Chemical characterization of A beta 17-42 peptide, a component of diffuse amyloid deposits of Alzheimer disease.

A peptide corresponding to the amino acid sequence of A beta 17-42 (LVFFAEDVGSNKGAIIGLMVGGVVIA) was isolated from Alzheimer Disease patient brains containing large deposits of diffuse-type amyloid. Brain homogenates were lysed in SDS and submitted to high speed centrifugations. A beta peptides were purified by size exclusion chromatography on Superose 12 and TSK 3000 SW columns.

Synthesis and structure-activity studies of N,N'-diarylguanidine derivatives. N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine: a new, selective noncompetitive NMDA receptor antagonist.

Diarylguanidines, acting as NMDA receptor ion channel site ligands, represent a new class of potential neuroprotective drugs. Several diarylguanidines structurally related to N,N'-di-o-tolylguanidine (DTG), a known selective sigma receptor ligand, were synthesized and evaluated in in vitro radioligand displacement assays, with rat or guinea pig brain membrane homogenates, using the NMDA receptor ion channel site specific radioligand [3H]-(+)-5(S)-methyl-10(R),11-dihydro-5H-dibenzo[a,d]cyclohepten-5 ,10- imine (MK-801, 3), and the sigma receptor-specific radioligand [3H]-di-o-tolylguanidine (DTG, 5). This paper presents the structure-activity relationships leading to novel tri- and tetrasubstituted guanidines, which exhibit high selectivity for NMDA receptor ion channel sites and weak or negligible affinity for sigma receptors.

beta-Amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease.

Reinvestigation of the chemical structure of beta-amyloid peptide (A beta) deposits in the vascular tissue of Alzheimer disease brains revealed that the 42-residue form A beta-(1-42), rather than the more soluble A beta-(1-40) form, is the predominant peptide. Following removal of the surrounding tissue with SDS and collagenase, A beta was solubilized in formic acid and purified by Superose 12 chromatography. Peptides generated by enzymatic and chemical digestion of the A beta were purified by HPLC and characterized by amino acid analysis, sequence analysis, and mass spectrometry.

Chlorinated phenyl azides as photolabeling reagents. Synthesis of an ortho,ortho'-dichlorinated arylazido PCP receptor ligand.

The enhanced photolabeling properties of chlorinated phenyl azides are demonstrated by the synthesis and photolysis of methyl 4-azido-2,3,5,6-tetrachlorobenzoate (3) and methyl 4-azido-3,5-dichlorobenzoate (4). Photolysis of azide 3 in 1 M diethylamine/cyclohexane as the trapping medium gave 34% NH-insertion product. Similar photolysis of azide 4 gave 35% NH insertion product.

A curved-field reflectron for improved energy focusing of product ions in time-of-flight mass spectrometry.

A new type of curved-field reflectron has been developed for the energy focusing of ions formed after initial acceleration in time-of-flight (TOF) mass spectrometers. These include ions generated in the dissociation region of a tandem TOF, as well as metastable decay products formed in the field-free drift region prior to their reflection. Unlike conventional linear-field reflectrons, which focus product ions to different focal lengths that are proportional to the mass (energy) of the fragment, the new curved-field reflectron energy focuses all ions to within a small region near the exit of the reflectron.

Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M.

Neurofilaments are neuronal intermediate filaments that play an important role in the growth and maintenance of large myelinated axons. Mammalian neurofilaments are composed of three polypeptide subunits, designed as NF-L, NF-M, and NF-H, all of which are phosphorylated. Here, we demonstrate by several criteria that neurofilament polypeptides are also modified by an abundant type of intracellular protein glycosylation in which single N-acetylglucosamine monosaccharides are O-glycosidically (O-GlcNAc) linked to serine or threonine residues.

10,5-(Iminomethano)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene and derivatives. Potent PCP receptor ligands.

IDDC (3, 10,5-(iminomethano)-10,11-dihydro-5H-dibenzo[a,d]cycloheptene++ +) and a series of substituted derivatives were synthesized and evaluated in vitro for their ability to displace tritiated MK-801 ([3H]-2) from its specific binding site in guinea pig brain homogenate. Substitution at the 3-position of 3 with bromine, chlorine, and fluorine led to increased binding affinity. In contrast, substitution of donor groups at the 3-position gave decreased binding affinities, as did all substitutions at the 7-position and on nitrogen.

Tandem time-of-flight mass spectrometer.

A compact, laser desorption tandem time-of-flight mass spectrometer is described. The instrument incorporates two dual-stage reflectron analyzers and a collision region for producing product ions by collision-induced dissociation. Selection of ions of a particular mass is accomplished by deflection of ions from stable trajectory angles entering the second reflectron, while the use of a pulsed valve for introduction of the collision gas obviates the need for differential pumping of the collision region.

Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease.

The structure of beta-amyloid (beta A) from Alzheimer disease brains was examined to determine if post-translational modifications might be linked to the abnormal deposition of this peptide in the diseased tissue. The beta A peptides were isolated from the compact amyloid cores of neuritic plaques and separated from minor glycoprotein components by size-exclusion high-pressure liquid chromatography (HPLC). This parenchymal beta A has a maximal length of 42 residues, but shorter forms with "ragged" NH2 termini are also present.

Mass spectrometric analysis of proteins.

The past year has seen greatly increased acceptance and application of the analytical capabilities of mass spectrometry by the biochemical community. The technique has been used to provide accurate mass determinations of non-covalently bound protein complexes, rapid mapping of molecular weights of altered peptides in protease digests, sequencing by collisional activation in tandem mass spectrometry, characterization of glycosylation and other modifications, and quantitation of peptides used in clinical diagnostics.

Effects of a novel NMDA antagonist on experimental stroke rapidly and quantitatively assessed by diffusion-weighted MRI.

We employed diffusion-weighted MRI (DWI) to identify regions of focal brain ischemia during the first 3 hours after permanent occlusion of the middle cerebral artery in rats. Using DWI as early as 30 minutes after the onset of ischemia, it was possible to identify the areas of brain destined to progress to infarction over the next 24 hours in untreated animals, as demonstrated by postmortem evaluation. DWI studies revealed the cerebroprotective effects of a noncompetitive N-methyl-D-aspartate receptor antagonist, CNS 1102, administered 15 minutes postocclusion, both on the cortical and caudoputaminal regions during the initial 3 hours of ischemia.

Preparation and characterization of biologically active 6'-O-(6-aminocaproyl)-4'-O-monophosphoryl lipid A and its conjugated derivative.

N-tert-butyloxycarbonyl (t-Boc) protected 6-aminocaproic (Cap) anhydride was reacted with unprotected hexaacyl-4'-O-monophosphoryl lipid A (MLA) obtained from the lipopolysaccharide of Escherichia coli J5 to yield t-Boc-Cap-MLA. After a column purification step, the t-Boc group was removed by incubating the sample at low temperature in the presence of acid to yield Cap-MLA. This product was analyzed by californium plasma desorption mass spectrometry (PDMS).

Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl3 with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl2 moiety. The present study reveals that BrCCl3 also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCL2 or heme-CCl3 were bound to cysteine residue 93 of the beta-chain of hemoglobin.

A compact time-of-flight mass spectrometer for the structural analysis of biological molecules using laser desorption.

A compact laser-desorption time-of-flight mass spectrometer using a 600 ps nitrogen laser and 1 Gsample/s transient recorder is described. The instrument incorporates two electrically-isolatable, reflecting flight tubes designed for subsequent configuration of the mass spectrometer as a tandem instrument. In this first report, we compare mass resolution in the laser-desorption mass spectra of an organic dye, sinapinic and caffeic acid matrices, and several small peptides.

A herpesvirus maturational proteinase, assemblin: identification of its gene, putative active site domain, and cleavage site.

A herpesvirus proteinase activity has been identified and partially characterized by using the cloned enzyme and substrate genes in transient transfection assays. Evidence is presented that the proteinase gene of cytomegalovirus strain Colburn encodes a 590-amino acid protein whose N-terminal 249 residues contain the proteolytic activity and two domains that are highly conserved in the homologous protein of other herpesviruses. Insertion of a short amino acid sequence between these domains abolished proteinase activity, suggesting that this region constitutes part or all of the enzyme active site.

A matrix-assisted laser desorption time-of-flight mass spectrometer based on a 600 ps, 1.2 mJ nitrogen laser.

The design and performance of a short-pulse UV laser desorption time-of-flight mass spectrometer is described. The instrument combines the advantages of a 600 ps, 337 nm pulsed nitrogen laser with a compact, high-voltage extraction linear time-of-flight analyzer. A number of peptides and proteins have been analyzed to demonstrate sensitivity, high mass range, resolution and mixture analysis.

Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021-7.

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021-7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR-type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A.

Secretion of the beta/A4 amyloid precursor protein. Identification of a cleavage site in cultured mammalian cells.

Alzheimer's disease, a progressive neurodegenerative disorder, affects greater than 10% of the population of individuals greater than 65 years of age. A principal neuropathological feature of this disease is the senile plaque, a fibrillar extracellular deposit primarily composed of a approximately 4-kDa peptide, beta/A4, derived from the amyloid precursor protein (APP). Studies in cultured cells have documented that APP matures through a constitutive secretory pathway and is cleaved at or near the cell surface to release a large ectodomain into the extracellular space.

Endothermic ion molecule reactions.

Endothermic ion-molecule reactions in a tandem mass spectrometer have been used for a number of years for determining thermodynamic quantities, such as heats of formation and proton affinities, for gaseous ions. Recently, the reactive, endothermic collision has been exploited as an analytical technique for the structural analysis of peptides and other biomolecules. The technique is based upon the endothermic transfer of protons associated with amide bonds to ammonia.

Chemical reduction of 3-oxo and unsaturated groups in fatty acids of diphosphoryl lipid A from the lipopolysaccharide of Rhodopseudomonas sphaeroides. Comparison of biological properties before and after reduction.

Unlike the diphosphoryl lipid A (DPLA) derived from toxic lipopolysaccharide of Escherichia coli and Salmonella strains, the DPLA from nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 is biologically inactive. This could be due to the presence of 3-oxotetradecanoic and delta 7-tetradecenoic acids. These two fatty acids in R.

Scrapie prion rod formation in vitro requires both detergent extraction and limited proteolysis.

Scrapie prion infectivity can be enriched from hamster brain homogenates by using limited proteolysis and detergent extraction. Purified fractions contain both scrapie infectivity and the protein PrP 27-30, which is aggregated in the form of prion rods. During purification, PrP 27-30 is produced from a larger membrane protein, PrPSc, by limited proteolysis with proteinase K.

High-performance liquid chromatography-time-of-flight mass spectrometry and its application to peptide analyses.

High-performance liquid chromatography (HPLC) has been successfully interfaced on-line with liquid secondary-ion time-of-flight mass spectrometry, utilizing a continuous-flow interface. Time-of-flight mass spectrometry (TOF-MS) is a low-resolution, high-mass-range technique, compatible with extremely rapid data acquisition rates. Thus a TOF-MS system is extremely well suited for coupling with HPLC.

Medium-chain-triglyceride lipid emulsion: metabolism and tissue distribution.

The utilization and distribution of radioactively labeled lipid emulsions were evaluated in Sprague-Dawley rats. Animals received one of three lipid emulsions. Group 1 received [14C]medium-chain-triglyceride (MCT) lipid emulsion, group 2 received a 75%:25% (vol:vol) admixture of [14C]MCT: unlabeled long-chain-triglyceride (LCT) lipid emulsion, and group 3 received only [14C]LCT.