositol 3-OH kinase (PI3'K) signaling and has shown to be hyperactivated through the loss of the PTEN tumor suppressor. In Drosophila, insulin signaling as studied using the Drosophila IRS-4 homolog (Chico) has been shown to be a crucial regulator of cell size. We have studied Drosophila Akt (Dakt1) and have shown that it is also involved in the regulation of cell size. Furthermore we have performed genetic epistasis tests to demonstrate that in Drosophila, PI3'K, PTEN and Akt comprise a signaling cassette that is utilized during multiple stages of development. In addition, we show that this signaling cassette is also involved in the regulation of cell survival during embryogenesis. This study therefore establishes the evolutionary conservation of this signaling pathway in Drosophila. Oncogene (2000) 19, 3971 - 3977.
Subject(s)
Drosophila melanogaster/physiology , Insect Proteins/physiology , Insulin/physiology , Phosphatidylinositol 3-Kinases/physiology , Phosphoric Monoester Hydrolases/physiology , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins , Signal Transduction/physiology , Tumor Suppressor Proteins , Animals , Cell Size , Cell Survival , Drosophila Proteins , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Epistasis, Genetic , Eye/embryology , Female , Genes, Insect , Male , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/genetics , Phosphoproteins/genetics , Phosphoproteins/physiology , Phosphoric Monoester Hydrolases/genetics , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins c-akt , TransfectionABSTRACT
We describe the use of image software programs available for both PC and Macintosh computers to quantify the accumulation and distribution of gold-labeled constructs within two-dimensional cell sections. The compartmentalization of a biotinylated-peptide was visualized in radiation-induced fibrosarcoma cells by transmission electron microscopy, using a gold particle-streptavidin conjugate. This study illustrates the ease of tabulating gold particles observed in scanned electron micrographs, using Adobe Photoshop in conjunction with the public domain NIH Image program (Version 1.61). Quantitative information regarding the localization of molecules inside cells is crucial in defining their sites of action and in developing more effective therapeutic agents.
Subject(s)
Gold/analysis , Image Processing, Computer-Assisted , Animals , Biotinylation , Fibrosarcoma/chemistry , Fibrosarcoma/pathology , Fibrosarcoma/ultrastructure , Microscopy, Electron , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/ultrastructureABSTRACT
The stability of a total nutrient admixture (TNA) has been postulated to be less than 7 days in refrigerated storage. When a TNA destabilizes, lipid particles coalesce and enlarge. Liposomes larger than 6 microns can obstruct pulmonary capillaries. A TNA containing 1500 ml of 7% Vamin, 1000 ml of 50% dextrose and 500 ml of 10% Intralipid, including the usual electrolytes, minerals and vitamins, was studied. Liposome size was measured in the original Intralipid and the TNA at intervals up to 14 days at 4 degrees C followed by 2 days at 22 degrees C. There was a small increase in liposome size up to 16 days. However, the number of particles larger than 6 microns was insignificant (by light microscopy, 3.9 +/- 2.4 [+/- SD] per 20 high-power fields; by Coulter counter, 99.8% smaller than 1.9 microns, with 0% larger than 6 microns; and by electron microscopy, 100% smaller than 2.0 microns). The osmolality and pH of the TNA were 1472 +/- 31 mOsm/kg and 5.5 +/- 0.1 respectively (mean +/- SD), with no significant change during the study times. The authors concluded that this TNA remains physically stable when refrigerated for 14 days and at room temperature for a further 2 days.
