45 results on '"Michael R. Nichols"'
Search Results
2. Development of a Simple and Effective Lipid-A Antagonist Based on Computational Prediction
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Olivia Slater, Kapur B. Dhami, Ganesh Shrestha, Maria Kontoyianni, Michael R. Nichols, and Alexei V. Demchenko
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Inflammation ,Lipopolysaccharides ,Toll-Like Receptor 4 ,Infectious Diseases ,Binding Sites ,Lipid A ,Humans ,Article - Abstract
Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. In an effort to identify compounds that block lipopolysaccharide (LPS)-induced inflammation reported herein is the development of simple Lipid-A analogues that lack a disaccharide core yet still possess potent antagonistic activity against LPS. The structure of the new lead compound was developed based on predictive computational experiments. LPS antagonism by the lead compound was not straightforward, and a biphasic effect was observed suggesting a possibility of more than one binding site. An IC(50) value of 13 nM for the new compound was determined for the possible high affinity site. The combination of computational, synthetic, and biological studies revealed new structural determinants of these simplified analogues. It is expected that the acquired information will aid future design of LPS targeting glycopharmaceuticals.
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- 2022
3. Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and - independent cellular responses in Alzheimer’s disease
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Yingyue Zhou, Wilbur M. Song, Prabhakar Andhey, Amanda Swain, Tyler Levy, Kelly R Miller, Pietro L. Poliani, Manuela Cominelli, Shikha Grover, Susan Gilfillan, Marina Cella, Tyler K Ulland, Konstantin Zaitsev, Akinori Miyashita, Takeshi Ikeuchi, Makoto Sainouchi, Akiyoshi Kakita, David A Bennett, Julie A Schneider, Michael R Nichols, Sean A Beausoleil, Jason Ulrich, David M Holtzman, Maxim Artyomov, and Marco Colonna
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nervous system ,Immunology ,Immunology and Allergy ,Article - Abstract
Alzheimer’s disease (AD) is the most common form of dementia that is initiated by extracellular plaque deposition and intraneuronal hyperphosphorylated tau aggregates. Reactive astrocytosis and microgliosis are secondary cellular responses to pathology that have gained increasing attention. Variants of the microglia receptor TREM2 increase AD risk and activation of “disease-associated microglia” (DAM) is dependent on TREM2 in mouse models of AD. To investigate global transcriptomic changes during AD pathology, we surveyed gene expression changes associated with AD pathology and TREM2 in Ab-driven 5XFAD mouse model and human AD by single nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a novel Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less palpable in TREM2 R47H and R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.
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- 2020
4. Effect of mesoporous silica nanoparticles loaded with α-tomatine on HepG2 cancer cells studied in vitro
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Bishal Nepal, Jay K. Bhattarai, Kapur B. Dhami, Michael R. Nichols, and Keith J. Stine
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Pharmaceutical Science - Published
- 2023
5. Development of β-sheet structure in Aβ aggregation intermediates diminishes exposed hydrophobic surface area and enhances proinflammatory activity
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Kapur B. Dhami, Sanjib Karki, Antanisha Parks, Cameron G. Nichols, and Michael R. Nichols
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Amyloid beta-Peptides ,Alzheimer Disease ,Biophysics ,Humans ,Protein Conformation, beta-Strand ,Hydrophobic and Hydrophilic Interactions ,Molecular Biology ,Biochemistry ,Peptide Fragments ,Analytical Chemistry - Abstract
Three decades of research, both in vitro and in vivo, have demonstrated the conformational heterogeneity that is displayed by the amyloid β peptide (Aβ) in Alzheimer's disease (AD). Understanding the distinct properties between Aβ conformations and how conformation may impact cellular activity remain open questions, yet still continue to provide new insights into protein misfolding and aggregation. In particular, there is interest in the group of soluble oligomeric prefibrillar Aβ species comprising lower molecular weight oligomers up to larger protofibrils. In the current study, a number of strategies were utilized to separate Aβ protofibrils and oligomers and show that the smaller Aβ oligomers have a much different conformation than Aβ protofibrils. The differences were consistent for both Aβ40 and Aβ42. Protofibrils bound thioflavin T to a greater extent than oligomers, and were highly enriched in β-sheet secondary structure. Aβ oligomers possessed a more open structure with significant solvent exposure of hydrophobic domains as determined by tryptophan fluorescence and bis-ANS binding, respectively. The protofibril-selective antibody AbSL readily discerned conformational differences between protofibrils and oligomers. The more developed structure for Aβ protofibrils ultimately proved critical for provoking the release of tumor necrosis factor α from microglial cells. The findings demonstrated a dependency on β-sheet structure for soluble Aβ aggregates to cause a microglial inflammatory response. The Aβ aggregation process yields many conformationally-varied species with different levels of β-structure and exposed hydrophobicity. The conformation elements likely determine biological activity and pathogenicity.
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- 2022
6. Disentangling aggregation‐prone proteins: a new method for isolating α‐synuclein species
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Michael R. Nichols
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0301 basic medicine ,Alpha-synuclein ,Circular dichroism ,Protein aggregation ,Fibril ,Biochemistry ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,030104 developmental biology ,0302 clinical medicine ,Monomer ,chemistry ,Protein purification ,Centrifugation ,Protein folding ,030217 neurology & neurosurgery - Abstract
Protein aggregation plays a central role in numerous neurodegenerative diseases. The key proteins in these diseases are of significant importance, but their investigation can be challenging due to unique properties of protein misfolding and oligomerization. Alpha-synuclein protein (α-Syn) is the predominant component of Lewy Bodies in Parkinson's disease (PD) and is a member of this class of proteins. Many α-Syn studies are limited by the inability to separate various monomeric, oligomeric, and fibrillar forms of the protein from heterogeneous mixtures. This Editorial Highlight summarizes the impact of a study published in the current issue of Journal of Neurochemistry, in which Lashuel and colleagues developed a simple, rapid centrifugation- and filter-based method for separating, isolating, and quantifying different forms of α-Syn. The researchers used electron microscopy, SDS-PAGE, circular dichroism, and protein assays to carefully validate the method and quantitate α-Syn yields and loss. The publication of this new method will not only aid in future studies of α-Syn, but will likely extend to other proteins that underlie a variety of neurodegenerative diseases.
- Published
- 2020
7. Expression of NLRP3 inflammasome proteins in ExpiCHO-S mammalian cells reveals oligomerization properties that are highly sensitive to solution conditions
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Adela Redzic, Evan C. Garrad, Michael R. Nichols, and Nyasha J. Makoni
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Mammals ,Mutation ,Innate immune system ,Chemistry ,Inflammasomes ,animal diseases ,Interleukin-1beta ,Caspase 1 ,chemical and pharmacologic phenomena ,hemic and immune systems ,Inflammasome ,medicine.disease_cause ,Fusion protein ,eye diseases ,law.invention ,Cell biology ,law ,Protein purification ,NLR Family, Pyrin Domain-Containing 3 Protein ,medicine ,Recombinant DNA ,Animals ,Intracellular ,Biotechnology ,medicine.drug - Abstract
The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-β. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.
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- 2021
8. The intricate biophysical puzzle of caspase-1 activation
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Michael R. Nichols and Nyasha J. Makoni
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0301 basic medicine ,Inflammasomes ,Biophysics ,Caspase 1 ,Biochemistry ,Article ,Proinflammatory cytokine ,03 medical and health sciences ,Protein structure ,Cell Line, Tumor ,NLR Family, Pyrin Domain-Containing 3 Protein ,medicine ,Animals ,Humans ,Molecular Biology ,030102 biochemistry & molecular biology ,Chemistry ,Pyroptosis ,Inflammasome ,Cysteine protease ,Cell biology ,Enzyme Activation ,030104 developmental biology ,Proteolysis ,Protein Multimerization ,Inflammasome complex ,Intracellular ,medicine.drug - Abstract
This review takes a closer look at the structural components of the molecules involved in the processes leading to caspase-1 activation. Interleukins 1β and −18 (IL-1β, IL-18) are well-known pro-inflammatory cytokines that are produced following cleavage of their respective precursor proteins by the cysteine protease caspase-1. Active caspase-1 is the final step of the NLRP3 inflammasome, a three-protein intracellular complex involved in inflammation and induction of pyroptosis (a proinflammatory cell-death process). NLRP3 activators facilitate assembly of the inflammasome complex and subsequently, activation of caspase-1 by autoproteolysis. However, the definitive structural components of active caspase-1 are still unclear and new data add to the complexity of this process. This review outlines the historical and recent findings that provide supporting evidence for the structural aspects of caspase-1 autoproteolysis and activation.
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- 2020
9. The influence of gold surface texture on microglia morphology and activation
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Keith J. Stine, Yih Horng Tan, Michael R. Nichols, Shana E. Terrill, and Geeta S. Paranjape
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geography ,geography.geographical_feature_category ,Materials science ,Microglia ,Biocompatibility ,Nanoporous ,Scanning electron microscope ,Biomedical Engineering ,Biomaterial ,Nanotechnology ,medicine.anatomical_structure ,Drug delivery ,medicine ,Biophysics ,General Materials Science ,Monolith ,Neuroinflammation - Abstract
Microglial cells play a critical role in the propagation of neuroinflammation in the central nervous system. Microglia sense and respond to environmental signals including chemical, physical and biological cues from the surrounding cell/tissue components. In this project, our goal was to examine the effects of surface texture on BV-2 microglia morphology and function by comparing flat and nanoporous gold (np-Au) surfaces to the more conventional glass. The biocompatibility of np-Au with microglia was evaluated using functional cell assays and high resolution imaging with scanning electron microscopy (SEM). Microglia seeded on glass, ultra-flat gold (UF-Au), ultra-thin (UT) np-Au and np-Au monolith were adherent to all surfaces and their viability was not compromised as assessed by multiple toxicity assays. SEM revealed detailed morphological characteristics of adherent microglia and indicated few dramatic changes as a result of the different surfaces. Microglia proliferation was hampered by np-Au monolith but less by UT np-Au and not at all on UF-Au or glass. Microglial activation, measured by tumor necrosis factor α (TNFα) production, was fully functional (and equivalent) on all gold surfaces compared to glass. The present findings should help further the understanding of basic microglia biology on textured surfaces and more fully evaluate np-Au as a multi-functional biocompatible material. The knowledge obtained and technology developed will have a significant impact in the fabrication of nanoelectronic devices, chemical sensor development, porous nanostructured materials for BioMEMs/NEMs integration, and functional biomaterial coatings for drug delivery.
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- 2020
10. Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer’s disease
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Marco Colonna, Konstantin Zaitsev, David M. Holtzman, Makoto Sainouchi, Manuela Cominelli, Akiyoshi Kakita, David A. Bennett, Susan Gilfillan, Marina Cella, Sean A. Beausoleil, Takeshi Ikeuchi, Tyler K. Ulland, Shikha Grover, Prabhakar S. Andhey, Kelly R. Miller, Michael R. Nichols, Tyler Levy, Julie A. Schneider, Wilbur M. Song, Amanda Swain, Jason D. Ulrich, Akinori Miyashita, Maxim N. Artyomov, Yingyue Zhou, and Pietro Luigi Poliani
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0301 basic medicine ,Male ,Transcription, Genetic ,Inbred C57BL ,Transgenic ,Transcriptome ,Mice ,0302 clinical medicine ,Immunologic ,Receptors ,Receptors, Immunologic ,Receptor ,education.field_of_study ,Membrane Glycoproteins ,Microglia ,Brain ,General Medicine ,Middle Aged ,Phenotype ,Cell biology ,Oligodendroglia ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Transcription ,Astrocyte ,Population ,Mice, Transgenic ,Biology ,Aged ,Alzheimer Disease ,Amyloid beta-Peptides ,Animals ,Astrocytes ,Axons ,Cell Nucleus ,Humans ,Mice, Inbred C57BL ,Nerve Degeneration ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Genetic ,medicine ,education ,TREM2 ,Oligodendrocyte ,030104 developmental biology ,nervous system - Abstract
Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.
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- 2020
11. Aβ42 Protofibrils Interact with and Are Trafficked through Microglial-Derived Microvesicles
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Lisa K. Gouwens, David Osborn, Nathan T Zeller, Evan C. Garrad, Victoria A. Rogers, Mudar S Ismail, Fatima S. Amtashar, Michael R. Nichols, and Nyasha J. Makoni
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0301 basic medicine ,Physiology ,Amyloid beta ,Cognitive Neuroscience ,Cell ,Stimulation ,Biochemistry ,Extracellular Vesicles ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Alzheimer Disease ,Cell Movement ,medicine ,Animals ,Senile plaques ,Neuroinflammation ,Inflammation ,Amyloid beta-Peptides ,Microglia ,biology ,Tumor Necrosis Factor-alpha ,Chemistry ,Vesicle ,Brain ,Cell Biology ,General Medicine ,Peptide Fragments ,Microvesicles ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,030217 neurology & neurosurgery - Abstract
Microvesicles (MVs) and exosomes comprise a class of cell-secreted particles termed extracellular vesicles (EVs). These cargo-holding vesicles mediate cell-to-cell communication and have recently been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). The two types of EVs are distinguished by the mechanism of cell release and their size, with the smaller exosomes and the larger MVs ranging from 30 to 100 nm and 100 nm to 1 μm in diameter, respectively. MV numbers are increased in AD and appear to interact with amyloid-β peptide (Aβ), the primary protein component of the neuritic plaques in the AD brain. Because microglial cells play such an important role in AD-linked neuroinflammation, we sought to characterize MVs shed from microglial cells, better understand MV interactions with Aβ, and determine whether internalized Aβ may be incorporated into secreted MVs. Multiple strategies were used to characterize MVs shed from BV-2 microglia after ATP stimulation. Confocal images of isolated MVs bound to fluorescently labeled annexin-V via externalized phosphatidylserine revealed a polydisperse population of small spherical structures. Dynamic light scattering measurements yielded MV diameters ranging from 150 to 600 nm. Electron microscopy of resin-embedded MVs cut into thin slices showed well-defined uranyl acetate-stained ring-like structures in a similar diameter range. The use of a fluorescently labeled membrane insertion probe, NBD C
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- 2018
12. The conformational epitope for a new Aβ42 protofibril-selective antibody partially overlaps with the peptide N-terminal region
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Elizabeth A. Ridgway, Colin K. Combs, Victoria A. Rogers, Fatima S. Amtashar, Michael R. Nichols, Benjamin A. Colvin, and Joshua A. Kulas
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0301 basic medicine ,Amyloid beta ,Mice, Transgenic ,Peptide ,Fibril ,Biochemistry ,Antibodies ,Epitope ,Epitopes ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Alzheimer Disease ,Antibody Specificity ,Amyloid precursor protein ,Animals ,Antiserum ,chemistry.chemical_classification ,Amyloid beta-Peptides ,biology ,Chemistry ,Peptide Fragments ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,biology.protein ,Protein Conformation, beta-Strand ,Antibody ,030217 neurology & neurosurgery ,Conformational epitope - Abstract
Aggregation and accumulation of amyloid-β peptide (Aβ) is a key component of Alzheimer's disease (AD). While monomeric Aβ appears to be benign, oligomers adopt a biologically detrimental structure. These soluble structures can be detected in AD brain tissue by antibodies that demonstrate selectivity for aggregated Aβ. Protofibrils are a subset of soluble oligomeric Aβ species and are described as small (
- Published
- 2017
13. Amyloid-β42 protofibrils are internalized by microglia more extensively than monomers
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Nyasha J. Makoni, Lisa K. Gouwens, Michael R. Nichols, and Victoria A. Rogers
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0301 basic medicine ,Amyloid ,media_common.quotation_subject ,Peptide ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Extracellular ,Animals ,Internalization ,Molecular Biology ,Cells, Cultured ,media_common ,Alexa Fluor ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Microglia ,Tumor Necrosis Factor-alpha ,General Neuroscience ,Peptide Fragments ,Mice, Inbred C57BL ,Cytosol ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Biochemistry ,Biophysics ,Tumor necrosis factor alpha ,Neurology (clinical) ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
One pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid-β peptide (Aβ) in the affected brain. While there are numerous deleterious effects of Aβ accumulation, there is general agreement that a sustained inflammatory response to aggregated Aβ contributes to progressive neurodegeneration in AD and microglial cells play a significant role in this process. Our laboratory and others have shown that small soluble aggregates of Aβ activate a microglia-mediated inflammatory response. One component of the response involves internalization of extracellular Aβ, and this process is likely very sensitive to Aβ structure. In this study we analyzed the proclivity of microglia for internalization of Aβ42 monomers and protofibrils using fluorescently-labeled Aβ. Both Aβ42 species were labeled directly via amino linkage with an Alexa Fluor 488 tetrafluorophenyl ester (AF488-TFP) and then isolated individually by chromatography. Aβ42 protofibrils retained their size and morphological properties after labeling but monomers had a much higher stoichiometry of labeling compared to protofibrils. Primary murine microglia internalized AF488-Aβ42 protofibrils rapidly and in significant amounts compared to AF488-Aβ42 monomers. Microglial internalization of protofibrils was dependent on time and concentration, and corresponded with tumor necrosis factor α secretion. In competition studies, unlabeled Aβ42 protofibril internalization, detected by immunostaining, did not diminish AF488-protofibril uptake. Internalized AF488-Aβ42 protofibrils were found widely dispersed in the cytosol with some lysosomal accumulation but little degradation. These studies highlight the sensitivity that microglia exhibit to Aβ structure in the internalization process and emphasize their affinity for soluble Aβ protofibrils.
- Published
- 2016
14. APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease
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Kendra L. Puig, Keiko Rausch, Joshua A. Kulas, Brett A. McGregor, Lalida Rojanathammanee, Angela M. Floden, Colin K. Combs, Gunjan D. Manocha, Sanjib Karki, James E. Porter, Kelley R. Puig, Michael R. Nichols, and Diane C. Darland
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0301 basic medicine ,Journal Club ,Mice, Transgenic ,Microgliosis ,Presenilin ,Morpholinos ,Proinflammatory cytokine ,Amyloid beta-Protein Precursor ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Alzheimer Disease ,mental disorders ,Presenilin-1 ,medicine ,Amyloid precursor protein ,Animals ,Humans ,Cells, Cultured ,Neuroinflammation ,Cell Proliferation ,Amyloid beta-Peptides ,Microglia ,biology ,Adaptation, Ocular ,Chemistry ,General Neuroscience ,P3 peptide ,Articles ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,Phenotype ,030104 developmental biology ,medicine.anatomical_structure ,Astrocytes ,Mutation ,Exploratory Behavior ,biology.protein ,Cytokines ,Cytokine secretion ,Neuroscience ,030217 neurology & neurosurgery - Abstract
Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer9s disease. Although fibrillar amyloid β (Aβ)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP −/− ) microglial cultures, oligomeric Aβ was unable to stimulate increased secretion from mAPP −/− cells. This was consistent with an ability of oligomeric Aβ to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aβ produced less microgliosis in mAPP −/− mice compared with wild-type mice. The mAPP −/− mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aβ plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT A hallmark of Alzheimer9s disease (AD) brains is the accumulation of amyloid β (Aβ) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aβ stimulation of microglial activation is one source of brain inflammatory changes during disease. Aβ is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aβ are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aβ production to drive the microgliosis associated with AD brains.
- Published
- 2016
15. Author Correction: Human and mouse single-nucleus transcriptomics reveal TREM2-dependent and TREM2-independent cellular responses in Alzheimer’s disease
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Manuela Cominelli, Julie A. Schneider, Prabhakar S. Andhey, Sean A. Beausoleil, Michael R. Nichols, Konstantin Zaitsev, Akinori Miyashita, Takeshi Ikeuchi, Kelly R. Miller, Shikha Grover, Jason D. Ulrich, Tyler K. Ulland, Amanda Swain, Makoto Sainouchi, David A. Bennett, Yingyue Zhou, Maxim N. Artyomov, Pietro Luigi Poliani, Wilbur M. Song, David M. Holtzman, Susan Gilfillan, Akiyoshi Kakita, Marco Colonna, Tyler Levy, and Marina Cella
- Subjects
Transcriptome ,medicine.anatomical_structure ,TREM2 ,medicine ,General Medicine ,Disease ,Biology ,Neuroscience ,Nucleus ,General Biochemistry, Genetics and Molecular Biology - Published
- 2020
16. Inflammatory mechanisms in neurodegeneration
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Marie-Ève Tremblay, Colin K. Combs, Ann-Christin Wendeln, Nyasha J. Makoni, Mona Sohrabi, Jonas J. Neher, Marie-Kim St-Pierre, Lisa K. Gouwens, Evan C. Garrad, and Michael R. Nichols
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0301 basic medicine ,Amyloid beta ,immunology [Nerve Degeneration] ,Inflammation ,Disease ,metabolism [Microglia] ,Biochemistry ,Article ,pathology [Alzheimer Disease] ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,immunology [Inflammation] ,0302 clinical medicine ,Alzheimer Disease ,medicine ,Animals ,Humans ,ddc:610 ,Neuroinflammation ,Microglia ,biology ,business.industry ,Neurodegeneration ,pathology [Nerve Degeneration] ,medicine.disease ,immunology [Microglia] ,immunology [Alzheimer Disease] ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,Nerve Degeneration ,biology.protein ,medicine.symptom ,business ,Neuroscience ,030217 neurology & neurosurgery - Abstract
This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer’s disease (AD)(). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are research areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies.
- Published
- 2018
17. Amyloid-β(1-42) protofibrils stimulate a quantum of secreted IL-1β despite significant intracellular IL-1β accumulation in microglia
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Shana E. Terrill-Usery, Michael J. Mohan, and Michael R. Nichols
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Innate immune system ,Microglia ,Inflammation ,Inflammasome ,Biology ,Amyloid-beta protein ,NLRP3 inflammasome ,Article ,Toll-like receptors ,Cell biology ,medicine.anatomical_structure ,Interleukin 1-beta ,Immunology ,medicine ,Molecular Medicine ,Tumor necrosis factor alpha ,Senile plaques ,Protofibril ,medicine.symptom ,Molecular Biology ,Intracellular ,Neuroinflammation ,medicine.drug - Abstract
Neuroinflammation is a characteristic feature of the Alzheimer’s disease (AD) brain. Significant inflammatory markers such as activated microglia and cytokines can be found surrounding the extracellular senile plaques predominantly composed of amyloid-β protein (Aβ). Several innate immune pathways, including Toll-like receptors (TLRs) and the NLRP3 inflammasome, have been implicated in AD inflammation. Aβ plays a primary role in activating these pathways which likely contributes to the progressive neurodegeneration in AD. In order to better understand the complexities of this interaction we investigated the inflammatory response of primary microglia to Aβ(1-42) protofibrils. Aβ(1-42) protofibrils triggered a time- and MyD88-dependent process that produced tumor necrosis factor alpha (TNFα) and interleukin-1β (IL-1β) mRNA, and intracellular pro and mature forms of IL-1β protein. The accumulation of both IL-1β forms indicated that Aβ(1-42) protofibrils were able to prime and activate the NLRP3 inflammasome. Surprisingly, Aβ-induced accumulation of intracellular mature IL-1β did not translate into greater IL-1β secretion. Instead, we found that Aβ elicited a quantized burst of secreted IL-1β and this process occurred even prior to Aβ priming of the microglia suggesting a basal level of either pro or mature IL-1β in the cultured primary microglia. The IL-1β secretion burst was rapid but not sustained, yet could be re-evoked with additional Aβ stimulation. The findings from this study demonstrated multiple sites of IL-1β regulation by Aβ(1-42) protofibrils including TLR/MyD88-mediated priming, NLRP3 inflammasome activation, and modulation of the IL-1β secretory process. These results underscore the wide-ranging effects of Aβ on the innate immune response.
- Published
- 2014
18. Stability of early-stage amyloid-β(1–42) aggregation species
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Sanjib Karki, Geeta S. Paranjape, Kelley A. Coalier, and Michael R. Nichols
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Amyloid ,Amyloid beta-Peptides ,Protein Stability ,Size-exclusion chromatography ,Biophysics ,Sodium Dodecyl Sulfate ,Biochemistry ,Antibodies ,Peptide Fragments ,Article ,In vitro ,Analytical Chemistry ,chemistry.chemical_compound ,chemistry ,Humans ,Urea ,Thioflavin ,Denaturation (biochemistry) ,Sodium dodecyl sulfate ,Guanidine ,Molecular Biology ,Incubation - Abstract
Accumulation of aggregated amyloid-β protein (Aβ) is an important feature of Alzheimer’s disease. There is significant interest in understanding the initial steps of Aβ aggregation due to the recent focus on soluble Aβ oligomers. In vitro studies of Aβ aggregation have been aided by the use of conformation-specific antibodies which recognize shape rather than sequence. One of these, OC antiserum, recognizes certain elements of fibrillar Aβ across a broad range of sizes. We have observed the presence of these fibrillar elements at very early stages of Aβ incubation. Using a dot blot assay, OC-reactivity was found in size exclusion chromatography (SEC)-purified Aβ(1-42) monomer fractions immediately after isolation (early-stage). The OC-reactivity was not initially observed in the same fractions for Aβ(1-40) or the aggregation-restricted Aβ(1-42) L34P but was detected within 1–2 weeks of incubation. Stability studies demonstrated that early-stage OC-positive Aβ(1-42) aggregates were resistant to 4M urea or guanidine hydrochloride but sensitive to 1% sodium dodecyl sulfate (SDS). Interestingly, the sensitivity to SDS diminished over time upon incubation of the SEC-purified Aβ(1-42) solution at 4° C. Within 6–8 days the OC-positive Aβ42 aggregates were resistance to SDS denaturation. The progression to, and development of, SDS resistance for Aβ(1-42) occurred prior to thioflavin T fluorescence. In contrast, Aβ(1-40) aggregates formed after 6 days of incubation were sensitive to both urea and SDS. These findings reveal information on some of the earliest events in Aβ aggregation and suggest that it may be possible to target early-stage aggregates before they develop significant stability.
- Published
- 2013
19. A comparative first-principles study of structural and electronic properties among memantine, amantadine and rimantadine
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Kirsten Middleton, Guoping Zhang, Michael R. Nichols, and Thomas F. George
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Rimantadine ,Stereochemistry ,Chemistry ,Adamantane ,Biophysics ,Memantine ,Amantadine ,Condensed Matter Physics ,chemistry.chemical_compound ,medicine ,Physical and Theoretical Chemistry ,Molecular Biology ,medicine.drug ,Electronic properties - Abstract
Memantine, amantadine and rimantadine are structurally derived from the same diamondoid, adamantane. These derivatives demonstrate therapeutic efficacy in human diseases: memantine for Alzheimer's disease and amantadine and rimantadine for influenza. In order to better understand some of the properties that distinguish these three compounds, we conduct first-principles calculations on their structure and electronic properties. Our results indicate that protonation has a significant effect on the dipole moment, where the dipole moment in protonated memantine is over eight times larger than in the deprotonated form.
- Published
- 2012
20. Aβ40 has a subtle effect on Aβ42 protofibril formation, but to a lesser degree than Aβ42 concentration, in Aβ42/Aβ40 mixtures
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Michael R. Nichols, Benjamin A. Colvin, Shana E. Terrill-Usery, and Richard E. Davenport
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Biophysics ,Peptide ,Protein aggregation ,Fibril ,Biochemistry ,Peptide Fragments ,Protein Structure, Secondary ,Article ,03 medical and health sciences ,Crystallography ,chemistry.chemical_compound ,Protein Aggregates ,030104 developmental biology ,0302 clinical medicine ,Monomer ,chemistry ,Humans ,Senile plaques ,Molecular Biology ,030217 neurology & neurosurgery - Abstract
Recent findings suggest that the senile plaques in Alzheimer’s disease may contain soluble amyloid-β peptide (Aβ) fibril precursors along with insoluble fibrils.. These soluble Aβ species, including oligomers and protofibrils, have been well-studied in vitro and are formed via non-covalent self-assembly of Aβ monomers. While both 40- and 42-residue forms of Aβ are observed in the human body, the majority of the Aβ aggregation work has been conducted on Aβ42 or Aβ40 separately, with relatively few investigations of mixtures. In order to study the effect of different combinations of Aβ40 and Aβ42 on protofibril formation, mixtures of either dry solid peptide, or purified Aβ40 and Aβ42 monomer solutions were mixed together and protofibril/monomer distributions were quantified. Increases in the Aβ42/Aβ40 ratio increased protofibril formation but the presence of Aβ40 in the mixed Aβ solutions had a significant negative impact on protofibril formation compared to equivalent solutions of pure Aβ42. Protofibril size was less affected, but β-sheet structure increased with protofibrils formed from higher Aβ42/Aβ40 ratio solutions. Direct measurement of Aβ42/Aβ40 ratios by C-terminal-selective ELISA found very little Aβ40 incorporated into protofibrils. The cumulative data emphasizes the critical importance of Aβ42, yet establishes Aβ40 as a regulator of Aβ42 aggregation.
- Published
- 2015
21. Substituted tryptophans at amyloid-β(1–40) residues 19 and 20 experience different environments after fibril formation
- Author
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Geeta S. Paranjape, Ryan T. McDonough, Fabio Gallazzi, and Michael R. Nichols
- Subjects
Amyloid beta-Peptides ,Quenching (fluorescence) ,Protein Conformation ,Tryptophan ,Biophysics ,P3 peptide ,macromolecular substances ,Fibril ,Biochemistry ,Fluorescence ,Peptide Fragments ,In vitro ,chemistry.chemical_compound ,Monomer ,chemistry ,Alzheimer Disease ,Humans ,Senile plaques ,Hydrophobic and Hydrophilic Interactions ,Molecular Biology - Abstract
Amyloid-β protein (Aβ) is the principal component of the neuritic plaques found in Alzheimer’s disease. The predominant Aβ morphology in the plaques is fibrillar which has prompted substantial in vitro work to better understand the molecular organization of Aβ fibrils. In the current study, tryptophan substitutions were made at Aβ(1–40) position 19 (F19W) or 20 (F20W) to ascertain environmental differences between the two residues in the fibril structure. Kinetic studies revealed similar rates of fibril formation between Aβ(1–40) F19W and F20W and both peptides formed typical amyloid fibril structures. Aβ(1–40) F19W fibrils displayed a significant tryptophan fluorescence blue-shift in λmax (33 nm) compared to monomer while Aβ(1–40) F20W fibrils had a much smaller shift (9 nm). Fluorescence quenching experiments with water-soluble acrylamide and KI demonstrated that both W19 and W20 were much less accessible to quenching in fibrils compared to monomer. Lipid-soluble TEMPO quenched the fluorescence of Aβ(1–40) F19W fibrils more effectively than F20W fibrils in agreement with the fluorescence blue-shift results. These findings demonstrate distinct environments between Aβ(1–40) residues 19 and 20 fibrils and indicate that while W20 accessibility is compromised in Aβ fibrils it resides in a much less hydrophobic environment than W19.
- Published
- 2011
22. Probing the amyloid-β(1–40) fibril environment with substituted tryptophan residues
- Author
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Jillienne C. Touchette, Michael R. Nichols, Fabio Gallazzi, Deepa Ajit, and Laura L. Williams
- Subjects
Amyloid β ,Molecular Sequence Data ,Kinetics ,Biophysics ,macromolecular substances ,Fibril ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Tryptophan fluorescence ,Amino Acid Sequence ,Benzothiazoles ,Molecular Biology ,Amyloid beta-Peptides ,Chemistry ,Tryptophan ,Inner core ,Fluorescence ,Peptide Fragments ,Thiazoles ,Crystallography ,Spectrometry, Fluorescence ,Amino Acid Substitution ,Thioflavin ,Hydrophobic and Hydrophilic Interactions - Abstract
A signature feature of Alzheimer's disease is the accumulation of plaques, composed of fibrillar amyloid-beta protein (Abeta), in the brain parenchyma. Structural models of Abeta fibrils reveal an extensive beta-sheet network with a hydrophobic core extending throughout the fibril axis. In this study, phenylalanines in the Abeta(1-40) sequence were substituted with tryptophan residues at either position 4 (F4W) or 19 (F19W) to probe the fibril environment. The F4W substitution did not alter self-assembly kinetics, while the F19W change slightly lengthened the lag phase without hindering fibril formation. The tryptophan fluorescence of Abeta(1-40) F19W, but not Abeta(1-40) F4W, underwent a marked blue shift during fibril formation and this shift was temporally correlated with thioflavin T binding. Isolated Abeta(1-40) F19W fibrils exhibited the largest fluorescence blue shifts consistent with W19 insertion into the Abeta(1-40) fibril inner core and direct probing of the substantially hydrophobic environment therein.
- Published
- 2010
23. Biophysical comparison of soluble amyloid-β(1-42) protofibrils, oligomers, and protofilaments
- Author
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Michael R. Nichols, Elizabeth Anne Hood, Geeta S. Paranjape, David Osborn, Benjamin A. Colvin, and Shana E. Terrill-Usery
- Subjects
Amyloid ,Amyloid beta-Peptides ,Amyloid β ,Circular Dichroism ,Fibril ,Biochemistry ,Oligomer ,Peptide Fragments ,Protein Structure, Secondary ,chemistry.chemical_compound ,Microscopy, Electron ,Thiazoles ,Spectrometry, Fluorescence ,chemistry ,Solubility ,Chromatography, Gel ,Scattering, Radiation ,Senile plaques ,Benzothiazoles - Abstract
Some of the pathological hallmarks of the Alzheimer's disease brain are senile plaques composed of insoluble amyloid-β protein (Aβ) fibrils. However, much of the recent emphasis in research has been on soluble Aβ aggregates in response to a growing body of evidence that shows that these species may be more neurotoxic than fibrils. Within this subset of soluble aggregated Aβ are protofibrils and oligomers. Although each species has been widely investigated separately, few studies have directly compared and contrasted their physical properties. In this work, we examined well-recognized preparations of Aβ(1-42) oligomers and protofibrils with multiangle (MALS) and dynamic (DLS) light scattering in line with, or following, size-exclusion chromatography (SEC). Multiple SEC-MALS analyses of protofibrils revealed molecular weight (Mw) gradients ranging from 200 to 2600 kDa. Oligomeric Aβ species are generally considered to be a smaller and more nascent than protofibrils. However, oligomer Mw values ranged from 225 to 3000 kDa, larger than that for protofibrils. Root-mean-square radius (Rg) values correlated with the Mw trends with protofibril Rg values ranging from 16 to 35 nm, while oligomers produced one population at 40-43 nm with a more disperse population from 22 to 39 nm. Hydrodynamic radius (RH) measurements by DLS and thioflavin T fluorescence measurements indicated that protofibrils and oligomers had commonalities, yet electron microscopy revealed morphological differences between the two. SEC-purified Aβ(1-42) monomer at lower concentrations was slower to nucleate but formed protofibrils (1500 kDa) or soluble protofilaments (3000 kDa) depending on the buffer type. The findings from these studies shed new light on the similarities and differences between distinct soluble aggregated Aβ species.
- Published
- 2015
24. Amyloid-β Protofibrils Differ from Amyloid-β Aggregates Induced in Dilute Hexafluoroisopropanol in Stability and Morphology
- Author
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Melissa A. Moss, Jan H. Hoh, Terrone L. Rosenberry, Dana Kim Reed, Michael R. Nichols, and Stephanie Cratic-McDaniel
- Subjects
Circular dichroism ,Time Factors ,Light ,Amyloid ,Propanols ,Protein Conformation ,Size-exclusion chromatography ,Kinetics ,Microscopy, Atomic Force ,Fibril ,Biochemistry ,chemistry.chemical_compound ,Protein structure ,Alzheimer Disease ,mental disorders ,Humans ,Scattering, Radiation ,Benzothiazoles ,Molecular Biology ,Ions ,Chromatography ,Amyloid beta-Peptides ,Circular Dichroism ,Cell Biology ,Peptide Fragments ,Microscopy, Electron ,Thiazoles ,Crystallography ,Monomer ,chemistry ,Biophysics ,Thioflavin - Abstract
The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-beta (Abeta) peptides. Several lines of evidence indicate that fibrillar Abeta and especially soluble Abeta aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Abeta-(1-40) can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant approximately 1 h(-1)) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Abeta aggregates was generated rapidly (
- Published
- 2005
25. Amyloid-β aggregates formed at polar-nonpolar interfaces differ from amyloid-β protofibrils produced in aqueous buffers
- Author
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Melissa A. Moss, Dana Kim Reed, Jan H. Hoh, Terrone L. Rosenberry, and Michael R. Nichols
- Subjects
Circular dichroism ,Histology ,Buffers ,Microscopy, Atomic Force ,Fibril ,Phase Transition ,Protein Structure, Secondary ,law.invention ,chemistry.chemical_compound ,Protein structure ,law ,mental disorders ,Humans ,Senile plaques ,Instrumentation ,Amyloid beta-Peptides ,Aqueous solution ,Chemistry ,Circular Dichroism ,Water ,Peptide Fragments ,Microscopy, Electron ,Medical Laboratory Technology ,Crystallography ,Monomer ,Thioflavin ,Anatomy ,Electron microscope - Abstract
The deposition of aggregated amyloid-beta (Abeta) peptides in the brain as senile plaques is a pathological hallmark of Alzheimer's disease (AD). Several lines of evidence indicate that fibrillar and, in particular, soluble aggregates of these 40- and 42-residue peptides are important in the etiology of AD. Recent studies also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we review our recent reports that Abeta(1-40) in vitro can form soluble aggregates with predominant beta-structures that differ in stability and morphology. One class of aggregates involved soluble Abeta protofibrils, prepared by vigorous overnight agitation of monomeric Abeta(1-40) in low ionic strength buffers. These aggregates were quite stable and disaggregated to only a limited extent on dilution. A second class of soluble Abeta aggregates was generated at polar-nonpolar interfaces. Aggregation in a two-phase system of buffer over chloroform occurred more rapidly than in buffer alone. In buffered 2% hexafluoroisopropanol (HFIP), microdroplets of HFIP were formed and the half-time for aggregation was less than 10 minutes. Like Abeta protofibrils, these interfacial aggregates showed increased thioflavin T fluorescence and were rich in beta-structure by circular dichroism. However, electron microscopy and atomic force microscopy revealed very different morphologies. The HFIP aggregates formed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP these aggregates initially were very unstable and disaggregated completely within 2 minutes. However, their stability increased as they progressed to fibers. It is important to determine whether similar interfacial Abeta aggregates are produced in vivo.
- Published
- 2005
26. The Peptide KLVFF-K6Promotes β-Amyloid(1–40) Protofibril Growth by Association but Does Not Alter Protofibril Effects on Cellular Reduction of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT)
- Author
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Michael R. Nichols, Dana Kim Reed, Terrone L. Rosenberry, Jan H. Hoh, and Melissa A. Moss
- Subjects
Pharmacology ,chemistry.chemical_classification ,Amyloid beta-Peptides ,Amyloid ,Peptide ,Fibrillogenesis ,Tritium ,Fibril ,Peptide Fragments ,chemistry.chemical_compound ,Monomer ,chemistry ,Biochemistry ,Bromide ,Toxicity ,Humans ,Molecular Medicine ,Thioflavin ,Peptides ,Oligopeptides ,Oxidation-Reduction - Abstract
The peptide KLVFF-K6 was observed by Lowe et al. to simultaneously enhance amyloid beta-protein (Abeta) fibrillogenesis and decrease cellular toxicity, as measured in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. It was postulated that accelerated Abeta aggregation and precipitation induced by KLVFF-K6 may lead to an increase in less toxic insoluble fibrils at the expense of more toxic soluble protofibrils. In a previous study, we distinguished between two modes of protofibril growth: elongation by monomer deposition and direct protofibril-protofibril association. These growth mechanisms could be resolved by varying Abeta monomer and NaCl concentrations. Using assays designed to isolate these distinct modes of protofibril growth, we report here that larger Abeta aggregates formed in the presence of KLVFF-K6 resulted from enhanced protofibril association. 3H-Radiomethylated KLVFF-K6 bound to associated protofibrils with an apparent Kd of 180 nM, and concentrations of free [3H]KLVFF-K6 in this range were sufficient to convert soluble protofibrils to sedimentable fibrils. However, promotion of Abeta protofibril association by KLVFF-K6 had no effect on Abeta-induced decreases in cellular MTT reduction. Therefore, our data do not support the proposal that insoluble fibrils formed with KLVFF-K6 are less toxic than soluble protofibrils. KLVFF-K6 did not alter rates of protofibril elongation by monomer deposition. In contrast, when added to Abeta monomers isolated with the use of size-exclusion chromatography, KLVFF-K6 inhibited fibrillogenesis, as measured by thioflavin T fluorescence, and this inhibition was paralleled by a failure to alter cellular MTT reduction.
- Published
- 2003
27. CD47 Does Not Mediate Amyloid-β(1-42) Protofibril-Stimulated Microglial Cytokine Release
- Author
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Sanjib Karki and Michael R. Nichols
- Subjects
Amyloid ,medicine.medical_treatment ,Interleukin-1beta ,Biophysics ,CD47 Antigen ,Biology ,Biochemistry ,Article ,Proinflammatory cytokine ,Mice ,Alzheimer Disease ,medicine ,Animals ,Humans ,Secretion ,Senile plaques ,Molecular Biology ,Neuroinflammation ,Cells, Cultured ,Inflammation ,Mice, Knockout ,Amyloid beta-Peptides ,Microglia ,Tumor Necrosis Factor-alpha ,Cell Biology ,Antibodies, Neutralizing ,Peptide Fragments ,Cell biology ,Mice, Inbred C57BL ,Disease Models, Animal ,Cytokine ,medicine.anatomical_structure ,Immunology ,Tumor necrosis factor alpha ,Cytokine secretion ,Oligopeptides - Abstract
Neuroinflammation triggered by accumulation of amyloid-β protein (Aβ) is a significant component of the Alzheimer's disease (AD) brain. Senile plaques composed of Aβ attract and activate microglia cells resulting in cytokine secretion and a proinflammatory environment. The mechanism by which Aβ activates microglia is complex and involves numerous cellular components. One receptor potentially involved in Aβ recognition and the ensuing microglia proinflammatory response is CD47. Since there is significant interest in soluble aggregated Aβ species, we sought to determine if CD47 plays a key role in microglia cytokine release stimulated by soluble Aβ(1-42) protofibrils. Pretreatment of primary murine microglia with the CD47 antagonist peptide 4N1K significantly and potently inhibited both tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) secretion stimulated by Aβ(1-42) protofibrils. 4N1K displayed toxicity to the microglia but only at concentrations much higher than the observed inhibition. Surprisingly, 4N1K also potently inhibited TNFα secretion triggered by lipopolysaccharide which is not known to signal through CD47. Treatment of the microglia with a neutralizing anti-CD47 antibody failed to block the Aβ protofibril response even though comparable samples were completely inhibited by 4N1K. Finally, Aβ(1-42) protofibrils stimulated similar levels of secreted TNFα production in both wild-type and CD47(-/-) microglia and 4N1K still potently inhibited the Aβ protofibril response even in the CD47(-/-) microglia. The overall findings demonstrated that the microglial proinflammatory response to Aβ(1-42) protofibril is not dependent on CD47 and that 4N1K exhibits CD47-independent inhibitory activity.
- Published
- 2014
28. Characterization of Recombinant, Soluble β-Secretase from an Insect Cell Expression System
- Author
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Michael R. Nichols, Kumar Sambamurti, Debra Yager, Lisa M. Kopcho, Jovita Marcinkeviciene, Terrone L. Rosenberry, Luisa Onstead, Christopher B. Eckman, Robert A. Copeland, and William D. Mallender
- Subjects
Sequence analysis ,Cathepsin D ,Enzyme-Linked Immunosorbent Assay ,Peptide ,Biology ,Transfection ,law.invention ,Protein structure ,Sequence Analysis, Protein ,law ,Endopeptidases ,Amyloid precursor protein ,Animals ,Aspartic Acid Endopeptidases ,Humans ,Enzyme Inhibitors ,Cells, Cultured ,Chromatography, High Pressure Liquid ,Pharmacology ,chemistry.chemical_classification ,P3 peptide ,Molecular biology ,Recombinant Proteins ,Drosophila melanogaster ,Enzyme ,Solubility ,chemistry ,Biochemistry ,Recombinant DNA ,biology.protein ,Molecular Medicine ,Amyloid Precursor Protein Secretases ,Peptide Hydrolases - Abstract
The beta-site amyloid precursor protein-cleaving enzyme (BACE) cleaves the amyloid precursor protein to produce the N terminus of the amyloid beta peptide, a major component of the plaques found in the brains of Alzheimer's disease patients. Sequence analysis of BACE indicates that the protein contains the consensus sequences found in most known aspartyl proteases, but otherwise has only modest homology with aspartyl proteases of known three-dimensional structure (i.e., pepsin, renin, or cathepsin D). Because BACE has been shown to be one of the two proteolytic activities responsible for the production of the Abeta peptide, this enzyme is a prime target for the design of therapeutic agents aimed at reducing Abeta for the treatment of Alzheimer's disease. Toward this ultimate goal, we have expressed a recombinant, truncated human BACE in a Drosophila melanogaster S2 cell expression system to generate high levels of secreted BACE protein. The protein was convenient to purify and was enzymatically active and specific for cleaving the beta-secretase site of human APP, as demonstrated with soluble APP as the substrate in novel sandwich enzyme-linked immunosorbent assay and Western blot assays. Further kinetic analysis revealed no catalytic differences between this recombinant, secreted BACE, and brain BACE. Both showed a strong preference for substrates that contained the Swedish mutation, where NL is substituted for KM immediately upstream of the cleavage site, relative to the wild-type sequence, and both showed the same extent of inhibition by a peptide-based inhibitor. The capability to produce large quantities of BACE enzyme will facilitate protein structure determination and inhibitor development efforts that may lead to the evolution of useful Alzheimer's disease treatments.
- Published
- 2001
29. A Clinical Study of AlloDerm in Lip Augmentation
- Author
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Peter D. Waite, Melanie S. Lang, Jon D. Holmes, and Michael R. Nichols
- Subjects
Lip augmentation ,Clinical study ,030207 dermatology & venereal diseases ,03 medical and health sciences ,0302 clinical medicine ,business.industry ,Dentistry ,Medicine ,030230 surgery ,business - Abstract
Introduction: Lip augmentation is a frequently requested facial cosmetic procedure. The use of AlloDerm cosmetically has been documented since 1994, with clinical observations of some degree of resorption. The purpose of this prospective study was to objectively and subjectively assess the degree of labial change with AlloDerm implantation and its long-term clinical stability. Materials and Methods: Nineteen patients were treated with AlloDerm lip augmentation between January 1999 and October 1999. These patients were then followed up at 3, 6, and 12 months after surgery with a combination of physical evaluation, patient questionnaires, photographs, and lateral cephalometric radiographs. The data obtained were then evaluated objectively and subjectively. Objective analysis included computer analysis of photographs and lateral cephalometric radiographs. Subjective analysis included patient questionnaires and visual photographic assessment by professional independent evaluators. Results: Of the 19 patients initially enrolled in the AlloDerm lip study, 74% were seen on 3-month follow-up, 79% on 6-month follow-up, and 58% at 12-month follow-up. At 3 months, the compiled National Institutes of Health computer-analyzed photographic data showed an overall mean increase in the collective vermilion surface area of 16% on frontal and 39% on profile from the preoperative status. By 1 year, the overall mean increase in vermilion surface area was 8% on frontal and 36% on profile. The compiled lateral cephalometric radiographic data showed an overall mean increase in lip projection of 16% in the upper lip and 7% in the lower lip at 3 months. However, by 1 year, the overall mean increase in lip projection from preoperative status had dropped to 6% in the upper lip and 2% in the lower lip. Preoperatively, only 16% of the patients perceived their lips as looking about right or younger; this improved to 91% at 1 year. Independent evaluators viewed 54.5% of the patient's lips as looking about right or younger before surgery, as compared to 82% at 1 year. Conclusions: The objective results clearly indicate that there is substantial mean labial volumetric loss from 3 to 6 months after lip augmentation with AlloDerm. From 6 to 12 months, further volumetric loss is less substantial, with more stable clinical results. Despite documented resorption, there is an overall improvement in the subjective perception at all assessed points by both the patients and independent evaluators.
- Published
- 2000
30. Tyrosine Kinase-Independent Inhibition of Cyclic-AMP Phosphodiesterase by Genistein and Tyrphostin 51
- Author
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Bruce H. Morimoto and Michael R. Nichols
- Subjects
medicine.drug_class ,Phosphodiesterase 3 ,Biophysics ,Genistein ,PDE1 ,Biochemistry ,Tyrosine-kinase inhibitor ,Mice ,Neuroblastoma ,chemistry.chemical_compound ,Tumor Cells, Cultured ,medicine ,Animals ,Enzyme Inhibitors ,Tyrosine ,Molecular Biology ,Rolipram ,Phosphodiesterase ,Protein-Tyrosine Kinases ,Tyrphostins ,Cyclic Nucleotide Phosphodiesterases, Type 1 ,Enzyme Activation ,Kinetics ,chemistry ,3',5'-Cyclic-AMP Phosphodiesterases ,Tyrosine kinase ,medicine.drug - Abstract
The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.
- Published
- 1999
31. Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia
- Author
-
Geeta S. Paranjape, Benjamin M. Ruck, Michael R. Nichols, Lisa K. Gouwens, and Shana E. Terrill
- Subjects
Amyloid ,Immunology ,Size-exclusion chromatography ,Neuroscience (miscellaneous) ,Enzyme-Linked Immunosorbent Assay ,Fibril ,Article ,Mice ,Alzheimer Disease ,medicine ,Immunology and Allergy ,Bicinchoninic acid assay ,Animals ,Benzothiazoles ,Cerebrospinal Fluid ,Fluorescent Dyes ,Pharmacology ,Amyloid beta-Peptides ,Microglia ,Chemistry ,Tumor Necrosis Factor-alpha ,Macrophage Activation ,In vitro ,Peptide Fragments ,Culture Media ,Microscopy, Electron ,Thiazoles ,medicine.anatomical_structure ,Membrane ,Biochemistry ,Ionic strength ,Biophysics ,Chromatography, Gel ,Quinolines - Abstract
Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils
- Published
- 2012
32. Isolated amyloid-β(1-42) protofibrils, but not isolated fibrils, are robust stimulators of microglia
- Author
-
Michael R. Nichols, David Osborn, Lisa K. Gouwens, and Geeta S. Paranjape
- Subjects
Amyloid ,Physiology ,Cell Survival ,Cognitive Neuroscience ,Context (language use) ,Fibril ,Biochemistry ,Proinflammatory cytokine ,chemistry.chemical_compound ,Mice ,medicine ,Animals ,Senile plaques ,Cell Line, Transformed ,Amyloid beta-Peptides ,Microglia ,Cell Biology ,General Medicine ,Peptide Fragments ,Mice, Inbred C57BL ,medicine.anatomical_structure ,chemistry ,Animals, Newborn ,Biophysics ,Thioflavin ,Tumor necrosis factor alpha - Abstract
Senile plaques composed of amyloid-β protein (Aβ) are an unshakable feature of the Alzheimer's disease (AD) brain. Although there is significant debate on the role of the plaques in AD progression, there is little disagreement on their role in stimulating a robust inflammatory response within the context of the disease. Significant inflammatory markers such as activated microglia and cytokines are observed almost exclusively surrounding the plaques. However, recent evidence suggests that the plaque exterior may contain a measurable level of soluble Aβ aggregates. The observations that microglia activation in vivo is selectively stimulated by distinct Aβ deposits led us to examine what specific form of Aβ is the most effective proinflammatory mediator in vitro. We report here that soluble prefibrillar species of Aβ(1-42) were better than fibrils at inducing microglial tumor necrosis factor α (TNFα) production in either BV-2 and primary murine microglia. Reconstitution of Aβ(1-42) in NaOH followed by dilution into F-12 media and isolation with size exclusion chromatography (SEC) revealed classic curvilinear β-sheet protofibrils 100 nm in length. The protofibrils, but not monomers, markedly activated BV-2 microglia. Comparisons were also made between freshly isolated protofibrils and Aβ(1-42) fibrils prepared from SEC-purified monomer. Surprisingly, while isolated fibrils had a much higher level of thioflavin T fluorescence per mole, they were not effective at stimulating either primary or BV-2 murine microglia compared to protofibrils. Furthermore, SEC-isolated Aβ(1-40) protofibrils exhibited significantly less activity than concentration-matched Aβ(1-42). This report is the first to demonstrate microglial activation by SEC-purified protofibrils, and the overall findings indicate that small, soluble Aβ(1-42) protofibrils induce much greater microglial activation than mature insoluble fibrils.
- Published
- 2011
33. Special issue on Alzheimer's disease
- Author
-
Colin K. Combs and Michael R. Nichols
- Subjects
Pharmacology, Toxicology and Pharmaceutics(all) ,Amyloid β ,business.industry ,Biochemistry, Genetics and Molecular Biology(all) ,Immunology ,Medicine ,General Medicine ,Disease ,General Pharmacology, Toxicology and Pharmaceutics ,business ,General Biochemistry, Genetics and Molecular Biology - Published
- 2011
- Full Text
- View/download PDF
34. The Chinese Communist Intervention in the Korean War: An Exercise in Analyzing Documents
- Author
-
Michael R. Nichols
- Subjects
History ,business.industry ,World history ,Public relations ,Social studies ,Education ,Test (assessment) ,History and Philosophy of Science ,Political science ,Analytical skill ,Development economics ,ComputingMilieux_COMPUTERSANDEDUCATION ,Advanced Placement ,business ,China ,Lesson plan ,Communism - Abstract
Objectives T? his exercise seeks to analyze four documents pertaining to the Korean War, in order to assist students preparing for the Document-Based Questions (DBQs) of the College Board Advanced Placement Test. The discussion questions are designed to stimulate students to ask certain questions when examining the documents. The documents in this lesson plan allow students to see how the United States, the Soviet Union, and the People's Republic of China (PRC) all perceived the Korean War. These documents are not meant to represent a DBQ as a whole. Instead, they are designed to get students to analyze documents for their importance so that they can quickly interpret other documents during the Advanced Placement Test. Students should be able to apply these analytic skills to the broader question of the DBQ.
- Published
- 2000
35. The Role of Humor in Transforming Stressful Life Events
- Author
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Clifford C. Kuhn, Barbara L. Belew, and Michael R. Nichols
- Subjects
Face-to-face ,History ,Aesthetics ,Natural Killer Cell Activity ,Life events ,Sorrow - Abstract
There often seems to be only one constant in life – change! And change comes in all sizes, shapes, and colors. Life changes can be so major that they overwhelm us, or so minor as to be hardly noticeable. They can be very subtle or dramatic, happy or sad, welcomed or feared. Change can be a cause for celebration or sorrow. Change can provide new opportunities to reach for our greatest potential, or change can diminish our capacities and cause us to redefine ourselves. Change sometimes brings us face to face with the prospect of death – our own or a loved one’s. Change can take many forms
- Published
- 2009
36. Amyloid-β(1-42) Fibrillar Precursors are Optimal for Inducing Tumor Necrosis Factor-α Production in the THP-1 Human Monocytic Cell Line†
- Author
-
Michael R. Nichols, Maria L. D. Udan, Deepa Ajit, and Geeta S. Paranjape
- Subjects
Biology ,Microscopy, Atomic Force ,Biochemistry ,Article ,Monocytes ,Proinflammatory cytokine ,Cell Line ,Alzheimer Disease ,mental disorders ,medicine ,Humans ,THP1 cell line ,Senile plaques ,Protein Precursors ,Protein Structure, Quaternary ,Amyloid beta-Peptides ,Microglia ,Tumor Necrosis Factor-alpha ,P3 peptide ,In vitro ,Peptide Fragments ,Cell biology ,medicine.anatomical_structure ,Cell culture ,Immunology ,Tumor necrosis factor alpha ,Inflammation Mediators - Abstract
Pathological studies have determined that fibrillar forms of amyloid-beta protein (Abeta) comprise the characteristic neuritic plaques in Alzheimer's disease (AD). These studies have also revealed significant inflammatory markers such as activated microglia and cytokines surrounding the plaques. Although the plaques are a hallmark of AD, they are only part of an array of Abeta aggregate morphologies observed in vivo. Interestingly, not all of these Abeta deposits provoke an inflammatory response. Since structural polymorphism is a prominent feature of Abeta aggregation both in vitro and in vivo, we sought to clarify which Abeta morphology or aggregation species induces the strongest proinflammatory response using human THP-1 monocytes as a model system. An aliquot of freshly reconstituted Abeta(1-42) in sterile water (100 microM, pH 3.6) did not effectively stimulate the cells at a final Abeta concentration of 15 microM. However, quiescent incubation of the peptide at 4 degrees C for 48-96 h greatly enhanced its ability to induce tumor necrosis factor-alpha (TNFalpha) production, the level of which surprisingly declined upon further aggregation. Imaging of the Abeta(1-42) aggregation solutions with atomic force microscopy indicated that the best cellular response coincided with the appearance of fibrillar structures, yet conditions that accelerated or increased the level of Abeta(1-42) fibril formation such as peptide concentration, temperature, or reconstitution in NaOH/PBS at pH 7.4 diminished its ability to stimulate the cells. Finally, depletion of the Abeta(1-42) solution with an antibody that recognizes fibrillar oligomers dramatically weakened the ability to induce TNFalpha production, and size-exclusion separation of the Abeta(1-42) solution provided further characterization of an aggregated species with proinflammatory activity. The findings suggested that an intermediate stage Abeta(1-42) fibrillar precursor is optimal for inducing a proinflammatory response in THP-1 monocytes.
- Published
- 2009
37. Rapid assembly of amyloid-beta peptide at a liquid/liquid interface produces unstable beta-sheet fibers
- Author
-
Dana Kim Reed, Jan H. Hoh, Michael R. Nichols, Melissa A. Moss, and Terrone L. Rosenberry
- Subjects
chemistry.chemical_classification ,Circular dichroism ,Chromatography ,Amyloid beta-Peptides ,Binding Sites ,Beta sheet ,Aqueous two-phase system ,Peptide ,Fibril ,Microscopy, Atomic Force ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Kinetics ,Protein structure ,Freeze Drying ,chemistry ,Alzheimer Disease ,Amphiphile ,Biophysics ,Humans ,Thioflavin - Abstract
Accumulation of aggregated amyloid-beta peptide (Abeta) in the brain is a pathological hallmark of Alzheimer's disease (AD). In vitro studies indicate that the 40- to 42-residue Abeta peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Abeta peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Abeta aggregation, but detailed characterization of these aggregates has not been presented. In this study Abeta(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Abeta in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Abeta aggregates had a beta-sheet secondary structure. However, these aggregates were far less stable than Abeta(1-40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Abeta protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Abeta with beta-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic beta-sheet structure of an in vitro Abeta aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo.
- Published
- 2005
38. Nordihydroguaiaretic acid does not disaggregate beta-amyloid(1-40) protofibrils but does inhibit growth arising from direct protofibril association
- Author
-
Melissa A, Moss, Nicholas H, Varvel, Michael R, Nichols, Dana Kim, Reed, and Terrone L, Rosenberry
- Subjects
Thiazoles ,Amyloid beta-Peptides ,Alzheimer Disease ,Humans ,Masoprocol ,Benzothiazoles ,Antioxidants ,Fluorescence ,Peptide Fragments - Abstract
Nordihydroguaiaretic acid (NDGA) was observed by Ono et al. (J Neurochem 87:172-181, 2002) to decrease the fluorescence of thioflavin T associated with freshly extended amyloid beta-protein (Abeta) fibrils. They concluded that NDGA could disaggregate Abeta fibrils into aggregates that were larger than monomers or oligomers and did not bind thioflavin T. Such an effect could be of therapeutic importance in the treatment of Alzheimer's disease. In the current study, we confirmed that NDGA induces a decrease in the fluorescence of thioflavin T associated with Abeta(1-40) fibrils and extended this observation to Abeta(1-40) protofibrils. However, attempts to identify protofibril disaggregation products using dynamic light scattering, electron microscopy, and size exclusion chromatography failed to demonstrate any decrease in aggregate size or concentration or a parallel increase in Abeta monomers or small oligomers when protofibrils were incubated with excess NDGA. We propose instead that the decreases in thioflavin T fluorescence resulted from either displacement or conformational alteration of thioflavin T upon the binding of NDGA to these aggregates. In fact, the same equilibrium fluorescence values were observed regardless of the order in which NDGA, thioflavin T, and Abeta protofibrils were added to the incubation. Although NDGA failed to disaggregate Abeta protofibrils, it did inhibit direct protofibril-protofibril association but did not alter protofibril elongation by monomer addition. These results suggest that NDGA might bind along the lateral surface of Abeta protofibrils. In addition, the binding of NDGA to Abeta protofibrils increased their nonspecific adherence to Superdex 75 resin and diminished their effects on cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
- Published
- 2004
39. Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy
- Author
-
Dana Kim Reed, Jan H. Hoh, Michael R. Nichols, Melissa A. Moss, Rajendrani Mukhopadhyay, Terrone L. Rosenberry, and Wen Lang Lin
- Subjects
Amyloid ,Light ,Macromolecular Substances ,Proteolysis ,Size-exclusion chromatography ,In Vitro Techniques ,Microscopy, Atomic Force ,Biochemistry ,Methylation ,chemistry.chemical_compound ,Alzheimer Disease ,medicine ,Amyloid precursor protein ,Humans ,Scattering, Radiation ,Amyloid beta-Peptides ,medicine.diagnostic_test ,biology ,Fibrillogenesis ,In vitro ,Peptide Fragments ,Molecular Weight ,Crystallography ,Microscopy, Electron ,Monomer ,chemistry ,biology.protein ,Biophysics ,Elongation - Abstract
Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.
- Published
- 2002
40. Differential inhibition of multiple cAMP phosphodiesterase isozymes by isoflavones and tyrphostins
- Author
-
Michael R. Nichols and Bruce H. Morimoto
- Subjects
Phosphodiesterase 3 ,Tyrphostins ,PDE1 ,Isozyme ,chemistry.chemical_compound ,Structure-Activity Relationship ,3',5'-Cyclic-GMP Phosphodiesterases ,Structure–activity relationship ,Animals ,Humans ,Enzyme Inhibitors ,Cells, Cultured ,Pharmacology ,Cyclic nucleotide phosphodiesterase ,Chemistry ,Phosphoric Diester Hydrolases ,Phosphodiesterase ,Isoflavones ,Cyclic Nucleotide Phosphodiesterases, Type 1 ,Cyclic Nucleotide Phosphodiesterases, Type 3 ,Isoenzymes ,Biochemistry ,3',5'-Cyclic-AMP Phosphodiesterases ,Molecular Medicine ,Cattle ,sense organs ,hormones, hormone substitutes, and hormone antagonists - Abstract
A series of isoflavone and tyrphostin compounds were found to inhibit the degradation of cAMP by several cyclic nucleotide phosphodiesterase (PDE) isozymes. Specific hydroxyl groups on the isoflavone structure were critical for PDE isozyme-selective inhibition. Replacement of the C-7 hydroxyl group of the isoflavone with a methoxy group raised the IC(50) for PDE1, PDE3, and PDE4. The absence of the C-5 hydroxyl group raised the IC(50) from 5 to >100 microM for PDE4, but actually lowered the IC(50) for PDE3 and PDE1. Replacement of the C-4' hydroxyl group with a methoxy group raised the IC(50) for PDE3 and PDE1, yet only slightly changed the IC(50) for PDE4. Various tyrphostins were also potent inhibitors of PDE1, PDE3, and PDE4. The four-carbon side chained tyrphostins were much less potent; however, a very interesting pattern was observed in which removal of phenolic hydroxyls on the tyrphostin structure increased the potency for PDE1 and PDE3, but not PDE4. These results may help to explain some of the therapeutic and intracellular signaling effects of isoflavones and tyrphostins. Moreover, the isozyme selectivity demonstrated by the isoflavones and tyrphostins can serve as a pharmacophore for the design of specific PDE inhibitors.
- Published
- 2000
41. Introduction
- Author
-
Michael R. Nichols and Colin K. Combs
- Subjects
Amyloid ,business.industry ,P3 peptide ,General Medicine ,Disease ,medicine.disease ,General Biochemistry, Genetics and Molecular Biology ,Biochemistry of Alzheimer's disease ,Cancer research ,medicine ,General Pharmacology, Toxicology and Pharmaceutics ,Alzheimer's disease ,Beta (finance) ,business ,Intracellular - Published
- 2012
42. Development of LPS antagonistic therapeutics: synthesis and evaluation of glucopyranoside-spacer-amino acid motifs
- Author
-
Maria L. D. Udan, Aileen F. G. Bongat, Sophon Kaeothip, Alexei V. Demchenko, Shana E. Terrill, Michael R. Nichols, Geeta S. Paranjape, Teerada Kamkhachorn, and Hope L. Johnson
- Subjects
chemistry.chemical_classification ,Innate immune system ,Lipopolysaccharide ,Chemistry ,General Chemical Engineering ,CD14 ,Inflammation ,General Chemistry ,Amino acid ,Proinflammatory cytokine ,Lipid A ,chemistry.chemical_compound ,Biochemistry ,medicine ,Macrophage ,medicine.symptom - Abstract
Sepsis is a serious medical condition characterized by bacterial infection and a subsequent massive systemic inflammatory response. The release of proinflammatory products and mediators from responding innate immune cells, such as mononuclear phagocytes, directly contributes to the pathogenesis of sepsis. The primary bacterial trigger of inflammation is lipopolysaccharide (LPS), which interacts with the germline-encoded macrophage receptor cluster of differentiation 14 (CD14) via its Lipid A moiety. In an effort to identify compounds that block LPS-induced inflammation we investigated a series of Lipid A analogs that lack a disaccharide core yet still possess potent antagonistic activity against LPS. We found it beneficial to develop molecules that contain the following: a glucopyranoside core, hydrophobic ether substituents, and an amino acid to provide an ionic character to the constructs. Here we report an efficient synthesis of molecules of this type and the ensuing biological studies thereof.
- Published
- 2011
43. A Historical Reevaluation of America's Role in the Kuril Islands Dispute
- Author
-
Michael R. Nichols, Matthew J. Ouimet, and Bruce A. Elleman
- Subjects
Sociology and Political Science ,media_common.quotation_subject ,Geography, Planning and Development ,World War II ,Possession (law) ,Territorial dispute ,Peace treaty ,Negotiation ,Political science ,Economic history ,Treaty ,Annexation ,On the Peace ,media_common - Abstract
A S RECENTLY as December 1995, Japanese Minister of State Masaki Nakayama announced "that, from a legal standpoint, Russia and Japan are still in a state of war."' A formal peace treaty ending World War II has continued to elude Moscow and Tokyo, even though both sides have expressed confidence that a treaty terminating Russo-Japanese hostilities will be signed before the turn of the century. One of the primary factors impeding progress on the peace talks has been the territorial dispute over which islands actually constitute the Kuril Island chain. As background to the Kuril Islands dispute, this article will first examine the early history of the Kurils, when Japan initially gained legal possession over them, and then the World War II negotiations. These negotiations led to the Soviet annexation of the entire Kuril Island chain as well as the Habomai Islands and Shikotan Island, formerly considered an indivisible part of the island of Hokkaido. The article will then consider the SovietAmerican exchanges between Stalin and Truman over the exact wording of the United States' General Order No. 1. Finally, it will discuss the pitfalls such as the so-called "Dulles Threat Incident" surrounding the Kuril negotiations between Moscow and Tokyo during the 1950s, negotiations that have yet to be completed. Based on declassified U.S. government documents from the W. Averell Harriman collection at the Library of Congress and the John Foster Dulles collection at Princeton University, this article will conclude that the United States never condoned the permanent cession of all of the Kuril Islands to the Soviet Union. Rather, Washington's policy from the Yalta Conference onward merely agreed that Moscow could negotiate directly with Tokyo to arrive at a mutually acceptable solution. In an attempt to assist Japan in these negotiations, John Foster Dulles even suggested in 1956 that Japan linkAmerica's planned return of Okinawa to the Soviet return of the disputed Kuril Islands. According to the American viewpoint, in the absence of a Russo
- Published
- 1998
44. Increased central serotonergic activity associated with nocturnal anorexia induced by Walker 256 carcinoma
- Author
-
George K.W. Yim, Roger P. Maickel, and Michael R. Nichols
- Subjects
Male ,Serotonin ,medicine.medical_specialty ,Food intake ,Anorexia ,Nocturnal ,Biology ,Serotonergic ,General Biochemistry, Genetics and Molecular Biology ,Walker 256 carcinoma ,Feeding and Eating Disorders ,Eating ,Internal medicine ,medicine ,Animals ,Humans ,Carcinoma 256, Walker ,General Pharmacology, Toxicology and Pharmaceutics ,Tryptophan ,Brain ,Rats, Inbred Strains ,General Medicine ,Hydroxyindoleacetic Acid ,Serotonin metabolism ,Circadian Rhythm ,Rats ,Endocrinology ,medicine.symptom - Abstract
The role of brain serotonin levels in Walker 256 tumor induced anorexia was investigated. Total and free plasma tryptophan, regional brain serotonin and 5-hydroxyindoleacetic acid were determined at night, and their relationship to nocturnal anorexia assessed by linear regression analysis. No significant difference in tryptophan, serotonin, or 5-hydroxyindoleacetic acid levels was detected between pair fed and tumor bearing rats exhibiting a 20% reduction of nighttime food intake. Tumor bearing rats with a 40% reduction in food intake had higher nighttime plasma free tryptophan and regional 5-hydroxyindoleacetic acid levels than their pair fed malnourished controls. These results indicate that increased plasma free tryptophan and elevated serotonin metabolism may not be the initial dysfunction responsible for nocturnal anorexia. However, it may contribute to the decreasing nocturnal food intake in severely anorexic tumor rats.
- Published
- 1983
45. A Model of Job Stress and Burnout
- Author
-
Eileen Berlin Ray, Lea J. Perritt, and Michael R. Nichols
- Subjects
Nursing staff ,Job stress ,Health Policy ,Applied psychology ,Hospices ,Public Health, Environmental and Occupational Health ,Models, Psychological ,Burnout ,medicine.disease_cause ,medicine ,Humans ,Psychological stress ,Nursing Staff ,Psychology ,Burnout, Professional ,Stress, Psychological - Abstract
Because hospice is still considered a fairly new and innovative concept in the United States (Munley, 1983; Torrens, 1984), attempts to provide a systematic method of identifying factors le...
- Published
- 1987
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