scholarly journals A Toxic Synergy between Aluminium and Amyloid Beta in Yeast

2021 ◽  
Vol 22 (4) ◽  
pp. 1835
Author(s):  
Jamieson B. Mcdonald ◽  
Sudip Dhakal ◽  
Ian Macreadie
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Growth Inhibition ◽  
Cell Viability ◽  
Amyloid Beta ◽  
Fluorescent Protein ◽  
Late Onset ◽  
Model Organism ◽  
Age Related ◽  
Green Fluorescent

Alzheimer’s disease (AD), the most prevalent, age-related, neurodegenerative disease, is associated with the accumulation of amyloid beta (Aβ) and oxidative stress. However, the sporadic nature of late-onset AD has suggested that other factors, such as aluminium may be involved. Aluminium (Al3+) is the most ubiquitous neurotoxic metal on earth, extensively bioavailable to humans. Despite this, the link between Al3+ and AD has been debated for decades and remains controversial. Using Saccharomyces cerevisiae as a model organism expressing Aβ42, this study aimed to examine the mechanisms of Al3+ toxicity and its interactions with Aβ42. S. cerevisiae cells producing Aβ42 treated with varying concentrations of Al3+ were examined for cell viability, growth inhibition, and production of reactive oxygen species (ROS). Al3+ caused a significant reduction in cell viability: cell death in yeast producing green fluorescent protein tagged with Aβ42 (GFP–Aβ42) was significantly higher than in cells producing green fluorescent protein (GFP) alone. Additionally, Al3+ greatly inhibited the fermentative growth of yeast producing GFP–Aβ42, which was enhanced by ferric iron (Fe3+), while there was negligible growth inhibition of GFP cells. Al3+- induced ROS levels in yeast expressing native Aβ42 were significantly higher than in empty vector controls. These findings demonstrate Al3+ has a direct, detrimental toxic synergy with Aβ42 that can be influenced by Fe3+, causing increased oxidative stress. Thus, Al3+ should be considered as an important factor, alongside the known characteristic hallmarks of AD, in the development and aetiology of the disease.

scholarly journals Simvastatin Efficiently Reduces Levels of Alzheimer’s Amyloid Beta in Yeast

2019 ◽  
Vol 20 (14) ◽  
pp. 3531 ◽  
Author(s):  
Sudip Dhakal ◽  
Mishal Subhan ◽  
Joshua M. Fraser ◽  
Kenneth Gardiner ◽  
Ian Macreadie
Keyword(s):  
Fusion Protein ◽  
Amyloid Beta ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Dependent Manner ◽  
Green Fluorescence ◽  
Convenient Means ◽  
Ergosterol Synthesis ◽  
Green Fluorescent

A large-scale epidemiology study on statins previously showed that simvastatin was unique among statins in reducing the incidence of dementia. Since amyloid beta (Aβ42) is the protein that is most associated with Alzheimer’s disease, this study has focused on how simvastatin influences the turnover of native Aβ42 and Aβ42 fused with green fluorescent protein (GFP), in the simplest eukaryotic model organism, Saccharomyces cerevisiae. Previous studies have established that yeast constitutively producing Aβ42 fused to GFP offer a convenient means of analyzing yeast cellular responses to Aβ42. Young cells clear the GFP fusion protein and do not have green fluorescence while the older population of cells retains the fusion protein and exhibits green fluorescence, offering a fast and convenient means of studying factors that affect Aβ42 turnover. In this study the proportion of cells having GFP fused to Aβ after exposure to simvastatin, atorvastatin and lovastatin was analyzed by flow cytometry. Simvastatin effectively reduced levels of the cellular Aβ42 protein in a dose-dependent manner. Simvastatin promoted the greatest reduction as compared to the other two statins. A comparison with fluconazole, which targets that same pathway of ergosterol synthesis, suggests that effects on ergosterol synthesis do not account for the reduced amounts of Aβ42 fused to GFP. The levels of native Aβ42 following treated with simvastatin were also examined using a more laborious approach, quantitative MALDI TOF mass spectrometry. Simvastatin efficiently reduced levels of native Aβ42 from the population. This work indicates a novel action of simvastatin in reducing levels of Aβ42 providing new insights into how simvastatin exerts its neuroprotective role. We hypothesize that this reduction may be due to protein clearance.

scholarly journals Green Fluorescent Protein as a Novel Indicator of Antimicrobial Susceptibility in Aureobasidium pullulans

2001 ◽  
Vol 67 (12) ◽  
pp. 5614-5620 ◽  
Author(s):  
Jeremy S. Webb ◽  
Sarah R. Barratt ◽  
Hristo Sabev ◽  
Marianne Nixon ◽  
Ian M. Eastwood ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Viability ◽  
Aureobasidium Pullulans ◽  
Fluorescent Protein ◽  
Fungal Species ◽  
Antimicrobial Compounds ◽  
Viable Cells ◽  
Gfp Fluorescence ◽  
Highly Correlated ◽  
Green Fluorescent

ABSTRACT Presently there is no method available that allows noninvasive and real-time monitoring of fungal susceptibility to antimicrobial compounds. The green fluorescent protein (GFP) of the jellyfishAequoria victoria was tested as a potential reporter molecule for this purpose. Aureobasidium pullulans was transformed to express cytosolic GFP using the vector pTEFEGFP (A. J. Vanden Wymelenberg, D. Cullen, R. N. Spear, B. Schoenike, and J. H. Andrews, BioTechniques 23:686–690, 1997). The transformed strain Ap1 gfp showed bright fluorescence that was amenable to quantification using fluorescence spectrophotometry. Fluorescence levels in Ap1 gfp blastospore suspensions were directly proportional to the number of viable cells determined by CFU plate counts (r 2 > 0.99). The relationship between cell viability and GFP fluorescence was investigated by adding a range of concentrations of each of the biocides sodium hypochlorite and 2-n-octylisothiozolin-3-one (OIT) to suspensions of Ap1gfp blastospores (pH 5 buffer). These biocides each caused a rapid (<25-min) loss of fluorescence of greater than 90% when used at concentrations of 150 μg of available chlorine ml−1 and 500 μg ml−1, respectively. Further, loss of GFP fluorescence from A. pullulanscells was highly correlated with a decrease in the number of viable cells (r 2 > 0.92). Losses of GFP fluorescence and cell viability were highly dependent on external pH; maximum losses of fluorescence and viability occurred at pH 4, while reduction of GFP fluorescence was absent at pH 8.0 and was associated with a lower reduction in viability. When A. pullulanswas attached to the surface of plasticized poly(vinylchloride) containing 500 ppm of OIT, fluorescence decreased more slowly than in cell suspensions, with >95% loss of fluorescence after 27 h. This technique should have broad applications in testing the susceptibility of A. pullulans and other fungal species to antimicrobial compounds.

High Throughput Studies of Gene Expression Using Green Fluorescent Protein-Oxidative Stress Promoter Probe Constructs The Potential for Living Chips

2001 ◽  
Vol 6 (6) ◽  
pp. 421-428
Author(s):  
C. Renee Albano ◽  
Canghai Lu ◽  
William E. Bentley ◽  
Govind Rao
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Green Fluorescent Protein ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chip Technology ◽  
Great Specificity ◽  
Green Fluorescent ◽  
Stress Promoter

Green fluorescent protein fusions were constructed with several oxidative stress promoters from Escherichia coli. These promoters were chosen for their induction by reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and hydroxyl radicals. When exposed to various free radical insults, the cells fluoresced with great specificity based on the corresponding ROS. In this work, we propose a way in which these constructs could be used to study the mode of action of a variety of antitumor drugs. This approach offers the possibility of complementing gene chip technology by the creation of living chips for high throughput screening as well as studying differential gene expression.

Non-viral genetic transfection of rat Schwann cells with FuGENE HD© lipofection and AMAXA© nucleofection is feasible but impairs cell viability

2010 ◽  
Vol 6 (4) ◽  
pp. 225-230 ◽  
Author(s):  
Armin Kraus ◽  
Joachim Täger ◽  
Konrad Kohler ◽  
Max Haerle ◽  
Frank Werdin ◽  
...  
Keyword(s):  
Cell Culture ◽  
Green Fluorescent Protein ◽  
Cell Viability ◽  
Schwann Cells ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Cell Counting ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Viral Transfection

Purpose:To determine transfection efficiency of FuGENE HD© lipofection and AMAXA© nucleofection on rat Schwann cells (SC).Methods:The ischiadic and median nerves of 6-8 week old Lewis rats were cultured in modified melanocyte-growth medium. SCs were genetically transfected with green fluorescent protein (GFP) as reporter gene using FuGENE HD© lipofection and AMAXA© nucleofection. Transfection rates were determined by visualization of GFP fluorescence under fluorescence microscopy and cell counting. Transfected cell to non-transfected cell relation was determined.Results:Purity of Schwann cell culture was 88% as determined by immunohistologic staining. Transfection rate of FuGENE HD© lipofection was 2%, transfection rate of AMAXA© nucleofection was 10%. With both methods, Schwann cells showed pronounced aggregation behavior which made them unfeasible for further cultivation. Settling of Schwann cells on laminin and poly-l-ornithine coated plates was compromised by either method.Conclusion:Non-viral transfection of rat SC with FuGENE HD© lipofection and AMAXA© nucleofection is basically possible with a higher transfection rate for nucleofection than for lipofection. As cell viability is compromised by either method however, viral transfection is to be considered if higher efficiency is required.

Eel green fluorescent protein is associated with resistance to oxidative stress

2016 ◽  
Vol 181-182 ◽  
pp. 35-39 ◽  
Author(s):  
Aki Funahashi ◽  
Masaharu Komatsu ◽  
Tatsuhiko Furukawa ◽  
Yuki Yoshizono ◽  
Hikari Yoshizono ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

Monitoring Oxidative Stress and DNA Damage Induced by Heavy Metals in Yeast Expressing a Redox-Sensitive Green Fluorescent Protein

2009 ◽  
Vol 58 (5) ◽  
pp. 504-510 ◽  
Author(s):  
Shanshan Yu ◽  
Wei Qin ◽  
Guoqiang Zhuang ◽  
Xianen Zhang ◽  
Guanjun Chen ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heavy Metals ◽  
Dna Damage ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals A single-wavelength flow cytometric approach using redox-sensitive green fluorescent protein probes for measuring redox stress in live cells

BioTechniques ◽  
2021 ◽  
Author(s):  
Elizabeth R Denn ◽  
Joseph M Schober
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recovery Phase ◽  
Live Cells ◽  
Redox Stress ◽  
Green Fluorescent

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.

Quantitative and kinetic study of oxidative stress regulons using green fluorescent protein

2005 ◽  
Vol 89 (5) ◽  
pp. 574-587 ◽  
Author(s):  
Canghai Lu ◽  
C. Renee Albano ◽  
William E. Bentley ◽  
Govind Rao
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Kinetic Study ◽  
Fluorescent Protein ◽  
Green Fluorescent
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