ABSTRACT
Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons.
Subject(s)
Cell Death , Cerebral Cortex/cytology , Epidermal Growth Factor/toxicity , Neurons/cytology , Oxidative Stress , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Cells, Cultured , Dizocilpine Maleate/pharmacology , Embryo, Mammalian , Enzyme Inhibitors/pharmacology , Epidermal Growth Factor/administration & dosage , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/physiology , Excitatory Amino Acid Antagonists/pharmacology , Mice , Microscopy, Electron , Phosphorylation , Protein Kinase Inhibitors , Protein Kinases/metabolism , Time FactorsSubject(s)
Amidohydrolases/biosynthesis , Escherichia coli/growth & development , Amidohydrolases/chemistry , Amidohydrolases/genetics , Cloning, Molecular , Enzyme Stability , Escherichia coli/enzymology , Escherichia coli/genetics , Glycerol/metabolism , Hot Temperature , Protein Engineering , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Thermodynamics , beta-Lactamases/biosynthesisABSTRACT
D-Hydantoinase is an industrial enzyme widely used for the synthesis of optically active D-amino acids. A gene encoding thermostable D-hydantoinase of Bacillus stearothermophilus SD-1 has previously been cloned and constitutively expressed by its native promoter in Escherichia coli XL1-Blue (Lee et al., 1996b). In this work, we attempted mass production of the D-hydantoinase by batch culture of the recombinant E. coli using glycerol as a carbon source. The plasmid content in cells increased in proportion to the culture temperature, which resulted in a two- or three-fold increase of the specific D-hydantoinase activity at 37 degrees C compared with that at 30 degrees C. The plasmid was stably maintained over 80 generations. When glycerol was initially added to a concentration of 100 g/L, the final biomass concentration reached about 50 g-dry cell weight/L in a 50 L-scale fermentation, resulting in the specific enzyme production of 3.8 x 10(4) unit/g-dry cell weight in a soluble form. Glycerol-using batch cultivation of recombinant E. coli was found to be a cost-effective process for the mass production of industrially useful D-hydantoinase. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 449-455, 1997.
