scholarly journals Systematic Probing of the Sequence Selectivity of Exonuclease III with a Photosensitization Colorimetric Assay

ACS Omega ◽  
2019 ◽  
Vol 4 (8) ◽  
pp. 13382-13387
Author(s):  
Yu Ding ◽  
Xianming Li ◽  
Xinfeng Zhang ◽  
Feng Li ◽  
Xiandeng Hou ◽  
...  
Keyword(s):  
Colorimetric Assay ◽  
Exonuclease Iii ◽  
Sequence Selectivity

Gold nanoparticle-based exonuclease III signal amplification for highly sensitive colorimetric detection of folate receptor

Nanoscale ◽  
2014 ◽  
Vol 6 (6) ◽  
pp. 3055-3058 ◽  
Author(s):  
Xinjian Yang ◽  
Zhiqiang Gao
Keyword(s):  
Gold Nanoparticles ◽  
Small Molecule ◽  
Signal Amplification ◽  
Colorimetric Assay ◽  
Folate Receptor ◽  
Colorimetric Detection ◽  
Label Free ◽  
Exonuclease Iii ◽  
Highly Sensitive ◽  
Highly Sensitive Detection

By combining terminal protection of small molecule (folate)-capped DNA probes, exonuclease III signal amplification and gold nanoparticles, we developed a simple and label-free colorimetric assay for highly sensitive detection of folate receptor.

Electron microscopic assays of restriction endonuclease cutting, exonuclease digestion, and hybridization

1984 ◽  
Vol 42 ◽  
pp. 686-687
Author(s):  
Mark Hannibal ◽  
Jacob Varkey ◽  
Michael Beer
Keyword(s):  
Electron Microscope ◽  
Low Temperature ◽  
Restriction Endonuclease ◽  
Double Helix ◽  
Test Material ◽  
Electron Microscopic ◽  
Exonuclease Iii ◽  
Dna Molecule ◽  
Linear Molecules ◽  
Exonuclease Digestion

Workman and Langmore have recently proposed a procedure for isolating particular chromatin fragments. The method requires restriction endonuclease cutting of the chromatin and a probe, their digestion with two exonucleases which leave complimentary single strand termini and low temperature hybridization of these. We here report simple electron microscopic monitoring of the four reactions involved.Our test material was ϕX-174 RF DNA which is cut once by restriction endonuclease Xho I. The conversion of circles to linear molecules was followed in Kleinschmidt spreads. Plate I shows a circular and a linear DNA molecule. The rate of cutting is shown in Figure 1.After completion of the endonuclease cutting, one portion of the DNA was treated with exonuclease III, an enzyme known to digest the 3' terminals of double helical DNA. Aliquots when examined in the electron microscope reveal a decreasing length of double helix and increasing bushes at the ends.

Synchrony of Digestion of SV40 DNA by Exonuclease III Determined by EM

1975 ◽  
Vol 33 ◽  
pp. 674-675
Author(s):  
Ray Wu ◽  
G. Ruben ◽  
B. Siegel ◽  
P. Spielman ◽  
E. Jay
Keyword(s):  
Dark Field ◽  
Uranyl Acetate ◽  
Enzyme Digestion ◽  
Dna Molecules ◽  
Exonuclease Iii ◽  
Aluminum Films ◽  
Double Stranded Dna ◽  
Before And After ◽  
Exo Iii ◽  
Sv40 Dna

A method for determining long nucleotide sequences of double-stranded DNA is being developed. It involves (a) the synchronous digestion of the DNA from the 3' ends with EL coli exonuclease III (Exo III) followed by (b) resynthesis with labeled nucleotides and DNA polymerase. A crucial factor in the success of this method is the degree to which the enzyme digestion proceeds synchronously under proper conditions of incubation (step a). Dark field EM is used to obtain accurate measurements on the lengths and distribution of the DNA molecules before and after digestion with Exo III, while gel electrophoresis is used in parallel to obtain a mean length for these molecules. It is the measurements on a large enough sample of individual molecules by EM that provides the information on how synchronously the digestion proceeds. For length measurements, the DNA molecules were picked up on 20-30 Å thick carbon-aluminum films, using the aqueous Kleinschmidt technique and stained with 7.5 x 10-5M uranyl acetate in 90% ethanol for 3 minutes.

Development of a colorimetric protein phosphorylation assay for detecting cyanobacterial toxins

1995 ◽  
Vol 31 (5-6) ◽  
pp. 47-49 ◽  
Author(s):  
C. Ash ◽  
C. MacKintosh ◽  
R. MacKintosh ◽  
C. R. Fricker
Keyword(s):  
Protein Phosphorylation ◽  
Colorimetric Assay ◽  
Cyanobacterial Toxins ◽  
Sensitive Determination

A new colorimetric assay is described, based on inhibition of protein phosphotases, that enables the rapid, simple and sensitive determination of the concentration of toxins from cyanobacteria.

Design, Synthesis, In vitro and In silico Evaluation of New Hydrazonebased Antitumor Agents as Potent Akt Inhibitors

2020 ◽  
Vol 17 (11) ◽  
pp. 1380-1392
Author(s):  
Emine Merve Güngör ◽  
Mehlika Dilek Altıntop ◽  
Belgin Sever ◽  
Gülşen Akalın Çiftçi
Keyword(s):  
Cell Line ◽  
Anticancer Agents ◽  
In Silico ◽  
Antitumor Agents ◽  
Colorimetric Assay ◽  
Fibroblast Cell ◽  
Lipinski’S Rule ◽  
In Silico Docking ◽  
Embryonic Fibroblast

Background: Akt is overexpressed or activated in a variety of human cancers, including gliomas, lung, breast, ovarian, gastric and pancreatic carcinomas. Akt inhibition leads to the induction of apoptosis and inhibition of tumor growth and therefore extensive efforts have been devoted to the discovery of potent antitumor drugs targeting Akt. Objectives: The objective of this work was to identify potent anticancer agents targeting Akt. Methods: New hydrazone derivatives were synthesized and investigated for their cytotoxic effects on 5RP7 H-ras oncogene transformed rat embryonic fibroblast and L929 mouse embryonic fibroblast cell lines. Besides, the apoptotic effects of the most active compounds on 5RP7 cell line were evaluated using flow cytometry. Their Akt inhibitory effects were also investigated using a colorimetric assay. In silico docking and Absorption, Distribution, Metabolism and Excretion (ADME) studies were also performed using Schrödinger’s Maestro molecular modeling package. Results and Discussion: Compounds 3a, 3d, 3g and 3j were found to be effective on 5RP7 cells (with IC50 values of <0.97, <0.97, 1.13±0.06 and <0.97 μg/mL, respectively) when compared with cisplatin (IC50= 1.87±0.15 μg/mL). It was determined that these four compounds significantly induced apoptosis in 5RP7 cell line. Among them, N'-benzylidene-2-[(4-(4-methoxyphenyl)pyrimidin- 2-yl)thio]acetohydrazide (3g) significantly inhibited Akt (IC50= 0.5±0.08 μg/mL) when compared with GSK690693 (IC50= 0.6±0.05 μg/mL). Docking studies suggested that compound 3g showed good affinity to the active site of Akt (PDB code: 2JDO). According to in silico ADME studies, the compound also complies with Lipinski's rule of five and Jorgensen's rule of three. Conclusion: Compound 3g stands out as a potential orally bioavailable cytotoxic agent and apoptosis inducer targeting Akt.

An Effort to Making a Colorimitric Nano-Biosensor for Vibrio cholera Detection

2020 ◽  
Vol 16 (5) ◽  
pp. 793-804
Author(s):  
Naimeh Mahheidari ◽  
Jamal Rashidiani ◽  
Hamid Kooshki ◽  
Khadijeh Eskandari
Keyword(s):  
Gold Nanoparticles ◽  
Colorimetric Assay ◽  
Au Nanoparticle ◽  
Bacteria Detection ◽  
Great Promise ◽  
Bacterial Cells ◽  
Vibrio Cholera ◽  
Minimum Concentration ◽  
Vis Spectroscopy ◽  
Fast Procedure

Background: Today, nanoparticles hold great promise in biomedical researches and applications including bacteria detection. The rapid and sensitive outcomes of bacteria detection strategies using nanoparticle conjugates become determinative, especially in bacterial outbreaks. In the current research, we focused on detecting V. cholera bacteria and its toxin using a thiocyanate/Au nanoparticle. Thiocyanate adsorbed strongly on the surface of gold nanoparticles and changed the surface by enhancing surface plasmon resonance of gold nanoparticles. Objective: This method is tried to introduce a simple and fast procedure to assay vibrio cholera. So, it is observed by the naked eyes as well. Methods: We used two antibodies (Ab) for V. cholera detection: a) a primary antibody conjugated to magnetic nanoparticles (MNPs) for trapping V. cholera bacterial cells, and b) a secondary Abconjugated thiocyanate-GNPs as a colorimetric detector. Then, an immuno-magnetic separation system connected to a colorimetric assay was designed based on the GNPs. The results were measured by ultraviolet-visible (UV-Vis) spectroscopy. Results: The results showed that gold nanoparticles are an appropriate optical assay for detecting biological samples in a minimum concentration and also it can be easily seen by the naked eyes. The linear range of this biosensor is 3.2×104 to 28×104 cells per ml. Conclusion: In this research, a colorimetric immune assay based on gold nanoparticles was designed to improve the sensitivity of V. cholera detection. Also, this method can be used for the detection of other biological agents.

scholarly journals Suitability of the Cyclic Voltammetry Measurements and DPPH• Spectrophotometric Assay to Determine the Antioxidant Capacity of Food-Grade Oenological Tannins

Molecules ◽  
2019 ◽  
Vol 24 (16) ◽  
pp. 2925 ◽  
Author(s):  
Arianna Ricci ◽  
Giuseppina Paola Parpinello ◽  
Nemanja Teslić ◽  
Paul Andrew Kilmartin ◽  
Andrea Versari
Keyword(s):  
Cyclic Voltammetry ◽  
Colorimetric Assay ◽  
Radical Scavenging ◽  
Calibration Graph ◽  
Spectrophotometric Assay ◽  
Experimental Conditions ◽  
Fast Analysis ◽  
Cv Measurements ◽  
And Control ◽  
Analytical Approaches

Twenty commercially available oenological tannins (including hydrolysable and condensed) were assessed for their antiradical/reducing activity, comparing two analytical approaches: The 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging spectrophotometric assay and the cyclic voltammetry (CV) electrochemical method. Electrochemical measurements were performed over a −200 mV–500 mV scan range, and integrated anodic currents to 500 mV were used to build a calibration graph with (+)-catechin as a reference standard (linear range: From 0.0078 to 1 mM, R2 = 0.9887). The CV results were compared with the DPPH• assay (expressed as % of radical scavenged in time), showing high correlation due to the similarity of the chemical mechanisms underlying both methods involving polyphenolic compounds as reductants. Improved correlation was observed by increasing the incubation time with DPPH• to 24 h (R2 = 0.925), demonstrating that the spectrophotometric method requires a long-term incubation to complete the scavenging reaction when high-molecular weight tannins are involved; this constraint has been overcome by using instant CV measurements. We concluded that the CV represents a valid alternative to the DPPH• colorimetric assay, taking advantage of fast analysis and control on the experimental conditions and, because of these properties, it can assist the quality control along the supply chain.

The Need for Using An Enzymatic Colorimetric Assay in Creatinine Determination of Peritoneal Dialysis Solutions

1990 ◽  
Vol 10 (1) ◽  
pp. 89-92 ◽  
Author(s):  
Liliane Larpent ◽  
Christian Verger
Keyword(s):  
Peritoneal Dialysis ◽  
Reliable Method ◽  
Colorimetric Assay ◽  
Colorimetric Method ◽  
Peritoneal Membrane ◽  
Creatinine Kinetics ◽  
Dialysis Solutions ◽  
Peritoneal Dialysis Solutions ◽  
Enzymatic Test

The fate of the peritoneal membrane on continuous ambulatory peritoneal dialysis (CAPD) is usually evaluated through the modification of its permeability to various solutes as glucose, creatinine, and urea. Therefore, the accuracy of the methods used for measurements of creatinine is of great importance. A particular problem does exist for creatinine determination as it may be influenced by the presence of glucose. We studied a new enzymatic colorimetric method for creatinine determination in peritoneal dialysis solutions which contain high dextrose concentrations. Creatinine was measured in plasma, urine, and dialysate from 18 patients on CAPD and in pure dextrose solutions, with an enzymatic test (Boehringer Mannheim) and with Jaffe's reaction on two different analyzers: Astra (Beckman) and Eris (Merck). Creatinine results were similar with both assays (Jaffe's reaction and enzymatic test) when measured in blood and urine. However the Jaffe's reaction gave higher creatinine results than the enzymatic test (p < 0.001), when assays were performed in peritoneal dialysis solutions and in pure glucose solutions. In addition, it appeared that other components of dialysis solutions, mainly calcium chloride, influenced unpredictably the results of creatinine with the Jaffe's reaction. We conclude that specific enzymatic test is a more accurate and reliable method to evaluate creatinine kinetics through the peritoneal membrane when determined in CAPD solutions.

scholarly journals Validating Anti-Infective Activity of Pleurotus Opuntiae via Standardization of Its Bioactive Mycoconstituents through Multimodal Biochemical Approach

Coatings ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 484
Author(s):  
Aprajita Tiwari Pandey ◽  
Ishan Pandey ◽  
Anurag Kanase ◽  
Amita Verma ◽  
Beatriz Garcia-Canibano ◽  
...  
Keyword(s):  
Bioactive Compounds ◽  
High Performance ◽  
Colorimetric Assay ◽  
Diffusion Method ◽  
Therapeutic Effects ◽  
Medication Regimen ◽  
Methanol Extracts ◽  
Retention Factors ◽  
Before And After ◽  
Solvent Systems

Mushrooms produce a variety of bioactive compounds that are known to have anti-pathogenic properties with safer and effective therapeutic effects in human disease prognosis. The antibacterial activity of ethanol and methanol extracts of Pleurotus opuntiae were checked against pathogenic microorganisms viz. Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis NCIM 2300, Proteus vulgaris NCIM 5266, Serratia marcescens NCIM 2078, Shigella flexeneri NCIM 5265, Moraxella sp. NCIM 2795, Staphylococcus aureus ATCC 25923 by agar well diffusion method at different concentrations of the extracts. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the extracts was determined by INT (Iodonitrotetrazolium chloride) colorimetric assay. Extracts were standardized by thin layer chromatography (TLC) in different solvent systems. The Retention factors (Rf) of different compounds were calculated by high performance TLC (HPTLC) fingerprinting at UV 254, 366, and 540 nm before and after derivatization. The ethanol and methanol extracts of P. opuntiae showed bactericidal activity against all the test pathogens at MIC values of 15.6 to 52.08 mg/mL and 20.81 to 52.08 mg/mL respectively. Whereas the MBC values for ethanol and methanol extract of P. opuntiae against all pathogens were recorded as 26.03 to 62.5 mg/mL and 125 mg/mL respectively. Preliminary mycochemical screening of both the extracts revealed high contents of bioactive compounds. Amongst all the solvent systems used in TLC, the best result was given by chloroform + hexane (8:2) which eluted out 5 different compounds (spots). HPTLC results revealed spots with different Rf values for all the 24 compounds present. Thus, it can be inferred from the present investigation that the mycoconstituents could be an alternative medication regimen and could play a role in new drug discoveries against different infections. Further, the antimicrobial components of these mushrooms can be transformed to bioengineered antimicrobial coatings for surfaces, drug and other hybrid systems for public health implications in combating persistent infections.

Ultrasensitive fluorometric biosensor based on Ti3C2 MXenes with Hg2+-triggered exonuclease III-assisted recycling amplification

The Analyst ◽  
2021 ◽  
Author(s):  
Liling Lu ◽  
Xiao Han ◽  
Jingwen Lin ◽  
Yingxin Zhang ◽  
Minghao Qiu ◽  
...  
Keyword(s):  
Two Dimensional ◽  
Exonuclease Iii ◽  
Exo Iii ◽  
Recycling Amplification ◽  
Fluorescence Quencher

Herein a rapid and sensitive fluorometric bioanalysis platform for mercury(II) (Hg2+) detection was innovatively developed using ultrathin two-dimensional MXenes (Ti3C2) as fluorescence quencher and Hg2+-induced exonuclease III (Exo III)-assisted target...

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