scholarly journals A novel class of polymeric fluorescent dyes assembled using a DNA synthesizer

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243218
Author(s):  
Tracy Matray ◽  
Sharat Singh ◽  
Hesham Sherif ◽  
Kenneth Farber ◽  
Erin Kwang ◽  
...  
Keyword(s):  
Rational Design ◽  
Tertiary Structure ◽  
Fluorescent Dyes ◽  
Polymer System ◽  
Fluorescence Emission ◽  
Proper Function ◽  
Molecular Architecture ◽  
Primary Sequence ◽  
Phosphoramidite Chemistry ◽  
And Control

In the pursuit of a novel class of fluorescent dyes we have developed a programmable polymer system that enables the rational design and control of macromolecular constructs through simple control of polymer primary sequence. These polymers are assembled using standard phosphoramidite chemistry on a DNA synthesizer which allows for extremely rapid prototyping and enables many permutations due to the large selection of phosphoramidite monomers presently available on the market. This programmability to some extent allows us to control the interactions/spacing of payload molecules distributed along the designed polymeric backbone. Control of molecular architecture using this technology has allowed us to address the long-standing technical issue of contact quenching between fluorescent dyes offering new possibilities in the life sciences arena. Much like peptidic sequences coding for enzymes, cofactors, and receptors (all needing control of tertiary structure for proper function via primary sequence) our programmable system approaches a similar endpoint using a phosphate based polymeric backbone assembled in a completely automated fashion. Using this novel technology, we have efficiently synthesized several types of fluorescent dyes and demonstrated the programmability in molecule design, including the increases in brightness of the fluorescence emission.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Abstract 66: Ascorbate Prevents Hypertension, Proteinuria and Concentric Hypertrophy by Balancing the Nitroso-redox System in a Model of Preeclampsia, the S-nitrosoglutathione Reductase (gsnor) Deficient Mice

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Shathiyah Kulandavelu ◽  
Raul A Dulce ◽  
Rosemeire K Takeuchi ◽  
Wayne Balkan ◽  
Joshua M Hare
Keyword(s):  
Nitrosative Stress ◽  
Fluorescent Dyes ◽  
Protein S ◽  
Redox System ◽  
Fetal Mortality ◽  
Mortality And Morbidity ◽  
Redox Imbalance ◽  
Concentric Hypertrophy ◽  
Maternal Hypertension ◽  
And Control

Introduction: Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc). Mice lacking S-nitrosoglutathione reductase (GSNOR –/– ), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, proteinuria, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesize that the PE-like phenotype is mediated by nitroso-redox imbalance and nitrosative stress and can be rescued with ascorbate treatment. Methods: Pregnant GSNOR –/– and control (WT) mice (n=5-7) were provided drinking water ± Asc beginning at day 0.5 of gestation (E0.5). We determined blood pressure using a Millar catheter, relative wall thickness (RWT) by echocardiography, and placental vascularization by isolectin staining. Cardiomyocytes (CM) were isolated at late stage pregnancy (E17.5) and fluorescent dyes used to determine levels of ROS (2’7’-dichlorofluorescein), nitric oxide (NO, diaminofluorescin) and peroxynitrite (dihydrorhodamine 123). Results: Isolated CMs from pregnant GSNOR –/– hearts, exhibited elevated levels of ROS (2.48±0.39 vs. 1.58±0.18 ΔF/F 0 ), free NO (6.65±0.43 vs. 5.59±0.26 ΔF/F 0 ) and peroxynitrite (0.75±0.04 vs. 0.39±0.03 ΔF/F 0 ) compared to WT. These increases were prevented with Asc treatment (P<0.01), which completely rescued the PE phenotype in GSNOR –/– mothers, including hypertension (105±2 mmHg vs. 95± mmHg in Asc-treated, P<0.05), proteinuria (P<0.05) and RWT (0.56±0.04 vs. 0.45±0.03 in Asc-treated (P<0.05). Placental vascularization was also significantly improved with Asc treatment in GSNOR –/– mothers. Asc had no significant effect in WT mice. Conclusion: Our findings indicate that nitroso-redox imbalance and nitrosative stress contributes to PE in mice. Asc treatment balanced the nitroso-redox system and rescued the pathological phenotypes in GSNOR –/– mice, suggesting that it can be used therapeutically to treat or prevent preeclampsia.

scholarly journals Inhaled Anesthetics Promote Albumin Dimerization through Reciprocal Exchange of Subdomains

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Benjamin J. Pieters ◽  
Eugene E. Fibuch ◽  
Joshua D. Eklund ◽  
Norbert W. Seidler
Keyword(s):  
Fluorescence Emission ◽  
Size Exclusion ◽  
Inhaled Anesthetics ◽  
Protein Protein Interaction ◽  
Reciprocal Exchange ◽  
Proposed Model ◽  
Exclusion Chromatography ◽  
Spectral Shifting ◽  
And Control ◽  
The Impact

Inhaled anesthetics affect protein-protein interaction, but the mechanisms underlying these effects are still poorly understood. We examined the impact of sevoflurane and isoflurane on the dimerization of human serum albumin (HSA), a protein with anesthetic binding sites that are well characterized. Intrinsic fluorescence emission was analyzed for spectral shifting and self-quenching, and control first derivatives (spectral responses to changes in HSA concentration) were compared against those obtained from samples treated with sevoflurane or isoflurane. Sevoflurane increased dimer-dependent self-quenching and both decreased oligomer-dependent spectral shifting, suggesting that inhaled anesthetics promoted HSA dimerization. Size exclusion chromatography and polarization data were consistent with these observations. The data support the proposed model of a reciprocal exchange of subdomains to form an HSA dimer. The open-ended exchange of subdomains, which we propose occuring in HSA oligomers, was inhibited by sevoflurane and isoflurane.

scholarly journals The molecular recognition of phosphatidic acid by an amphipathic helix in Opi1

2018 ◽  
Vol 217 (9) ◽  
pp. 3109-3126 ◽  
Author(s):  
Harald F. Hofbauer ◽  
Michael Gecht ◽  
Sabine C. Fischer ◽  
Anja Seybert ◽  
Achilleas S. Frangakis ◽  
...  
Keyword(s):  
Phosphatidic Acid ◽  
Rational Design ◽  
Acyl Chain ◽  
Membrane Biogenesis ◽  
Amphipathic Helix ◽  
New Feature ◽  
Dynamics Simulations ◽  
Membrane Recognition ◽  
And Control

A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important intermediate at the branch point of these pathways and is continuously monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. In this study, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for selective binding of PA over phosphatidylserine (PS). The insertion of the AH into the membrane core renders Opi1 sensitive to the lipid acyl chain composition and provides a means to adjust membrane biogenesis. By rational design of the AH, we tune the membrane-binding properties of Opi1 and control its responsiveness in vivo. Using extensive molecular dynamics simulations, we identify two PA-selective three-finger grips that tightly bind the PA phosphate headgroup while interacting less intimately with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.

Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride

2005 ◽  
Vol 83 (2) ◽  
pp. 109-114 ◽  
Author(s):  
Hong-Min Tang ◽  
Hong Yu
Keyword(s):  
Fluorescence Intensity ◽  
Tertiary Structure ◽  
Structural Characteristics ◽  
Molten Globule ◽  
Guanidine Hydrochloride ◽  
Arginine Kinase ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Blue Shift ◽  
Molten Globule State

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8–1.0 mol/L and 0.3–0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8–1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3–0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.Key words: arginine kinase, guanidine-denatured, refolding, intermediate, molten globule state.

scholarly journals Inverse Analysis and Modeling for Tunneling Thrust on Shield Machine

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Qian Zhang ◽  
Yilan Kang ◽  
Zheng Zheng ◽  
Lihui Wang
Keyword(s):  
Data Analysis ◽  
Inverse Analysis ◽  
Rational Design ◽  
Rapid Development ◽  
Scientific Basis ◽  
Measured Data ◽  
Engineering Systems ◽  
Shield Tunneling ◽  
Complex Engineering ◽  
And Control

With the rapid development of sensor and detection technologies, measured data analysis plays an increasingly important role in the design and control of heavy engineering equipment. The paper proposed a method for inverse analysis and modeling based on mass on-site measured data, in which dimensional analysis and data mining techniques were combined. The method was applied to the modeling of the tunneling thrust on shield machines and an explicit expression for thrust prediction was established. Combined with on-site data from a tunneling project in China, the inverse identification of model coefficients was carried out using the multiple regression method. The model residual was analyzed by statistical methods. By comparing the on-site data and the model predicted results in the other two projects with different tunneling conditions, the feasibility of the model was discussed. The work may provide a scientific basis for the rational design and control of shield tunneling machines and also a new way for mass on-site data analysis of complex engineering systems with nonlinear, multivariable, time-varying characteristics.

Rational design of a fluorescent poly(N-aryleneindole ether sulfone) switch by cation–π interactions

2015 ◽  
Vol 6 (5) ◽  
pp. 697-702 ◽  
Author(s):  
Guanjun Chang ◽  
Li Yang ◽  
Shenye Liu ◽  
Runxiong Lin ◽  
Jingsong You
Keyword(s):  
Rational Design ◽  
Fluorescence Emission ◽  
Pyridine Hydrochloride ◽  
Π Interactions

A fluorescence emission on–off switch is achieved by adjusting the assembly of poly(N-aryleneindole ether sulfone) (PESIN) and pyridine hydrochloride via the cation–π interactions.

Electro-optical Wavelength Selection Enables Confocal Ratio Imaging at Low Light Levels

1995 ◽  
Vol 1 (1) ◽  
pp. 1-11
Author(s):  
Roland Nitschke ◽  
Kenneth R. Spring
Keyword(s):  
Epithelial Cells ◽  
Fluorescent Dyes ◽  
Fluorescence Emission ◽  
Argon Laser ◽  
Tunable Filter ◽  
Transmitted Light ◽  
Light Levels ◽  
Mechanical Filter ◽  
Ratio Imaging ◽  
Illumination Wavelength

A confocal attachment (Odyssey) to an inverted microscope was modified to better study living cultured epithelial cells stained with fluorescent dyes. Improvements to the instrument included elimination of light leaks, improved electronic shielding, reduction of thermal effects, and use of low dark current detectors. In addition, rapid changes in illumination wavelength and power were accomplished by replacing the original mechanical filter changer by an acousto-optic tunable filter attached to the argon laser light source. The addition of a liquid crystal tunable filter to one of the two photomultiplier detectors also permitted rapid spectral scanning of the fluorescence emission. High-resolution, differential interference contrast transmitted light images were formed simultaneously by replacement of the photodiode-based transmitted light detector with a photomultiplier tube and dichroic mirror assembly. An illumination intensity of only 40 μW/cm2 at the back focal plane of the microscope objective allowed high-quality fluorescence and transmitted light images of living cells at video rates with minimal bleaching and photodynamic damage. Both excitation ratio imaging and emission spectral scanning of living epithelial cells were accomplished. The system performance was evaluated by optical sections of fluorescent beads and thin films.

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