theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholesterol, we prepared a theta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erythrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by either flow cytometry or laser confocal microscopy. The fluorescence intensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither trypan blue nor propidium iodide, indicating that BC stained just the outer surface of the plasma membrane of vital cells. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensity of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVEC. This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BC theta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on the topology and distribution of cholesterol.
The assembly of newly synthesized histones into chromatin during replication of MH-134SC cells was studied. Cells pulse-labeled with iododeoxyuridine and [3H]lysine were mixed with an equivalent number of normal cells labeled with [14C]lysine. Nuclease chromatin obtained from pooled cells was fractionated by buoyant-density centrifugation in a gradient containing Metrizamide and 3-iodo-1,2-propanediol. Histones extracted from heavy and normal chromatin regions of the gradient were fractionated by acid-urea gel electrophoresis, and 3H/14C ratios of individual histones were compared. The results showed highly preferential association of newly synthesized H3 and H4 with newly replicated DNA.
Alzheimer's disease (AD) is among the most common causes of progressive cognitive impairment in humans and is characterized by neurodegeneration in the brain. Lipid peroxidation is thought to play a role in the pathogenesis of AD. 4-hydroxynonenal (HNE) results from peroxidation of polyunsaturated fatty acids and it in turn gives evidence of lipid peroxidation in vivo. HNE reacts with protein histidine residue to form a stable HNE-histidine Michael adduct. To clarify the influence of lipid peroxidation on the pathogenesis of AD, we measured HNE-histidine Michael adduct in hippocampi from four AD patients and four age-matched controls by means of semiquantitative immunohistochemistry using a specific antibody to cyclic hemiacetal type of HNE-histidine Michael adduct. This antibody does not react with the ring-opened form of HNE-histidine Michael adduct and the pyrrole form of HNE-lysine Michael adduct. The HNE adduct was detected in the hippocampi of both AD and control donors, especially in the CA2, CA3 and CA4 sectors. Immunoreactive intensity of HNE adduct in these sectors were significantly higher in AD patients than in the controls. The HNE adduct was found in the perikarya of pyramidal cells in the hippocampus. These results show that the hippocampi of patients with AD undergo lipid peroxidation and imply that this activity underlies the production of cytotoxic products such as HNE that are responsible for the pathogenesis of AD.The progressive cognitive impairment of Alzheimer's disease (AD) is associated with neuronal loss as well as the formation of neurofibrillary tangles (NFTs) and senile plaques in the brain (25). Free radical-mediated oxidative damage, energy depletion, deposition of amyloids and NFTs, excitotoxicity, and vascular endothelial cell damage are all thought to participate in the pathogenesis of AD (13). Oxygen-derived free radicals, byproducts of respiration, cause oxidative damage to cellular biomolecules including lipids, proteins and nucleic acids. The brain seems to be especially vulnerable to lipid peroxidation by free radicals, because it consumes approximately one-fifth of humans' oxygen intake, has a relative paucity of antioxidant systems and contains high concentrations of polyunsaturated fatty acids (PUFAs) (11). Lipid peroxidation results in structural damage to membranes and generation of secondary products such as reactive aldehydes
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