Amina S. Woods

Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease

In the course of analyzing the chemical composition of Alzheimers disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Aβ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Aβ-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Aβ-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Aβ-(1-40/42) dimers and trimers from Alzheimers disease amyloid represent the soluble oligomers of Aβ recently described in Alzheimers disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens.

The development of many autoimmune diseases has been etiologically linked to exposure to infectious agents. For example, a subset of patients with a history of Salmonella infection develop reactive arthritis. The persistence of bacterial antigen in arthritic tissue and the isolation of Salmonella or Yersinia reactive CD8+ T cells from the joints of patients with reactive arthritis support the etiological link between Gram-negative bacterial infection and autoimmune disease. Models proposed to account for the link between infection and autoimmunity include inflammation-induced presentation of cryptic self-epitopes, antigen persistence and molecular mimicry. Several studies support molecular mimicry as a mechanism for the involvement of class II epitopes in infectious disease-induced self-reactivity. Here, we have identified an immunodominant epitope derived from the S. typhimurium GroEL molecule. This epitope is presented by the mouse H2-T23-encoded class Ib molecule Qa-1 and was recognized by CD8+ cytotoxic T lymphocytes induced after natural infection. S. typhimurium-stimulated cytotoxic T lymphocytes recognizing the GroEL epitope cross-reacted with a peptide derived from mouse heat shock protein 60 and recognized stressed macrophages. Our results indicate involvement of MHC class Ib molecules in infection-induced autoimmune recognition and indicate a mechanism for the etiological link between Gram-negative bacterial infection and autoimmunity.

Elevated Aβ42 in Skeletal Muscle of Alzheimer Disease Patients Suggests Peripheral Alterations of AβPP Metabolism

The levels of amyloid-β40 (Aβ40) and Aβ42 peptides were quantified in temporalis muscles and brain of neuropathologically diagnosed Alzheimer disease (AD) and of nondemented individuals. This was achieved by using a novel analytical approach consisting of a combination of fast-performance liquid chromatographic (FPLC) size exclusion chromatography developed under denaturing conditions and europium immunoassay on the 4.0- to 4.5-kd fractions. In the temporalis muscles of the AD and nondemented control groups, the average values for Aβ42 were 15.7 ng/g and 10.2 ng/g (P = 0.010), and for Aβ40 they were 37.8 ng/g and 29.8 ng/g (P = 0.067), respectively. Multiple regression analyses of the AD and control combined populations indicated that 1) muscle Aβ40 and muscle Aβ42 levels were correlated with each other (P < 0.001), 2) muscle Aβ40 levels were positively correlated with age (P = 0.036), and 3) muscle Aβ42 levels were positively correlated with Braak stage (P = 0.042). Other forms of the Aβ peptide were discovered by mass spectrometry, revealing the presence of Aβ starting at residues 1, 6, 7, 9, 10, and 11 and ending at residues 40, 42, 44, 45, and 46. It is possible that in AD the skeletal muscle may contribute to the elevated plasma pool of Aβ and thus indirectly to the amyloid deposits of the brain parenchyma and cerebral blood vessels. The increased levels of Aβ in the temporalis muscles of AD patients suggest that alterations in AβPP and Aβ metabolism may be manifested in peripheral tissues.

Identification of a ganglioside recognition domain of tetanus toxin using a novel ganglioside photoaffinity ligand.

Tetanus toxin entry into vertebrate motorneurons may involve binding of neuronal surface gangliosides containing the “1b” substructure (a NeuAcα2,8NeuAc group on an internal galactose residue). The domains of tetanus toxin involved in ganglioside binding are known to reside within the carboxyl-terminal half of the toxin’s heavy chain (“C fragment”). We developed a novel photoaffinity reagent based upon the structure of the 1b ganglioside GD1b(125I-azido-GD1b) to define the ganglioside-binding domains of tetanus toxin. Using this ligand, we observed radiolabeling of tetanus toxin C fragment which could be specifically inhibited by a ganglioside of the 1b series (GT1b), but not by a non-1b series ganglioside (GM3). When tetanus toxin C fragment was proteolyzed with clostripain, whether before or after reaction with125I-azido-GD1b, a radiolabeled band was observed by SDS-polyacrylamide gel electrophoresis autoradiography, which was selectively inhibited by GT1b. Protein sequencing of proteolyzed tetanus toxin C fragment co-migrating with that band revealed the carboxyl-terminal 34 amino acid residues of tetanus toxin. Matrix-assisted laser desorption/ionization mass spectrometry of a photoaffinity labeled synthetic polypeptide representing the 34-amino acid domain revealed modification at a single residue (His1293). We propose that this domain of tetanus toxin is sufficient for ganglioside binding.

Biologic response (antiviral) to recombinant human interferon alpha 2a as a aunction of dose and route of administration in healthy volunteers

The kinetics of the antiviral effect of intramuscular and intravenous injections of recombinant human interferon α2a were investigated in healthy volunteers. Cohorts of eight to 11 subjects received single intramuscular injections of either 0.3 × 106, 3 × 106, or 18 × 106 U or an intravenous infusion of 18 × 106 U over 30 minutes. Serial samples of peripheral blood mononuclear cells were analyzed for antiviral effects including both (2‐5) oligoadenylate synthetase activity and resistance to vesicular stomatitis virus infection in vitro. A dose‐response relationship was established between recombinant human interferon α2a dose and both vesicular stomatitis virus resistance and (2‐5) oligoadenylate synthetase activity. At the 0.3 × 106 U dose an antiviral effect occurred without clinical side effects. The presence of clinical side effects is not necessary for an antiviral effect.

Peptide Sequence Information Derived by Pronase Digestion and Ammonium Sulfate In-Source Decay Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

We present the use of Pronase digestion and in-source decay in the presence of ammonium sulfate as complementary techniques to confirm the amino acid sequence of a peptide. Pronase, a commercial preparation from Steptomyces griseus, is a combination of proteolytic enzymes. It produces carboxypeptidase and aminopeptidase ladders using a single Pronase digestion and represents an inexpensive, nonspecific, and fast supplement to traditional sequencing enzymes. However, N-terminal peptidase activity appears dependent on the terminal amino acid residue. We also introduce the use of saturated ammonium sulfate as an “on-slide” sample additive to promote in-source fragmentation of peptides. Use of saturated ammonium sulfate resulted in a simple way to increase peptide backbone fragmentation and essentially produced either a cn or ym ion series. Together these techniques provide useful supplements to existing methods for peptide sequence information.

Ferrichrome: Surprising stability of a cyclic peptide-FeIII complex revealed by mass spectrometry

Ferrichrome, a fungal siderophore that is also utilized by some bacterial species, was studied with liquid secondary ion mass spectrometry (LSIMS) and matrix-assisted laser desorption ionixation (MALDI) mass spectrometry. A strong ionic signal corresponding to a FeIII complex was observed with LSIMS in the positive ion mode. Switching the polarity of the mass spectrometer did not necessarily result in reduction of ferric ion, although certain conditions led to appearance of a FeII complex signal as well. The results of the structural studies of the metal ion-cyclic peptide complex with collisionally induced dissociation allowed unambiguous identification of the chelation sites. The action of the siderophore on FeIII was studied by in vitro chelation of ferric ion (from ferric citrate) by the iron-free ferrichrome. Effective chelation of ferric ion was compared to actions of the iron-free ferrichrome on other metal ions. Unlike LSIMS, desorption with MALDI did not form selectively molecular ions of intact ferrichrome: the spectra contained abundant peaks corresponding to the cyclic peptide itself and its nonspecific association with alkali metal ions.

Enzymatic digestion on the sample foil as a method for sequence determination by plasma desorption mass spectrometry: the primary structure of porpoise relaxin

Abstract Plasma desorption mass spectrometry (PDMS) was used to determine the C-terminus of the B-chain porpoise relaxin, a polypeptide hormone having three dimensional and disulfide homology with insulin. Trypsin was used to generate a series of peptide fragments for mapping by PDMS. The C-terminal fragments were purified and their sequences determined by on-foil digestion with carboxypeptidase Y.

Simple preparation of multi-valent cyclodextrin–carbohydrate conjugates

Abstract Several β-cyclodextrin (CD) derivatives conjugated with carbohydrates via aminohexyl linkages have been prepared. These CD-conjugates were demonstrated to be multi-valent ligands by lectin-binding assay.

Effects of prednisone, aspirin, and acetaminophen on an in vivo biologic response to interferon in humans

In healthy volunteers receiving a single intramuscular dose of 18 × 106 U interferon alone or after 24 hours of an 8‐day course of prednisone (40 mg/day), aspirin (650 mg every 4 hours), or acetaminophen (650 mg every 4 hours), the magnitude of the biologic response to interferon was quantified by measuring the time course of the induction of 2′‐5′‐oligoadenylate synthetase and resistance to vesicular stomatitis virus infection in human peripheral blood mononuclear cells. Prednisone decreased the AUC of 2′‐5′‐oligoadenylate synthetase activity (p < 0.05), whereas administration of aspirin or acetaminophen did not affect this biologic response. No measurable effect was seen during administration of prednisone, aspirin, or acetaminophen on the duration or intensity of vesicular stomatitis virus yield reduction. The side effects seen with interferon administration at the dose tested were not altered in a clinically meaningful manner by prednisone, aspirin, or acetaminophen.