The use of seldi proteinchip arrays to monitor production of Alzheimer's betaamyloid in transfected cells.

beta-Amyloid (Abeta), a 39-43 residue peptide, is the principal component of senile plaques found in the brains of patients with Alzheimer's disease (AD). There are two main lines of evidence that its deposition is the cause of neurodegeneration. First, mutations found in three genes in familial Alzheimer's cases give rise to increased production of the longest, most toxic, form, Abeta 1-42. Second. aggregated Abeta is toxic to neuronal cells in culture. Inhibitors of the proteases involved in its release from the amyloid precursor protein are, therefore, of major therapeutic interest. The best candidates for the releasing proteases are both aspartyl proteases, which are integrated into the membranes of the endoplasmic reticulum and Golgi network. A sensitive assay using Ciphergen's Seldi system has been developed to measure all the variants of Abeta in culture supernatants, which will be of great value in screening inhibitors of these proteases. With this assay, it has been shown that increasing intracellular cholesterol increases the activities of both beta-secretase, and gamma-secretase 42. Moreover, changing the intracellular targeting of amyloid precursor glycoprotein (APP) yields increased alpha-secretase cleavage, and increases in the amounts of oxidized/nitrated forms of Abeta.

Oligomerization of beta-amyloid of the Alzheimer's and the Dutch-cerebral-haemorrhage types.

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.

Screening neutral and acidic IgG N-glycans by high density electrophoresis.

IgG carries bi-antennary N-linked glycans which differ in degrees of galactosylation, core fucosylation and bisecting N-acetyl glucosamine. The majority of these are non-sialyated closely related neutral structures which can be resolved by HPLC analysis, but which are difficult to separate in techniques such as fluorophore-coupled carbohydrate electrophoresis. Derivatisation with the singly charged fluorophore, 2-amino benzoic acid and separation in gels with a 30% monomer content in tris/glycine buffer enabled separation of neutral glycans. In particular, agalactosyl glycans with either a core fucose substitution or bisecting N-acetyl galactosamine could be resolved. Good separation of mono- and di-galactosylated glycans was also achieved with this system. It was shown that IgG can be separated from serum by size-exclusion and anion exchange chromatography with minimal contamination, with complete glycan release accomplished by the enzyme peptide-N-glycosidase F (F. meningosepticum). This method of resolving IgG glycans could be used to monitor patients in which glycosylation changes may have a diagnostic value, as in rheumatoid arthritis. It could also be used to monitor recombinant IgG glycosylation where routine screening is required in the biotechnology industry.

The role of cholesterol in the biosynthesis of beta-amyloid.

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.

Inactivation of tissue inhibitor of metalloproteinase-1 by peroxynitrite.

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 metalloproteinase-1 (TIMP-1) in vitro. The inactivation of TIMP-1 by ONOO- was dose dependent with 50 microM ONOO- reducing the inhibitory activity of TIMP-1 towards gelatinase-A by 50%. High concentrations of ONOO- (500 microM-5 mM) caused protein fragmentation whilst lower concentrations (<250 microM) 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.

Holographic sensor for water in solvents.

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.