scholarly journals Stability of Nitroxide Biradical TOTAPOL in Biological Samples

2019 ◽  
Author(s):  
Kelsey McCoy ◽  
Rivkah Rogawski ◽  
Olivia Stovicek ◽  
Ann McDermott
Keyword(s):  
Solid State Nmr ◽  
Nuclear Polarization ◽  
Chemical Reduction ◽  
Paramagnetic Species ◽  
Whole Cells ◽  
Radical Degradation ◽  
Radical Reduction ◽  
The Stability ◽  
Nmr Signals

AbstractWe characterize chemical reduction of a nitroxide biradical, TOTAPOL, used in dynamic nuclear polarization (DNP) experiments, specifically probing the stability in whole-cell pellets and lysates, and present a few strategies to stabilize the biradicals for DNP studies. DNP solid-state NMR experiments use paramagnetic species such as nitroxide biradicals to dramatically increase NMR signals. Although there is considerable excitement about using nitroxide-based DNP for detecting the NMR spectra of proteins in whole cells, nitroxide radicals are reduced in minutes in bacterial cell pellets, which we confirm and quantify here. We show that addition of the covalent cysteine blocker N-ethylmaleimide to whole cells significantly slows the rate of reduction, suggesting that cysteine thiol radicals are important toin vivoradical reduction. The use of cell lysates rather than whole cells also slows TOTAPOL reduction, which suggests a possible role for the periplasm and oxidative phosphorylation metabolites in radical degradation. Reduced TOTAPOL in lysates can also be efficiently reoxidized with potassium ferricyanide. These results point to a practical and robust set of strategies for DNP of cellular preparations.

scholarly journals DNP-supported solid-state NMR studies of 13C,15N,29Si-enriched biosilica of Cyclotella cryptica and Thalassiosira pseudonana

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Helena Leona Ehren ◽  
Felicitas Kolbe ◽  
Alessandra Lucini Paioni ◽  
Eike Brunner ◽  
Marc Baldus
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Nuclear Polarization ◽  
Thalassiosira Pseudonana ◽  
Supramolecular Organization ◽  
Structural Elements ◽  
Nmr Studies ◽  
Amine Nitrogen ◽  
Nmr Signals ◽  
Cyclotella Cryptica

AbstractSolid-state NMR spectroscopy represents a powerful method for the investigation of diatom biosilica but detailed studies regarding its chemical composition and structural organization can be prohibited by insufficient spectroscopic sensitivity. Here, we used two-dimensional (2D) Dynamic Nuclear Polarization (DNP)-supported solid-state NMR experiments to obtain information about the molecular composition and supramolecular organization of proteins and carbohydrates in 13C,15N,29Si-labeled biosilica of C. cryptica. As a reference, we conducted DNP experiments on isotope-labeled biosilica of T. pseudonana. DNP-enhancement factors for different NMR signals, and thus, for different organic compounds, provide information about the supramolecular architecture of the biosilica. In addition, DNP-supported heteronuclear nitrogen-carbon correlation experiments allowed us to prove the presence of different structural elements of long chain polyamines (LCPAs) and revealed the occurrence of amine-nitrogen moieties exhibiting a correlation with carbonyl carbons that may indicate cross-linking of LCPAs to proteins as previously seen in studies on proteins extracted from other diatoms.

scholarly journals DNP-Supported Solid-State NMR Studies of 13C,15N,29Si-Enriched Biosilica of Cyclotella Cryptica and Thalassiosira Pseudonana

2020 ◽  
Author(s):  
Helena Ehren ◽  
Felicitas Kolbe ◽  
Alesandra Lucini Paioni ◽  
Eike Brunner ◽  
Marc Baldus
Keyword(s):  
Solid State ◽  
Solid State Nmr ◽  
Nuclear Polarization ◽  
Thalassiosira Pseudonana ◽  
Supramolecular Organization ◽  
Powerful Method ◽  
Nmr Studies ◽  
Amine Nitrogen ◽  
Nmr Signals ◽  
Cyclotella Cryptica

Abstract Solid-state NMR spectroscopy represents a powerful method for the investigation of diatom biosilica but detailed studies regarding its chemical composition and structural organization can be prohibited by insufficient spectroscopic sensitivity. Here, we used two-dimensional (2D) Dynamic Nuclear Polarization (DNP)-supported solid-state NMR experiments to obtain information about the molecular composition and supramolecular organization of proteins and carbohydrates in13C,15N,29Si-labeled biosilica of C. cryptica. As a reference, we conducted DNP experiments on isotope-labeled biosilica of T. pseudonana. DNP-enhancement factors for different NMR signals, and thus, for different organic compounds, provide information about the supramolecular architecture of the biosilica. In addition, DNP-supported heteronuclear nitrogen-carbon correlation experiments allowed us to prove the presence of different LCPAs (long chain polyamines) and revealed the occurrence of amine-nitrogen moieties exhibiting a correlation with carbonyl carbons that may indicate cross-linking of LCPAs to proteins as previously seen in studies on proteins extracted from other diatoms.

Stability differences in the NMR ensembles of amyloid β fibrils

2016 ◽  
Vol 15 (07) ◽  
pp. 1650059 ◽  
Author(s):  
Erik J. Alred ◽  
Malachi Phillips ◽  
Manikanthan Bhavaraju ◽  
Ulrich H. E. Hansmann
Keyword(s):  
Solid State Nmr ◽  
Amyloid Β ◽  
Test Cases ◽  
Binding Affinities ◽  
Drug Candidates ◽  
The Stability ◽  
Comparative Stability ◽  
The Brain ◽  
Nmr Signals

Alzheimer’s Disease is characterized by the formation of amyloid beta (A[Formula: see text] fibril plaques in the brain. These fibrils can be probed by solid state NMR (ssNMR), which leads to an ensemble of configurations that are compatible with the NMR signals. Typically, only the lowest energy conformer is considered in computer simulations that probe the stability of fibrils and their binding with drug candidates. This restriction could produce data that are not physiologically relevant if the NMR entries differ significantly in stability or binding affinities. In order to study this effect, we have investigated the variance in stability between members of NMR ensembles. Our test cases are a patient-derived A[Formula: see text]-fibril model and two in vitro A[Formula: see text]-fibril models from a previous study we performed on comparative stability. The latter two models allow us also to compare different staggering patterns. We observe significant variations in molecular flexibility, compactness and secondary structure, suggesting that the full NMR ensemble must be considered for a physiologically relevant description of A[Formula: see text] fibrils.

scholarly journals The Atomic-Level Structure of Cementitious Calcium Aluminate Silicate Hydrate Determined by NMR

2021 ◽  
Vol 75 (4) ◽  
pp. 272-275
Author(s):  
Pinelopi Moutzouri ◽  
Lyndon Emsley
Keyword(s):  
Solid State ◽  
Calcium Aluminate ◽  
First Principles ◽  
Solid State Nmr ◽  
Nuclear Polarization ◽  
Level Structure ◽  
Atomic Level ◽  
Two Dimensional ◽  
First Principles Calculations ◽  
Nmr Signals

We review our recent paper which resolves the long-standing dilemma of the location and nature of the six-fold coordinated aluminum in calcium aluminate silicate hydrate (C-A-S-H) samples. First principles calculations predict that at high Ca:Si and H2O ratios, aluminum is incorporated into the bridging sites of the linear silicate chains and that the stable coordination number is six. We confirm this hypothesis experimentally by one- and two-dimensional dynamic nuclear polarization enhanced 27 Al and 29 Si solid-state NMR experiments in which we correlate the distinctive six-fold coordinated aluminum NMR signal at 5 ppm to 29 Si NMR signals from silicates in C-A-S-H.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals Stability and ocular biodistribution of topically administered PLGA nanoparticles

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sean Swetledge ◽  
Renee Carter ◽  
Rhett Stout ◽  
Carlos E. Astete ◽  
Jangwook P. Jung ◽  
...  
Keyword(s):  
Uv Light ◽  
Light Exposure ◽  
Polymeric Nanoparticles ◽  
Plga Nanoparticles ◽  
Eye Disease ◽  
Control Group ◽  
Eye Tissues ◽  
Nanoparticle Morphology ◽  
The Stability

AbstractPolymeric nanoparticles have been investigated as potential delivery systems for therapeutic compounds to address many ailments including eye disease. The stability and spatiotemporal distribution of polymeric nanoparticles in the eye are important regarding the practical applicability and efficacy of the delivery system in treating eye disease. We selected poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with lutein, a carotenoid antioxidant associated with eye health, as our model ophthalmic nanodelivery system and evaluated its stability when suspended in various conditions involving temperature and light exposure. We also assessed the ocular biodistribution of the fluorescently labeled nanoparticle vehicle when administered topically. Lutein-loaded nanoparticles were stable in suspension when stored at 4 °C with only 26% lutein release and no significant lutein decay or changes in nanoparticle morphology. When stored at 25 °C and 37 °C, these NPs showed signs of bulk degradation, had significant lutein decay compared to 4 °C, and released over 40% lutein after 5 weeks in suspension. Lutein-loaded nanoparticles were also more resistant to photodegradation compared to free lutein when exposed to ultraviolet (UV) light, decaying approximately 5 times slower. When applied topically in vivo, Cy5-labled nanoparticles showed high uptake in exterior eye tissues including the cornea, episcleral tissue, and sclera. The choroid was the only inner eye tissue that was significantly higher than the control group. Decreased fluorescence in all exterior eye tissues and the choroid at 1 h compared to 30 min indicated rapid elimination of nanoparticles from the eye.

scholarly journals Effect of food processing on the antioxidant activity of flavones from Polygonatum odoratum (Mill.) Druce

2021 ◽  
Vol 16 (1) ◽  
pp. 92-101
Author(s):  
Guanghui Xia ◽  
Xinhua Li ◽  
Zhen Zhang ◽  
Yuhang Jiang
Keyword(s):  
Antioxidant Activity ◽  
High Pressure ◽  
Food Processing ◽  
Yeast Fermentation ◽  
Pressure Treatment ◽  
Processing Methods ◽  
High Pressure Treatment ◽  
Effect Of Food ◽  
The Stability

Abstract Polygonatum odoratum (Mill.) Druce (POD) is a natural plant widely used for food and medicine, thanks to its rich content of a strong antioxidant agent called homoisoflavones. However, food processing methods could affect the stability of POD flavones, resulting in changes to their antioxidant activity. This study attempts to evaluate the antioxidant activity of POD flavones subject to different processing methods and determines which method could preserve the antioxidant activity of POD flavones. Therefore, flavones were extracted from POD samples, which had been treated separately with one of the four processing methods: extrusion, baking, high-pressure treatment, and yeast fermentation. After that, the antioxidant activity of the flavones was subject to in vivo tests in zebrafish embryos. The results show that yeast fermentation had the least disruption to the antioxidant activity of POD flavones, making it the most suitable food processing method for POD. By contrast, extrusion and high-pressure treatment both slightly weakened the antioxidant activity of the flavones and should be avoided in food processing. The research results provide a reference for the development and utilization of POD and the protection of its biological activity.

scholarly journals Clinical Application and Efficacy of Silver Drug in Ophthalmology: A Literature Review and New Formulation of EYE Drops with Drug Silver (I) Complex of Metronidazole with Improved Dosage Form

Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 210
Author(s):  
Arleta Waszczykowska ◽  
Dominik Żyro ◽  
Justyn Ochocki ◽  
Piotr Jurowski
Keyword(s):  
Dosage Form ◽  
Complex Analysis ◽  
Bacterial Resistance ◽  
Eye Drops ◽  
Ophthalmic Diseases ◽  
The Stability ◽  
New Formulation ◽  
Additional Therapy ◽  
Antibacterial Potential

The use of silver preparations in medicine is becoming increasingly popular. The basic aim of this evaluation was to review the literature on the clinical (in vivo) and antibacterial potential of silver preparations in ophthalmic diseases. The second goal was to summarize the results of experimental research on the use of silver preparations in ophthalmology. The third objective was to present a method for stabilizing eye drops containing silver (I) complex. Analysis of the pH stability of the silver (I) complex with metronidazole in the prepared dosage form (eye drops) was carried out. Most silver preparations are clinically used for topical application. Few experimental results indicate the usefulness of intraocular or systemic administration of silver (I) preparations as an alternative or additional therapy in infectious and angiogenic eye diseases. The development of a new formulation increases the stability of the dosage form. New forms of silver (I) products will certainly find application in the treatment of many ophthalmic diseases. One of the most important features of the silver (I) complex is its capacity to break down bacterial resistance. The new eye drops formula can significantly improve comfort of use. Due to their chemical nature, silver (I) compounds are difficult to stabilize, especially in the finished dosage form.

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