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.

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.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

2001 ◽  
Vol 183 (23) ◽  
pp. 6752-6762 ◽  
Author(s):  
Johan H. J. Leveau ◽  
Steven E. Lindow
Keyword(s):  
Growth Rate ◽  
Green Fluorescent Protein ◽  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Proteolytic Degradation ◽  
Bacterial Cells ◽  
Prior Testing ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Best Fit

ABSTRACT We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-β-d-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfpfusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

scholarly journals A Rapid Method to Determine the Stress Status of Saccharomyces cerevisiae by Monitoring the Expression of a Hsp12:Green Fluorescent Protein (GFP) Construct under the Control of the Hsp12 Promoter

2005 ◽  
Vol 10 (3) ◽  
pp. 253-259 ◽  
Author(s):  
Robert J. Karreman ◽  
George G. Lindsey
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Stress ◽  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fusion Protein ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Time Dependent ◽  
Gfp Fluorescence ◽  
Green Fluorescent

The gene for the green fluorescent protein (GFP) was fused in-frame to the 3′ end of HSP12. This construct was regulated by the HSP12 promoter in a pYES2 yeast expression vector. No fluorescence was observed in yeast growing exponentially in glucose-containing medium, but fluorescence was observed when the yeast entered the stationary phase. Fluorescence microscopy indicated that the fusion protein was localized to the peripheral regions of the cell as well as to the cytoplasm and the tonoplast. Subjecting the yeast to a variety of stresses known to induce HSP12 transcription, including salt, osmotic, ethanol, and heat stress, resulted in a time-dependent increase in GFP fluorescence. The use of this system as a method to assess the general stress status of yeast growing in an industrial application is proposed.

scholarly journals Expression of Green Fluorescent Protein in Aureobasidium pullulans and Quantification of the Fungus on Leaf Surfaces

BioTechniques ◽  
1997 ◽  
Vol 23 (4) ◽  
pp. 686-690 ◽  
Author(s):  
A.J. Vanden Wymelenberg ◽  
D. Cullen ◽  
R.N. Spear ◽  
B. Schoenike ◽  
J.H. Andrews
Keyword(s):  
Green Fluorescent Protein ◽  
Aureobasidium Pullulans ◽  
Fluorescent Protein ◽  
Leaf Surfaces ◽  
Green Fluorescent

scholarly journals Identification of elements in the Smcp 5′ and 3′ UTR that repress translation and promote the formation of heavy inactive mRNPs in spermatids by analysis of mutations in transgenic mice

Reproduction ◽  
2010 ◽  
Vol 140 (6) ◽  
pp. 853-864 ◽  
Author(s):  
Jana Bagarova ◽  
Tamjid A Chowdhury ◽  
Mine Kimura ◽  
Kenneth C Kleene
Keyword(s):  
Green Fluorescent Protein ◽  
Early Detection ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Mrna Translation ◽  
Seminiferous Tubules ◽  
Cysteine Rich Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Reading Frames

The sperm mitochondria-associated cysteine-rich protein (Smcp) mRNA is transcribed in step 3 spermatids, and is stored in free mRNPs until translation begins ∼6 days later in step 11. To identify sequences that control the timing of Smcp mRNA translation, mutations in both UTRs were analyzed in transgenic mice using green fluorescent protein (GFP), squashes of seminiferous tubules, and quantification of polysomal loading in adult and 21 dpp testes in sucrose and Nycodenz gradients. GFP fluorescence is first detected in step 9 spermatids in lines harboring a transgene containing the Gfp 5′ UTR and Smcp 3′ UTR. Unexpectedly, this mRNA is stored in large, inactive mRNPs in early spermatids that sediment with polysomes in sucrose gradients, but equilibrate with the density of free mRNPs in Nycodenz gradients. Randomization of the segment 6–38 nt upstream of the first Smcp poly(A) signal results in early detection of GFP, a small increase in polysomal loading in 21 dpp testis, inactivation of the formation of heavy mRNPs, and loss of binding of a Y-box protein. GFP is first detected in step 5 spermatids in a transgene containing the Smcp 5′ UTR and Gfp 3′ UTR. Mutations in the start codons in the upstream reading frames eliminate translational delay by the Smcp 5′ UTR. Collectively, these findings demonstrate that Smcp mRNA translation is regulated by multiple elements in the 5′ UTR and 3′ UTR. In addition, differences in regulation between Smcp–Gfp mRNAs containing one Smcp UTR and the natural Smcp mRNA suggest that interactions between the Smcp 5′ UTR and 3′ UTR may be required for regulation of the Smcp mRNA.

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.

In vivo oxygen imaging using green fluorescent protein

2006 ◽  
Vol 291 (4) ◽  
pp. C781-C787 ◽  
Author(s):  
Eiji Takahashi ◽  
Tomohiro Takano ◽  
Yasutomo Nomura ◽  
Satoshi Okano ◽  
Osamu Nakajima ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Molecular Imaging ◽  
Oxygen Concentration ◽  
Fluorescent Protein ◽  
Red Shift ◽  
Coronary Artery Ligation ◽  
Oxygen Imaging ◽  
Gfp Fluorescence ◽  
Green Fluorescent

In vivo oxygen measurement is the key to understanding how biological systems dynamically adapt to reductions in oxygen supply. High spatial resolution oxygen imaging is of particular importance because recent studies address the significance of within-tissue and within-cell heterogeneities in oxygen concentration in health and disease. Here, we report a new technique for in vivo molecular imaging of oxygen in organs using green fluorescent protein (GFP). GFP-expressing COS-7 cells were briefly photoactivated with a strong blue light while lowering the oxygen concentration from 10% to <0.001%. Red fluorescence (excitation 520–550 nm, emission >580 nm) appeared after photoactivation at <2% oxygen (the red shift of GFP fluorescence). The red shift disappeared after reoxygenation of the cell, indicating that the red shift is stable as long as the cell is hypoxic. The red shift of GFP fluorescence was also demonstrated in single cardiomyocytes isolated from the GFP knock-in mouse (green mouse) heart. Then, we tried in vivo molecular imaging of hypoxia in organs. The red shift could be imaged in the ischemic liver and kidney in the green mouse using macroscopic optics provided that oxygen diffusion from the atmospheric air was prevented. In crystalloid-perfused beating heart isolated from the green mouse, significant spatial heterogeneities in the red shift were demonstrated in the epicardium distal to the coronary artery ligation. We conclude that the present technique using GFP as an oxygen indicator may allow in vivo molecular imaging of oxygen in organs.

scholarly journals The comparison of aggregation and folding of enhanced green fluorescent protein (EGFP) by spectroscopic studies

2010 ◽  
Vol 24 (3-4) ◽  
pp. 343-348 ◽  
Author(s):  
Joanna Krasowska ◽  
Monika Olasek ◽  
Agnieszka Bzowska ◽  
Patricia L. Clark ◽  
Beata Wielgus-Kutrowska
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Kinetic Studies ◽  
Spectroscopic Studies ◽  
Reducing Agents ◽  
Gfp Fluorescence ◽  
Reporter Proteins ◽  
Polypeptide Backbone ◽  
Green Fluorescent

GFP (Green Fluorescent Protein) is well known for its unique chromophore which is formed by autocatalytic cyclization of a polypeptide backbone of Ser65, Tyr66 and Gly67, and is able to emit green visible light. Due to unusual chromophore responsible for the fluorescence GFP and its mutants (e.g., EGFP) have become widely used reporter proteins in molecular biology and biotechnology. GFP can easily be fused to any protein of interest and co-expressed in cells; the GFP fluorescence is then used to visualize the distribution, transport and aggregation of the protein in the cell. However, GFP has a tendency to aggregate itself, and also formation of its chromophore critically depends on the presence of reducing agents. Therefore we have undertaken spectroscopic kinetic studies of EGFP folding and aggregation as a function of pH, and in the presence of various reducing agents, to study the competition between these two processes. The best conditions for folding of EGFP provides BME as a reducing agent. Aggregation of EGFP depends strongly on pH, and on the concentration of the protein. The careful control experiments must therefore be performed during investigations of proteins fused with EGFP, especially at pH lower than 7.

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