No description available.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

The alveoli of mammalian lungs are covered in a thin lipid film referred to as pulmonary surfactant. The primary purpose of pulmonary surfactant is to reduce the surface tension of the air/liquid interface allowing breathing with minimal effort required. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is inconsequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that for bothanionic DOPG and cationic DOTAP films which are in the fluid phase, amyloid-β interacts with the bilayer much quicker than in zwitterionic DPPC which is in the gel phase. Approaching 24 hours, we see small fibrils form on the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution. For fluid phase bilayer membrane, disruption is also observed. We investigated the effect of addition of cholesterol and amyloid-β peptide on structure and function of Bovine Lung Extract Surfactant (pulmonary surfactant BLES) and model lipid films. In our first experiment, we have demonstrated the effect of amyloid-β and cholesterol on lipid films of DPPC, DPPC-DOPG and BLES. We saw that cholesterol inhibits multilayer formation in all monolayers. Amyloid-β increases multilayer formation in DPPC and DPPC-DOPG, but reduced multilayer formation in BLES. When cholesterol and amyloid-β is added to BLES, 1% amyloid-β is in consequential, whereas 10% amyloid-β allows BLES to regain some of its surfactant function. In our second experiment, we observed that in anionic DOPG films, amyloid-β inserts into the bilayer much quicker than in zwitterionic DPPC. Approaching 24 hours, we see small fibrils form in the bilayer, but these fibrils are considerably smaller than those formed when amyloid-β is incubated in solution.

Diss. Linköping : Univ., 2001.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 81-91). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 81-91). Also available on the Internet.

Transthyretin-related amyloidosis (ATTR) is a disease involving the formation of a misfolded transthyretin (TTR) protein and resulting insoluble aggregates that deposit in extracellular regions of various tissues and organs. There are hereditary forms of the disease, referred to as ATTRm, and more than 100 TTR amyloid-forming mutants have been reported. The major goal of this work was to analyze the biochemical and biophysical properties of a unique and recently identified TTR mutant protein, TTR Glu51_Ser52dup, found in a patient with ATTRm. Unlike other single nucleotide replacements that have been described as amyloidogenic, the gene abnormality in the present case is the first identification of a TTR duplication mutation. The patient with TTR Glu51_Ser52dup exhibited an extremely aggressive form of ATTRm; clinical symptoms included peripheral neuropathy at baseline evaluation and rapid disease progression to early death from pneumonia and congestive heart failure. We hypothesized that the TTR Glu51_Ser52dup variant would be less stable than the wild-type protein and similar in stability to another highly amyloidogenic mutant, TTR L55P; moreover, the highly unstable nature of this TTR variant would provide a basis for understanding the extremely aggressive clinical phenotype observed in this case. Using Escherichia coli (E. coli) as an expression system and an appropriately modified expression vector, we produced histidine-tagged recombinant human TTR Glu51_Ser52dup protein in high yield and purified to homogeneity. Structural and stability studies were performed by circular dichroism (CD) spectroscopy and SDS-PAGE analysis. We demonstrated that TTR Glu51_Ser52dup was less stable than the wild-type or L55P proteins when measured under different types of denaturing conditions, including thermal and chemical stress. The presence of diflunisal, a drug that stabilizes tetrameric TTR and is currently approved for treatment of ATTRm, was also investigated; our results indicated that diflunisal stabilized the TTR Glu51_Ser52dup protein. Collectively, the data obtained from these studies suggest that Glu51_Ser52dup is one of the least stable and most amyloidogenic TTR variant described to date. Future investigations are necessary to determine which specific structural elements of the protein destabilize the TTR tetramer, and precisely characterize the binding of small molecules, including diflunisal, to the protein.

No / Beta-2-microglobulin (2m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (~1 m), straight morphology are produced by incubation of the protein at pH=<3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of 2m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.

No description available.

No description available.