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Full-Text Articles in Life Sciences
Green Fluorescent Protein Is Superior To Blue Fluorescent Protein As A Quantitative Reporter Of Promoter Activity In E-Coli., James Lissemore, Joshua Bays, Molly Calvey, Lucas Reineke, Anne Colaviagianni, Melissa Tscheiner, David Mascotti
Green Fluorescent Protein Is Superior To Blue Fluorescent Protein As A Quantitative Reporter Of Promoter Activity In E-Coli., James Lissemore, Joshua Bays, Molly Calvey, Lucas Reineke, Anne Colaviagianni, Melissa Tscheiner, David Mascotti
David P. Mascotti
Fluorescent proteins related to and derived from green fluorescent protein (GFP) are widely used as tools for investigating a wide range of biological processes. In particular, GFP and its relatives have been used extensively as qualitative reporters of gene expression in many different organisms, but relatively few studies have investigated fluorescent proteins as quantitative reporters of gene expression. GFP has some limitations as a reporter gene, including possible toxicity when expressed at high levels. Therefore, it would be useful if other fluorescent proteins could be identified for use as quantitative reporters. Toward this end, we investigated BFP as a quantitative …
A Simple Spectrophotometric Streptavidin-Biotin Binding Assay Utilizing Biotin-4-Fluorescein., Mark Waner, David Mascotti
A Simple Spectrophotometric Streptavidin-Biotin Binding Assay Utilizing Biotin-4-Fluorescein., Mark Waner, David Mascotti
David P. Mascotti
A new assay for biotin binding capacity of Streptavidin (SA) is presented in this work. The assay is based on the large decrease in the extinction coefficient at 493 nm that accompanies binding of biotin-4-fluorescein (B4F) to SA. This decrease is attributed to formation of a charge transfer complex between the B4F-donor and one or more SA residues. We show that one may observe the stoichiometric binding via monitoring the absorbance at 493 nm using either SA or B4F as the titrant. The sensitivity of the assay is at the lower end of similar fluorimetric and photometric assays. Though the …
Quantitative Nonisotopic Nitrocellulose Filter Binding Assays: Bacterial Manganese Superoxide Dismutase–Dna Interactions, Joshua Czerwinski, Stephanie Hovan, David Mascotti
Quantitative Nonisotopic Nitrocellulose Filter Binding Assays: Bacterial Manganese Superoxide Dismutase–Dna Interactions, Joshua Czerwinski, Stephanie Hovan, David Mascotti
David P. Mascotti
Nitrocellulose filter binding assays (NCFBAs) have been used for many years to qualitatively and quantitatively determine protein–nucleic acid affinities. While this technique can be robust thermodynamically and fairly simple to perform, the requirement of radiolabeled nucleic acids (typically 32P) has several major drawbacks. Some disadvantages are the short half-life of 32P, the inherent safety concerns, and the cost of working with radioisotopes. Another drawback is that over time the beta emissions cause fragmentation of the nucleic acids. We have modified standard NCFBAs by developing a quantitative nonisotopic chemiluminescent method using biotin-labeled DNA and a dual-filter format. The biotin tag is …
Thermal And Sodium Dodecylsulfate Induced Transitions Of Streptavidin, Mark Waner, Irina Navrotskaya, Amanda Bain, Edward Oldham, David Mascotti
Thermal And Sodium Dodecylsulfate Induced Transitions Of Streptavidin, Mark Waner, Irina Navrotskaya, Amanda Bain, Edward Oldham, David Mascotti
David P. Mascotti
The strong specific binding of streptavidin (SA) to biotin is utilized in numerous biotechnological applications. The SA tetramer is also known to exhibit significant stability, even in the presence of sodium dodecylsulfate (SDS). Despite its importance, relatively little is known about the nature of the thermal denaturation pathway for SA. This work uses a homogeneous SA preparation to expand on the data of previous literature reports, leading to the proposal of a model for temperature induced structural changes in SA. Temperature dependent data were obtained by SDS and native polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorescence and …
Quantitative Nonisotopic Nitrocellulose Filter Binding Assays: Bacterial Manganese Superoxide Dismutase–Dna Interactions., Joshua Czerwinski, Stephanie Hovan, David Mascotti
Quantitative Nonisotopic Nitrocellulose Filter Binding Assays: Bacterial Manganese Superoxide Dismutase–Dna Interactions., Joshua Czerwinski, Stephanie Hovan, David Mascotti
David P. Mascotti
Nitrocellulose filter binding assays (NCFBAs) have been used for many years to qualitatively and quantitatively determine protein–nucleic acid affinities. While this technique can be robust thermodynamically and fairly simple to perform, the requirement of radiolabeled nucleic acids (typically 32P) has several major drawbacks. Some disadvantages are the short half-life of 32P, the inherent safety concerns, and the cost of working with radioisotopes. Another drawback is that over time the beta emissions cause fragmentation of the nucleic acids. We have modified standard NCFBAs by developing a quantitative nonisotopic chemiluminescent method using biotin-labeled DNA and a dual-filter format. The biotin tag is …
Use Of Biotin-Labeled Nucleic Acids For Protein Purification And Agarose-Based Chemiluminescent Electromobility Shift Assays., Joseph Rodgers, Pinky Patel, Jason Hennes, Sara Bolognia, David Mascotti
Use Of Biotin-Labeled Nucleic Acids For Protein Purification And Agarose-Based Chemiluminescent Electromobility Shift Assays., Joseph Rodgers, Pinky Patel, Jason Hennes, Sara Bolognia, David Mascotti
David P. Mascotti
We have employed biotin-labeled RNA to serve two functions. In one, the biotin tethers the RNA to streptavidin–agarose beads, creating an affinity resin for protein purification. In the other, the biotin functions as a label for use in a modified chemiluminescent electromobility shift assay (EMSA), a technique used to detect the formation of protein–RNA complexes. The EMSA that we describe avoids the use not only of radioactivity but also of neurotoxic acrylamide by using agarose as the gel matrix in which the free nucleic acid is separated from protein–nucleic acid complexes. After separation of free from complexed RNA in agarose, …
Dependence Of The Extent Of Ion Association With Polynucleotides And Peptides As A Function Of Solution Dielectric., David Mascotti, A. Salwan
Dependence Of The Extent Of Ion Association With Polynucleotides And Peptides As A Function Of Solution Dielectric., David Mascotti, A. Salwan
David P. Mascotti
No abstract provided.
Involvement Of Heme In The Degradation Of Iron-Regulatory Protein 2, Lisa Goessling, David Mascotti, Robert Thach
Involvement Of Heme In The Degradation Of Iron-Regulatory Protein 2, Lisa Goessling, David Mascotti, Robert Thach
David P. Mascotti
ron-regulatory proteins (IRPs) recognize and bind to specific RNA structures called iron-responsive elements. Mediation of these binding interactions by iron and iron-containing compounds regulates several post-transcriptional events relevant to iron metabolism. There are two known IRPs, IRP1 and IRP2, both of which can respond to iron fluxes in the cell. There is ample evidence that IRP1 is converted by iron to cytoplasmic aconitase in vivo. It has also been shown that, under certain conditions, a significant fraction of IRP1 is degraded in cells exposed to iron or heme. Studies have shown that the degradation of IRP1 that is induced by …
Thermodynamics Of Oligoarginines Binding To Rna And Dna., David Mascotti, Timothy Lohman
Thermodynamics Of Oligoarginines Binding To Rna And Dna., David Mascotti, Timothy Lohman
David P. Mascotti
These results suggest that hydrogen bonding of arginine to the phosphate backbone of the nucleic acids contributes to the increased stability of these complexes.
Effects Of The Ferritin Open Reading Frame On Translational Induction By Iron., David Mascotti, Lisa Goessling, Diane Rup, Robert Thach
Effects Of The Ferritin Open Reading Frame On Translational Induction By Iron., David Mascotti, Lisa Goessling, Diane Rup, Robert Thach
David P. Mascotti
No abstract provided.
Thermodynamics Of Charged Oligopeptide-Heparin Interactions., David Mascotti, Timothy Lohman
Thermodynamics Of Charged Oligopeptide-Heparin Interactions., David Mascotti, Timothy Lohman
David P. Mascotti
No abstract provided.
Regulation Of Iron Metabolism: Translational Effects Mediated By Iron, Heme, And Cytokines, David Mascotti, Diane Rup, Robert Thach
Regulation Of Iron Metabolism: Translational Effects Mediated By Iron, Heme, And Cytokines, David Mascotti, Diane Rup, Robert Thach
David P. Mascotti
No abstract provided.
Irreversible Steps In The Ferritin Synthesis Induction Pathway.., L. Linggoess, David Mascotti, M. Bhattacharyya-Pakrasi, H. Gang, R. Thach
Irreversible Steps In The Ferritin Synthesis Induction Pathway.., L. Linggoess, David Mascotti, M. Bhattacharyya-Pakrasi, H. Gang, R. Thach
David P. Mascotti
The ability of cells to re-repress ferritin synthesis after removal of an inducing agent (iron or heme) was investigated. Re-repression was found to be a slow process, requiring approximately 4 (after iron removal) to 10 h (after heme removal) for completion. Desferrioxamine mesylate (Desferal) had only a slight effect on the rate of re-repression, whereas cycloheximide was strongly inhibitory, indicating that new protein synthesis is required for re-repression. Re-repression occurred at a slow but significant rate in the presence of both Desferal and cycloheximide. These results indicate that, in the absence of an iron chelator, the induction of ferritin synthesis …
Thermodynamics Of Single-Stranded Rna And Dna Interactions With Oligolysines Containing Tryptophan. Effects Of Base Composition., David Mascotti, Timothy Lohman
Thermodynamics Of Single-Stranded Rna And Dna Interactions With Oligolysines Containing Tryptophan. Effects Of Base Composition., David Mascotti, Timothy Lohman
David P. Mascotti
No abstract provided.
Thermodynamics Of Single-Standard Rna-Binding To Oligolysines Containing Tryptophan, David Mascotti, Timothy Lohman
Thermodynamics Of Single-Standard Rna-Binding To Oligolysines Containing Tryptophan, David Mascotti, Timothy Lohman
David P. Mascotti
These studies provide important comparative information for the interpretation of the thermodynamics of protein-ss nucleic acid interaction.
Interaction Of Single And Double Stranded Nucleic Acids With Charged Oligopeptides Containing Tryptophan, David Mascotti, T. Lohman
Interaction Of Single And Double Stranded Nucleic Acids With Charged Oligopeptides Containing Tryptophan, David Mascotti, T. Lohman
David P. Mascotti
No abstract provided.
Nonspecific Ligand-Dna Equilibrium Binding Parameters Determined By Fluorescence Methods., Timothy Lohman, David Mascotti
Nonspecific Ligand-Dna Equilibrium Binding Parameters Determined By Fluorescence Methods., Timothy Lohman, David Mascotti
David P. Mascotti
To understand the structures and dynamics of DNA and its interactions with ligands, a complete analysis of its confirmational flexibility is required.
Thermodynamics Of Ligand-Nucleic Acid Interactions, Timothy Lohman, David Mascotti
Thermodynamics Of Ligand-Nucleic Acid Interactions, Timothy Lohman, David Mascotti
David P. Mascotti
To understand the structures and dynamics of DNA and its interactions with ligands, a complete analysis of its confirmational flexibility is required.
Thermodynamics Of Charged Oligopeptide Nucleic Acid Interactions, David Mascotti, T. Lohman
Thermodynamics Of Charged Oligopeptide Nucleic Acid Interactions, David Mascotti, T. Lohman
David P. Mascotti
No abstract provided.
Thermodynamic Extent Of Counterion Release Upon Binding Oligolysines To Single-Stranded Nucleic Acids., David Mascotti, Timothy Lohman
Thermodynamic Extent Of Counterion Release Upon Binding Oligolysines To Single-Stranded Nucleic Acids., David Mascotti, Timothy Lohman
David P. Mascotti
A major contribution to the binding free energy associated with most protein-nucleic acid complexes is the increase in entropy due to counterion release from the nucleic acid that results from electrostatic interactions. To examine this quantitatively, we have measured the thermodynamic extent of counterion release that results from the interaction between single-stranded homopolynucleotides and a series of oligolysines, possessing net charges z = 2-6, 8, and 10. This was accomplished by measuring the salt dependence of the intrinsic equilibrium binding constants--i.e., (delta log Kobs/delta log[K+])--over the range from 6 mM to 0.5 M potassium acetate. These data provide a rigorous …
Ferroxidase Activity Of Mushroom Tyrosinase., Rodney Boyek, David Mascotti, Barbara Schori
Ferroxidase Activity Of Mushroom Tyrosinase., Rodney Boyek, David Mascotti, Barbara Schori
David P. Mascotti
Mushroom tyrosinase catalyses the oxidation of Fe(II) to Fe(III). Both the newly-discovered ferroxidase and the well-characterized diphenol oxidase activities of tyrosinase exhibit inhibition by cyanide and both activities co-purify during two preparation steps. The characteristics of tyrosinase-catalysed Fe(II) oxidation are compared with those of other ferroxidases.