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Tyler A Kokjohn,
Gregory D Van Vickle,
Chera L Maarouf,
Walter M Kalback,
Jesse M Hunter,
Ian D Daugs,
Dean C Luehrs,
John Lopez, Daniel Brune,
Lucia I Sue,
Thomas G Beach,
Eduardo M Castaño,
Alex E Roher
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ABSTRACT: Amyloid-β (Aβ) peptides are intimately involved in the inflammatory pathology of atherosclerotic vascular disease (AVD) and Alzheimer's disease (AD). Although substantial amounts of these peptides are produced in the periphery, their role and significance to vascular disease outside the brain requires further investigation. Amyloid-β peptides present in the walls of human aorta atherosclerotic lesions as well as activated and non-activated human platelets were isolated using sequential size-exclusion columns and HPLC reverse-phase methods. The Aβ peptide isolates were quantified by ELISA and structurally analyzed using MALDI-TOF mass spectrometry procedures. Our experiments revealed that both aorta and platelets contained Aβ peptides, predominately Aβ40. The source of the Aβ pool in aortic atherosclerosis lesions is probably the activated platelets and/or vascular wall cells expressing APP/PN2. Significant levels of Aβ42 are present in the plasma, suggesting that this reservoir makes a minor contribution to atherosclerotic plaques. Our data reveal that although aortic atherosclerosis and AD cerebrovascular amyloidosis exhibit clearly divergent end-stage manifestations, both vascular diseases share some key pathophysiological promoting elements and pathways. Whether they happen to be deposited in vessels of the central nervous system or atherosclerotic plaques in the periphery, Aβ peptides may promote and perhaps synergize chronic inflammatory processes which culminate in the degeneration, malfunction and ultimate destruction of arterial walls.
Biochimica et Biophysica Acta 07/2011; 1812(11):1508-14. · 4.66 Impact Factor
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ABSTRACT: Our purpose is to apply a fatty acid secretion strategy in photosynthetic microbial biofuel production, which will avoid the costly biomass recovery processes currently applied in algal biofuel systems. Starting with introducing acyl-acyl carrier protein thioesterases, we made five successive generations of genetic modifications into cyanobacterium Synechocystis sp. PCC 6803. The mutant strains were able to overproduce fatty acids (C10-C18) and secrete them into the medium at an efficiency of up to 133 +/- 12 mg/L of culture per day at a cell density of 1.5 x 10(8) cells/mL (0.23 g of dry weight/liter). Fatty acid secretion yields were increased by weakening the S layer and peptidoglycan layers. Although the fatty acid secreting strains had a long lag phase with many cells having damaged cell membranes when grown at low cell densities, these strains grew more rapidly in stationary phase and exhibited less cell damage than wild-type in a stationary culture. Our results suggest that fatty acid secreting cyanobacteria are a promising technology for renewable biofuel production.
Proceedings of the National Academy of Sciences 03/2010; · 9.68 Impact Factor
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Gregory D Van Vickle,
Chera L Esh,
Walter M Kalback,
R Lyle Patton,
Dean C Luehrs,
Tyler A Kokjohn,
Frederick G Fifield,
Paul E Fraser,
David Westaway,
Joanne McLaurin,
John Lopez, Daniel Brune,
Amanda J Newel,
Marissa Poston,
Thomas G Beach,
Alex E Roher
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ABSTRACT: We investigated the morphology and biochemistry of the amyloid-beta (Abeta) peptides produced in TgCRND8 Tg mice carrying combined amyloid precursor protein (APP) Swedish (K670M/N671L) and Indiana (V717F) mutations. Histological analyses employing amyloid-specific staining and electron microscopy revealed that the TgCRND8 Tg mice produce an aggressive pathology, evident as early as 3 months of age, that is a composite of core plaques and peculiar floccular diffuse parenchymal deposits. The Abeta peptides were purified using combined FPLC-HPLC, Western blots, and immunoprecipitation methods and characterized by MALDI-TOF/SELDI-TOF mass spectrometry. The C-terminal APP peptides, assessed by Western blot experiments and mass spectrometry, suggested an alteration in the order of secretase processing, yielding a C-terminal fragment pattern that is substantially different from that observed in sporadic Alzheimer's disease (AD). This modified processing pattern generated longer Abeta peptides, as well as those ending at residues 40/42/43, which may partially explain the early onset and destructive nature of familial AD caused by APP mutations. Despite an aggressive pathology that extended to the cerebellum and white matter, these animals tolerated the presence of an imposing amount of Abeta load. Abeta immunization resulted in an impressive 7-fold reduction in the number of amyloid core plaques and, as previously demonstrated, a significant memory recovery. However, given the phylogenetic distance and the differences in APP processing and Abeta chemistry between Tg mice and AD, caution should be applied in projecting mouse therapeutic interventions onto human subjects.
Biochemistry 10/2007; 46(36):10317-27. · 3.42 Impact Factor
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R Lyle Patton,
Walter M Kalback,
Chera L Esh,
Tyler A Kokjohn,
Gregory D Van Vickle,
Dean C Luehrs,
Yu-Min Kuo,
John Lopez, Daniel Brune,
Isidro Ferrer,
Eliezer Masliah,
Amanda J Newel,
Thomas G Beach,
Eduardo M Castaño,
Alex E Roher
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ABSTRACT: Experiments with amyloid-beta (Abeta)-42-immunized transgenic mouse models of Alzheimer's disease have revealed amyloid plaque disruption and apparent cognitive function recovery. Neuropathological examination of patients vaccinated against purified Abeta-42 (AN-1792) has demonstrated that senile plaque disruption occurred in immunized humans as well. Here, we examined tissue histology and quantified and biochemically characterized the remnant amyloid peptides in the gray and white matter and leptomeningeal/cortical vessels of two AN-1792-vaccinated patients, one of whom developed meningoencephalitis. Compact core and diffuse amyloid deposits in both vaccinated individuals were focally absent in some regions. Although parenchymal amyloid was focally disaggregated, vascular deposits were relatively preserved or even increased. Immunoassay revealed that total soluble amyloid levels were sharply elevated in vaccinated patient gray and white matter compared with Alzheimer's disease cases. Our experiments suggest that although immunization disrupted amyloid deposits, vascular capture prevented large-scale egress of Abeta peptides. Trapped, solubilized amyloid peptides may ultimately have cascading toxic effects on cerebrovascular, gray and white matter tissues. Anti-amyloid immunization may be most effective not as therapeutic or mitigating measures but as a prophylactic measure when Abeta deposition is still minimal. This may allow Abeta mobilization under conditions in which drainage and degradation of these toxic peptides is efficient.
American Journal Of Pathology 10/2006; 169(3):1048-63. · 4.89 Impact Factor
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Chera Esh,
Lyle Patton,
Walter Kalback,
Tyler A Kokjohn,
John Lopez, Daniel Brune,
Amanda J Newell,
Thomas Beach,
Dale Schenk,
Dora Games,
Steven Paul,
Kelly Bales,
Bernardino Ghetti,
Eduardo M Castaño,
Alex E Roher
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ABSTRACT: Central to the pathology of Alzheimer's disease (AD) is the profuse accumulation of amyloid-beta (Abeta) peptides in the brain of affected individuals, and several amyloid precursor protein (APP) transgenic (Tg) mice models have been created to mimic Abeta deposition. Among these, the PDAPP Tg mice carrying the familial AD APP 717 Val --> Phe mutation have been widely used to test potential AD therapeutic interventions including active and passive anti-Abeta immunizations. The structure and biochemistry of the PDAPP Tg mice Abeta-related peptides were investigated using acid and detergent lysis of brain tissue, ultracentrifugation, FPLC, HPLC, enzymatic and chemical cleavage of peptides, Western blot, immunoprecipitation, and MALDI-TOF and SELDI-TOF mass spectrometry. Our experiments reveal that PDAPP mice produce a variety of C-terminally elongated Abeta peptides in addition to Abeta n-40 and Abeta n-42, as well as N-terminally truncated peptides, suggesting anomalous proteolysis of both APP and Abeta. Important alterations in the overall APP degradation also occur in this model, resulting in a striking comparative lack of CT83 and CT99 fragments, which may be inherent to the strain of mice, a generalized gamma-secretase failure, or the ultimate manifestation of the overwhelming amount of expressed human transgene; these alterations are not observed in other strains of APP Tg mice or in sporadic AD. Understanding at the molecular level the nature of these important animal models will permit a better understanding of therapeutic interventions directed to prevent, delay, or reverse the ravages of sporadic AD.
Biochemistry 11/2005; 44(42):13807-19. · 3.42 Impact Factor
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Alex E Roher,
Tyler A Kokjohn,
Chera Esh,
Nicole Weiss,
Jennifer Childress,
Walter Kalback,
Dean C Luehrs,
John Lopez, Daniel Brune,
Yu-Min Kuo,
Martin Farlow,
Jill Murrell,
Ruben Vidal,
Bernardino Ghetti
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ABSTRACT: One of the familial forms of Alzheimer's disease (AD) encodes the amyloid-beta precursor protein (AbetaPP) substitution mutation V717F. This mutation is relevant to AD research, since it has been utilized to generate transgenic mice models to study AD pathology and therapeutic interventions. Amyloid beta (Abeta) peptides were obtained from the cerebral tissue of three familial AD subjects carrying the AbetaPP V717F mutation. A combination of ultracentrifugation, size-exclusion, and reverse-phase high performance liquid chromatography, tryptic and cyanogen bromide hydrolysis, amino acid analysis, and matrix-assisted laser desorption ionization and surface-enhanced laser desorption ionization mass spectrometry was used to characterize the familial AD mutant Abeta peptides. The AbetaPP V717F mutation, located 4-6 residues beyond the wild-type AbetaPP gamma-secretase cleavage site, yielded longer Abeta peptides with C termini between residues 43 and 54. In the cerebral cortex these peptides aggregated into thin water- and SDS-insoluble amyloid bundles that condensed into flocculent spherical plaques. In the leptomeningeal arteries the amyloid was deposited in moderate amounts and was primarily composed of the shorter and more soluble Abeta species ending at residues 40, 42, and 44. The single V717F mutation in AbetaPP results in distinctive and drastic changes in the length and tertiary structure of Abeta peptides, which appear to be responsible for the earlier clinical manifestations of dementia and death of these patients.
Journal of Biological Chemistry 03/2004; 279(7):5829-36. · 4.77 Impact Factor
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Alex E. Roher,
Tyler A. Kokjohn,
Chera Esh,
Nicole Weiss,
Jennifer Childress,
Walter Kalback,
Dean C. Luehrs,
John Lopez, Daniel Brune,
Yu-Min Kuo,
Martin Farlow,
Jill Murrell,
Ruben Vidal,
Bernardino Ghetti
[show abstract]
[hide abstract]
ABSTRACT: One of the familial forms of Alzheimer's disease (AD) encodes the amyloid-β precursor protein (AβPP) substitution mutation
V717F. This mutation is relevant to AD research, since it has been utilized to generate transgenic mice models to study AD
pathology and therapeutic interventions. Amyloid beta (Aβ) peptides were obtained from the cerebral tissue of three familial
AD subjects carrying the AβPP V717F mutation. A combination of ultracentrifugation, size-exclusion, and reverse-phase high
performance liquid chromatography, tryptic and cyanogen bromide hydrolysis, amino acid analysis, and matrix-assisted laser
desorption ionization and surface-enhanced laser desorption ionization mass spectrometry was used to characterize the familial
AD mutant Aβ peptides. The AβPP V717F mutation, located 4-6 residues beyond the wild-type AβPP γ-secretase cleavage site,
yielded longer Aβ peptides with C termini between residues 43 and 54. In the cerebral cortex these peptides aggregated into
thin water- and SDS-insoluble amyloid bundles that condensed into flocculent spherical plaques. In the leptomeningeal arteries
the amyloid was deposited in moderate amounts and was primarily composed of the shorter and more soluble Aβ species ending
at residues 40, 42, and 44. The single V717F mutation in AβPP results in distinctive and drastic changes in the length and
tertiary structure of Aβ peptides, which appear to be responsible for the earlier clinical manifestations of dementia and
death of these patients.
Journal of Biological Chemistry 02/2004; 279(7):5829-5836. · 4.77 Impact Factor
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ABSTRACT: Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD). Clearance of Abeta thus represents a potential therapeutic approach. Antibody-mediated clearance of Abeta by vaccination inhibited and cleared Abeta deposition in animal models; however, inflammatory side effects were observed in humans. An alternative potentially noninflammatory approach to facilitate clearance is to proteolytically cleave Abeta. We screened 12 proteolytic recombinant antibody fragments for potential alpha-secretase activity, a naturally occurring enzyme that cleaves between the Lys16 and Leu17 residues of the amyloid precursor protein (APP). We utilized the synthetic alpha-secretase substrate, benzyloxycarbonyl-l-lysine o-nitrophenyl ester (Z-lys-o-Np) as a preliminary screen for alpha-secretase activity. Two antibody light chain fragments that hydrolyzed Z-lys-o-Np were identified. Abeta hydrolysis was studied using mass spectrometry to identify the cleavage patterns of the antibodies. The recombinant antibody light chain antibody fragment, c23.5, showed alpha-secretase-like activity, producing the 1-16 and 17-40 amino acid fragments of Abeta. The second light chain antibody fragment, hk14, demonstrated carboxypeptidase-like activity, cleaving sequentially from the carboxyl terminal of Abeta. These antibody light chains provide a novel route toward engineering efficient therapeutic antibodies capable of cleaving Abeta in vivo.
Biochemistry 01/2004; 42(48):14328-34. · 3.42 Impact Factor
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Walter Kalback,
M Desiree Watson,
Tyler A Kokjohn,
Yu-Min Kuo,
Nicole Weiss,
Dean C Luehrs,
John Lopez, Daniel Brune,
Sangram S Sisodia,
Matthias Staufenbiel,
Mark Emmerling,
Alex E Roher
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ABSTRACT: The amyloid (Abeta) peptides generated in Hsiao's APP Tg2576 transgenic (Tg) mice are physically and chemically distinct from those characteristic of Alzheimer's disease (AD). Transgenic mouse Abeta peptides were purified using sequential size-exclusion and reverse-phase chromatographic systems and subjected to amino acid sequencing and mass spectrometry analyses. The mouse Abeta peptides lacked the extensive N-terminal degradations, posttranslational modifications, and cross-linkages abundant in the stable Abeta peptide deposits observed in AD. Truncated Abeta molecules appear to be generated in vivo by hydrolysis at multiple sites rather than by post-mortem C-terminal degradation. In contrast to AD amyloid cores, the Tg mice peptides were soluble in Tris-SDS-EDTA solutions, revealing both monomeric and SDS-stable oligomeric species of Abeta. In contrast to our report on Novartis Pharma APP23 Tg mice [Kuo et al. (2001) J. Biol. Chem. 276, 12991], which maintain high levels of soluble Abeta early on with later development of extensive vascular amyloid, Tg2576 mice exhibited an age-related elevation of soluble Abeta with relatively limited vascular amyloid deposition. The transgenic mouse levels of carboxy-terminal (CT) APP fragments were nearly 10-fold greater than those of human brains, and this condition may contribute to the unique pathology observed in these animals. Immunization of transgenic mice may act to prevent the pathological effects of betaAPP overproduction by binding CT molecules or halting their processing to toxic forms, in addition to having any effects on Abeta itself. Thus, differences in disease evolution and biochemistry must be considered when using transgenic animals to evaluate drugs or therapeutic interventions intended to reduce the Abeta burden in Alzheimer's disease.
Biochemistry 02/2002; 41(3):922-8. · 3.42 Impact Factor
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Yu-Min Kuo,
Tyler A. Kokjohn,
Thomas G. Beach,
Lucia I. Sue, Daniel Brune,
John C. Lopez,
Walter M. Kalback,
Dorothee Abramowski,
Christine Sturchler-Pierrat,
Matthias Staufenbiel,
Alex E. Roher
[show abstract]
[hide abstract]
ABSTRACT: We have undertaken an integrated chemical and morphological comparison of the amyloid-β (Aβ) molecules and the amyloid plaques
present in the brains of APP23 transgenic (tg) mice and human Alzheimer's disease (AD) patients. Despite an apparent overall
structural resemblance to AD pathology, our detailed chemical analyses revealed that although the amyloid plaques characteristic
of AD contain cores that are highly resistant to chemical and physical disruption, the tg mice produced amyloid cores that
were completely soluble in buffers containing SDS. Aβ chemical alterations account for the extreme stability of AD plaque
core amyloid. The corresponding lack of post-translational modifications such as N-terminal degradation, isomerization, racemization,
pyroglutamyl formation, oxidation, and covalently linked dimers in tg mouse Aβ provides an explanation for the differences
in solubility between human AD and the APP23 tg mouse plaques. We hypothesize either that insufficient time is available for
Aβ structural modifications or that the complex species-specific environment of the human disease is not precisely replicated
in the tg mice. The appraisal of therapeutic agents or protocols in these animal models must be judged in the context of the
lack of complete equivalence between the transgenic mouse plaques and the human AD lesions.
Journal of Biological Chemistry 04/2001; 276(16):12991-12998. · 4.77 Impact Factor
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ABSTRACT: The gene coding for the light-dependent NADPH :protochlorophyllide oxidoreductase (POR) was interrupted or deleted in a Synechocystis sp. PCC 6803 strain lacking photosystem I (PS I) as well as ChlL, which takes part in light-independent catalysis of protochlorophyllide reduction. Interruption of por by a kanamycin-resistance cartridge between the codons for M263 and V264 (about 83 % into the coding region) did not abolish POR activity, but resulted in a decrease in the protochlorophyllide-(PChlide)-binding capacity of POR. Deletion of por in the PS I-less/chlL− strain generated a mutant [PS I-less/chlL−/por (del)] which accumulated both monovinyl-PChlide and divinyl-PChlide and excreted PChlides into the medium. This mutant also synthesized small amounts of protochlorophyllide dihydrogeranylgeraniol ester (protochlorophyll) when it was grown under light-activated heterotrophic growth conditions. However, the mutant was still able to synthesize small amounts of normal chlorophyll a under weak continuous illumination, even though the quantum yield of chlorophyll a formation was reduced. Either protochlorophyll or PChlide reduction by an unspecific reductase or by a ChlB/ChlN complex could account for chlorophyll a synthesis in the PS I-less/chlL−/por (del) strain. Functional photosystem II (PS II) was assembled in this mutant, but the PS II/chlorophyll ratio was fourfold lower than in the PS I-less strain with normal chlorophyll synthesis. The PS I-less/chlL−/por (del) mutant had a 77-K fluorescence emission maximum at 685 nm but no peak or shoulder at 695 nm when the cells were excited at 435 nm. Much of the chlorophyll in the PS I-less/chlL−/por (del) mutant therefore seems to be associated with components other than PS II.
European Journal of Biochemistry. 03/1998; 253(1):161 - 172.
