Addition of the beta-hydroxy-beta-methylglutaryl-CoA (HmG-CoA) reductase inhibitor lovastatin to human HEK cells transfected with the amyloid precursor protein (APP) reduces intracellular cholesterol/protein ratios by 50%, and markedly inhibits beta-secretase cleavage of newly-synthesized APP. Exogenous water-solubilized cholesterol at 200 microg/ml concentration increases newly synthesized beta-amyloidogenic products four-fold. These intracellular changes are detectable by immunoprecipitation and immunofluorescent labelling. Analyses of the fragments captured from culture medium by an N-terminal anti-beta-amyloid antibody on ProteinChip arrays and detected using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry revealed that culture with cholesterol (200 microg/ml) increased secretion of beta-amyloid 1-40 by 1.8-fold, and increased secretion of beta-amyloid 1-42. Changes in APP processing by cholesterol may mediate the way in which the ApoE4 allele increases risk of developing Alzheimer's disease (AD) in western populations.
Peroxynitrite (ONOO-) has recently been implicated in connective tissue destruction in vivo. We have studied the effect of ONOO-on the activity of tissue inhibitor of metalloprotelnase-1 (TIMP-1) in vitro. The inactivation of TIMP-1 by ONOO-was dose dependent with 50 ~tM ONOOreducing the inhibitory activity of TIMP-1 towards gelatiuase-A by 50%. High concentrations of ONOO-(500 ~tM-5 mM) caused protein fragmentation whilst lower concentrations (< 250 ~tM) inactivated TIMP-1 without altering the molecular weight. Inactivation could be blocked by ONOO scavengers but not by hydroxyl radical scavengers. Our results show that ONOO-is capable of inactivating TIMP-1, a process which could potentiate metalloproteinase-mediated tissue breakdown.
The diffraction color of a gelatin holographic diffraction grating changed as a function of the water activity when immersed in a "wet" hydrophobic liquid. Quantification of the absorption maximum of the diffracted light showed that it was related, after calibration, to either the water content or the water activity of the solvent. The holographic diffraction grating measured water contents of hydrocarbon solvents at sensitivities comparable to that of the Karl Fischer coulometric titrator and over a wide range of water contents. A grating immersed in xylene revealed a visible color change when the water content was increased from 47 to 120 ppm. Conversely, the holographic grating responded to ethanol in water in the range 0-1% (w/w). The inexpensiveness and simplicity of silver halide holographic reflection gratings, combined with their relatively high sensitivity, suggests that these devices might find widespread application as immersible water activity sensors for hydrophobic liquids.
A novel ELISA has been developed which detects oligomerization of beta-amyloid (A beta). Oligomerization, fibrillization and neurotoxicity of native A beta associated with Alzheimer's disease (AD) type has been compared with E22Q A beta (amyloid beta-protein containing residues 1--40 with the native Glu at residue 22 changed to Gln) implicated in Dutch cerebral haemorrhage disease. Solutions of A beta rapidly yield soluble oligomers in a concentration-dependent manner, which are detected by the ELISA, and by size-exclusion gel chromatography. Conformational changes from disordered to beta-sheet occur more slowly than oligomerization, and fibrils are produced after prolonged incubation. The E22Q A beta oligomerizes, changes conformation and fibrillizes more rapidly than the native form and produces shorter stubbier fibrils. Aged fibrillar preparations of E22Q A beta are more potent than aged fibrils of native A beta in inducing apoptotic changes and toxic responses in human neuroblastoma cell lines, whereas low-molecular-mass oligomers in briefly incubated solutions are much less potent. The differences in the rates of oligomerization of the two A beta forms, their conformational behaviour over a range of pH values, and NMR data reported elsewhere, are consistent with a molecular model of oligomerization in which strands of A beta monomers initially overcome charge repulsion to form dimers in parallel beta-sheet arrangement, stabilized by intramolecular hydrophobic interactions, with amino acids of adjacent chains in register.
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