RESUMEN
Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis, a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD), an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally, we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels, with a concomitant increase of the RIP1 protein. Together, these two related proteins form the necrosome, the complex responsible for induction of necroptotic cell death. Interestingly, we found that caspase-8 mRNA, a known negative regulator of necroptosis, was transiently decreased following OGD. Importantly, we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia, specifically in the CA1 area of the hippocampus. Moreover, we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together, our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury.
Asunto(s)
Apoptosis/fisiología , Isquemia Encefálica/patología , Hipocampo/patología , Hipoxia/metabolismo , Neuronas/patología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Regulación hacia Arriba/fisiología , Animales , Anticuerpos/farmacología , Apoptosis/efectos de los fármacos , Isquemia Encefálica/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Maleato de Dizocilpina/farmacología , Embrión de Mamíferos , Glucosa/deficiencia , Hipocampo/citología , Hipoxia/patología , Imidazoles/farmacología , Indoles/farmacología , L-Lactato Deshidrogenasa/metabolismo , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-Dawley , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
In this study, we purified and characterized tetra- and triglycosyl glycolipids (GL-1 and GL-2, respectively) from two different colonial forms of Thermus scotoductus X-1, from T. filiformis Tok4 A2, and from T. oshimai SPS-11. Acid hydrolysis of the purified glycolipids liberated, in addition to the expected long-chain fatty acids, two components which were identified by gas chromatography-mass spectrometry as 16-methylheptadecane-1,2-diol and 15-methylheptadecane-1,2-diol. Fast atom bombardment mass spectrometry of the intact glycolipids indicated that a major proportion consisted of components with glycan head groups linked to long-chain 1,2-diols rather than to glycerol, although in all cases glycerol-linked compounds containing similar glycan head groups were also present. As in other Thermus strains, the polar head group of GL-1 from T. filiformis Tok4 A2 and from T. scotoductus X-1 colony type t2 was a glucosylgalactosyl-(N-acyl)glucosaminylglucosyl moiety. However, GL-2 from T. scotoductus X-1 colony type t1 and from T. oshimai SPS-11 was a truncated analog which lacked the nonreducing terminal glucose. Long-chain 1,2-diols have been previously reported in the polar lipids of Thermomicrobium roseum and (possibly) Chloroflexus aurantiacus, but to our knowledge, this is the first report of their detection in other bacteria and the first account of the structural determination of long-chain diol-linked glycolipids.
Asunto(s)
Glicerol/química , Glucolípidos/química , Glicoles/química , Thermus/química , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Polisacáridos/química , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The compositions of the major glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11, and Thermnus sp. strain CG-2 were examined by gas chromatography, gas chromatography-mass spectroscopy, fast atom bombardment-mass spectroscopy, and chemical methods. The results showed that, with the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was established as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. Glucosamine was present in GL-1 of T. oshimai SPS-11 and Thermus sp. strain CG-2, while galactosamine was present in the GL-1 of T. aquaticus and T. filiformis. The novel major glycolipid of T. aquaticus 15004 was identified as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-gluc - osyl diacylglycerol. The hydroxy fatty acids found in the T. aquaticus strains and in the type strain of T. filiformis were exclusively amide linked to the galactosamine of the major glycolipid. Ester-linked hydroxy fatty acids were not detected in the diacylglycerol moiety of GL-1 of these organisms. Hydroxy fatty acids were detected neither in the major glycolipid of T. oshimai SPS-11 and Thermnus sp. strain CG-2, in which glucosamine is present, nor in the major phospholipid of any of the strains examined.
Asunto(s)
Ácidos Grasos/química , Glucolípidos/química , Thermus/química , Amidas/química , Amino Azúcares/análisis , Galactosamina/química , Cromatografía de Gases y Espectrometría de Masas , Monosacáridos/análisis , Oxidación-Reducción , Especificidad de la Especie , Espectrometría de Masa Bombardeada por Átomos VelocesRESUMEN
The accumulation of intracellular organic solutes was examined in six species of the order Thermotogales by nuclear magnetic resonance spectroscopy. The newly discovered compounds di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate and di-myo-inositol-1,3'-phosphate were identified in Thermotoga maritima and Thermotoga neapolitana. In the latter species, at the optimum temperature and salinity the organic solute pool was composed of di-myo-inositol-1,1'(3,3')-phosphate, beta-glutamate, and alpha-glutamate in addition to di-myo-inositol-1,3'-phosphate and di-2-O-beta-mannosyl-di-myo-inositol-1,1'(3,3')-phosphate. The concentrations of the last two solutes increased dramatically at supraoptimal growth temperatures, whereas beta-glutamate increased mainly in response to a salinity stress. Nevertheless, di-myo-inositol-1,1'(3,3')-phosphate was the major compatible solute at salinities above the optimum for growth. The amino acids alpha-glutamate and proline were identified under optimum growth conditions in Thermosipho africanus, and beta-mannosylglycerate, trehalose, and glycine betaine were detected in Petrotoga miotherma. Organic solutes were not detected, under optimum growth conditions, in Thermotoga thermarum and Fervidobacterium islandicum, which have a low salt requirement or none.
Asunto(s)
Bacterias Anaerobias Gramnegativas/química , Fosfatos de Inositol/química , Bacterias Anaerobias Gramnegativas/efectos de los fármacos , Bacterias Anaerobias Gramnegativas/fisiología , Isomerismo , Espectroscopía de Resonancia Magnética , Manósidos/química , Modelos Moleculares , Presión Osmótica , Cloruro de Sodio/farmacología , Especificidad de la Especie , TemperaturaRESUMEN
The polar lipids and fatty acid composition of Thermus aquaticus YT-1 and YS 041, T. filiformis Wai33 A1 and eighteen isolates from New Zealand, several of which are attributed to T. filiformis, were compared to complement the taxonomy of these organisms. The polar lipid patterns were essentially similar in all strains and consisted of one major phospholipid and one major glycolipid. The fatty acid analysis produced three basic groups corresponding to T. filiformis Wai33 A1, T. aquaticus and the third to the other New Zealand strains. The presence of hydroxy fatty acids is reported in Thermus spp. for the first time.