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Quantitative Western Blotting (quantitative + western_blotting)
Selected AbstractsNucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cellsACTA PHYSIOLOGICA, Issue 2 2010I. Grimm Abstract Aim:, The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADP,S and UTP in neural stem cell migration. Methods:, Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results:, Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion:, Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB. [source] Individual differences in spatial memory among aged rats are related to hippocampal PKC, immunoreactivityHIPPOCAMPUS, Issue 2 2002Paul J. Colombo Abstract We reported previously that the extent of spatial memory impairment among aged rats was correlated positively with levels of protein kinase C, in hippocampal homogenates measured by quantitative Western blotting (Colombo et al., 1997). In the current study, immunocytochemistry was used to test whether the relationship between elevated PKC, and memory impairment among aged rats could be localized further within regions of the hippocampus. Six- and 24-month-old male Long-Evans rats were first trained in the water maze on a standard place-learning task and then trained 2 weeks later on a transfer task designed for rapid acquisition. In comparison with young rats, aged rats with impaired spatial memory had increased PKC,-immunoreactivity (PKC,-ir) in CA1 of the hippocampus, but not the dentate gyrus. In addition, PKC,-ir in CA1 was correlated positively with spatial memory impairment among aged rats on the standard place-learning and the transfer training tasks. The current results are consistent with our previous report of PKC, in hippocampal homogenates, and show further that the relationships between PKC,-ir and memory impairments among aged rats are most evident in area CA1. Thus age-related impairments of spatial memory, as well as deficits in the flexible use of previously acquired information, may result from dysregulation of PKC,. Hippocampus 2002;12:285,289. © 2002 Wiley-Liss, Inc. [source] Mercury stimulates rat liver glucocorticoid receptor association with Hsp90 and Hsp70JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2004Jelena Brklja Abstract The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes. © 2004 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:257,260, 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20032 [source] Ontogeny of human hepatic cytochromes P450JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 4 2007Ronald N. Hines Abstract Significant changes in drug-metabolizing enzyme (DME) expression occur during ontogeny. Such changes can have a profound effect on therapeutic efficacy in the fetus and child, as well as the risk for adverse drug reactions. To gain a better understanding of DME ontogeny, enzyme contents for six key cytochromes P450 were measured in 240 human liver samples representing ages from 8 weeks gestation to 18 years. Where possible, both quantitative western blotting and activity assays with probe substrates were performed. Although oversimplified, the DME can be grouped into one of three categories. As typified by CYP3A7, some enzymes are expressed at their highest level during the first trimester and either remain at high concentrations or decrease during gestation and are silenced or expressed at low levels within 1,2 years after birth. These data cause one to query whether these enzymes have an important endogenous function. Representatives of a second group, CYP3A5 and CYP2C19, are expressed at relatively constant levels throughout gestation. Postnatal increases in CYP2C19 are observed within the first year, but not for CYP3A5. CYP2C9, 2E1, and 3A4 are more typical of a third group of enzymes that are not expressed or are expressed at low levels in the fetus with the onset of expression generally in either the second or third trimester. Substantial increases in expression are observed within the first 1,2 years after birth; however, considerable interindividual variability is observed in the immediate postnatal (1,6 months) onset or increase in expression of these enzymes, often resulting in a window of hypervariability. © 2007 Wiley Periodicals, Inc. J Biochem Mol Toxicol 21:169,175, 2007; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20179 [source] | ||||||||||