Home About us Contact
|
Home About us Contact | ||
|
|
||
Cytosolic Extracts (cytosolic + extract)
Selected AbstractsUse of [125I]4,-iodoflavone as a tool to characterize ligand-dependent differences in Ah receptor behaviorJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 6 2002Hollie I. Swanson Abstract We have synthesized [125I]4,-iodoflavone to study Ah receptor (AhR),ligand interactions by a class of AhR ligands distinct from the prototypic ligand 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). This radioligand allows the comparison of AhR,ligand interactions using a ligand that differs in AhR affinity, and yet has the same radiospecific activity as [125I]2-iodo-7,8-dibromodibenzo- p -dioxin. Specific binding of [125I]4,-iodoflavone with the AhR was detected as a single radioactive peak (,9.7 S) following density sucrose gradient analysis. Cytosolic extracts from both Hepa 1 and HeLa cells were used as the source of mouse and human AhR, respectively. A ,6.7 S form of radioligand-bound Ah receptor was detected in the high salt nuclear extracts of both cell lines. In HeLa cells approximately twofold more [125I]4,-iodoflavone,AhR 6 S complex, compared with [125I]2-iodo-7,8-dibromodibenzo- p -dioxin, was recovered in nuclear extracts. A comparison of the ability of 4,-iodoflavone and TCDD to cause time-dependent translocation of AhR-yellow fluorescent protein revealed that 4,-iodoflavone was more efficient at enhancing nuclear accumulation of the receptor. These results suggest that [125I]4,-iodoflavone is a particularly useful and easily synthesized ligand for studying the AhR. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:298,310, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10053 [source] Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic miceFEBS JOURNAL, Issue 19 2008Judit Oláh Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered Vmax values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences. [source] Hypoxia-Induced Apoptotic Cell Death is Prevented by Oestradiol Via Oestrogen Receptors in the Developing Central Nervous SystemJOURNAL OF NEUROENDOCRINOLOGY, Issue 3 2008V. M. Pozo Devoto The neuroprotective effects of oestrogens have been demonstrated against a variety of insults, including excitotoxicity, oxidative stress and cerebral ischemia under certain conditions. However, the molecular mechanisms underlying oestrogen neuroprotection are still unclear. We aimed to determine whether 17,-oestradiol (E2) administration post-hypoxia (p-hx) was neuroprotective and whether these actions were mediated through oestrogen receptors (ER). For this purpose, 12-embyonic day-old chickens were subjected to acute hypoxia [8% (O2), 60 min], followed by different reoxygenation periods. To test the neuroprotective effect of E2 and its mechanism, embryos were injected 30 min after the end of hypoxia with E2 alone or with ICI 182 780, a competitive antagonist of ER. Cytochrome c (cyt c) release, an indicator of mitochondrial apoptotic pathway, was measured by western blot in optic lobe cytosolic extracts. DNA fragmentation by TUNEL fluorescence and caspase-3 fragmentation by immunofluorescence were detected on optic lobe sections. Acute hypoxia produces a significant increase in cyt c release from mitochondria at 4 h p-hx, followed by an increase in TUNEL positive cells 2 h later (6 h p-hx). Administration of E2 (0.5 mg/egg) produced a significant decrease in cytosolic cyt c levels at 4 h p-hx, in casapse-3 activation and in TUNEL positive cells at 6 h p-hx compared to vehicle treated embryos. In the E2 -ICI 182 780 treated embryos, cyt c release, caspase-3 fragmentation and TUNEL positive cells were similar to the hypoxic embryos, thus suggesting the requirement of an E2,ER interaction for E2 mediated neuroprotective effects. In conclusion, E2 prevents hypoxia-induced cyt c release and posterior cell death and these effects are mediated by oestrogen receptors. [source] Isolation, crystallization and preliminary X-ray analysis of a methanol-induced corrinoid protein from Moorella thermoaceticaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2005Weihong Zhou A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9,Ĺ resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54,Ĺ. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein. [source] Abnormal LTC4 synthase RNA degradation in neutrophils from CML patientsBRITISH JOURNAL OF HAEMATOLOGY, Issue 6 2004Cecilia Roos Summary Neutrophils from patients with chronic myeloid leukaemia (CML) have an aberrant expression of leukotriene (LT)C4 synthase. In order to learn more about the regulation of this abnormality, LTC4 synthase mRNA expression was determined by reverse transcription polymerase chain reaction. A digoxigenin (DIG)-labelled LTC4 synthase RNA was synthesized and incubated in cytsolic extracts from CML neutrophils, normal neutrophils and eosinophils. LTC4 synthase mRNA was detected in total but not cytoplasmic RNA from normal neutrophils. In contrast, LTC4 synthase mRNA was found in the cytoplasm of CML neutrophils and in normal eosinophils, which also express the enzyme. The DIG-labelled LTC4 synthase RNA was, as opposed to normal neutrophils, degraded in cytosolic extracts from CML neutrophils. The degradation was time dependent and cell concentration dependent. Degradation was also seen in eosinophils, indicating that degradation of LTC4 synthase RNA was correlated to the expression of the protein. This study showed that the difference in expression of LTC4 synthase in normal and CML neutrophils was not because of a total lack of LTC4 synthase mRNA in normal neutrophils. However normal neutrophils lack, in contrast to CML neutrophils, LTC4 synthase mRNA in the cytoplasm. This discrepancy is not caused by a stabilized LTC4 synthase RNA in the cytosol of CML neutrophils. Instead an abnormal degradation of LTC4 synthase RNA was found in the cytosol of CML neutrophils. [source] | |||||||||||||