Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca

1987 ◽  
Vol 252 (4) ◽  
pp. C441-C449 ◽  
Author(s):  
L. A. Levy ◽  
E. Murphy ◽  
R. E. London
Keyword(s):  
Chemical Shifts ◽  
Cell Types ◽  
Free Calcium ◽  
Tetraacetic Acid ◽  
Nmr Studies ◽  
Cytosolic Free Calcium ◽  
Fluorine Nmr ◽  
Dissociation Constant Kd

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.

DNA and RNA: NMR studies of conformations and dynamics in solution

1987 ◽  
Vol 20 (1-2) ◽  
pp. 35-112 ◽  
Author(s):  
Dinshaw J. Patel ◽  
Lawrence Shapiro ◽  
Dennis Hare
Keyword(s):  
Aqueous Solution ◽  
Data Analysis ◽  
Nucleic Acids ◽  
Nmr Studies ◽  
Magnet Design ◽  
Dna And Rna ◽  
Qualitative Nature ◽  
Abundant Supply

The early NMR research on nucleic acids was of a qualitative nature and was restricted to partial characterization of short oligonucleotides in aqueous solution. Major advances in magnet design, spectrometer electronics, pulse techniques, data analysis and computational capabilities coupled with the availability of pure and abundant supply of long oligonucleotides have extended these studies towards the determination of the 3-D structure of nucleic acids in solution.

Solid-State Carbon-13 NMR Studies of Vulcanized Elastomers. VII. Sulfur-Vulcanized Cis-1,4 Polybutadiene at 75.5 Mhz

1989 ◽  
Vol 62 (5) ◽  
pp. 908-927 ◽  
Author(s):  
R. S. Clough ◽  
J. L. Koenig
Keyword(s):  
Solid State ◽  
Nmr Spectroscopy ◽  
Chemical Shifts ◽  
Chemical Information ◽  
Chemical Probes ◽  
Nmr Studies ◽  
Nmr Characterization ◽  
The Individual ◽  
Insight Into

Abstract Solid-state C-13 NMR spectroscopy provides a wealth of chemical information concerning sulfur-vulcanized cis-l,4-polybutadiene. New resonances which appear upon curing have chemical shifts which are expected for crosslink and cyclic structures. The DEPT experiment with MAS worked well for the polybutadiene vulcanizate in the solid state. DEPT is very useful as an aid in the assignment of structures to resonances. The new resonances can be assigned to more than one type of structure given only chemical shift and DEPT information. Swelling measurements indicate the majority of the resonances are due to cyclics. Further work involving C-13 NMR characterization of polybutadiene vulcanizates treated with chemical probes and vulcanizates cured with organic accelerators should provide insight into the specific structures responsible for the individual resonances.

Aldehyde dehydrogenase in fresh primordial germ cells as a marker of cell ‘stemness’

Zygote ◽  
2019 ◽  
Vol 27 (1) ◽  
pp. 46-48
Author(s):  
Andrea Svoradová ◽  
Jaromír Vašíček ◽  
Alexander Ostró ◽  
Peter Chrenek
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Specific Inhibitor ◽  
Cell Types ◽  
Functional Marker ◽  
Aldh Activity

SummaryChicken primordial germ cells (PGCs) are the primary pluripotent stem cell types that will differentiate towards germ cells. High aldehyde dehydrogenase (ALDH) activity is considered as a functional marker for the detection of cell ‘stemness’. In our study the ALDEFLUOR™ kit was used for determination of ALDH activity in PGCs. PGCs were co-stained with diethylaminobenzaldehyde (DEAB) and ALDH and analyzed by flow cytometry. Our results showed a small cell population (8.0 ± 3.3%) upon preincubation of the cells with the specific inhibitor DEAB, however cells without inhibitor staining showed a fluorescence shift as an ALDH-positive population (70.5 ± 1.6%). These findings indicate higher expression of ALDH in PGCs and ALDH activity can therefore be used as a new functional marker for the detection of cell ‘stemness’ in chicken PGCs. These results may have importance for characterization of PGCs as a potential genetic resource in poultry. Further research is necessary to elucidate the role of this functional marker in these cells.

scholarly journals Cytosolic free calcium levels in sickle red blood cells

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1469-1474 ◽  
Author(s):  
E Murphy ◽  
LR Berkowitz ◽  
E Orringer ◽  
L Levy ◽  
SA Gabel ◽  
...  
Keyword(s):  
Mean Value ◽  
Ionized Calcium ◽  
Free Calcium ◽  
Tetraacetic Acid ◽  
Nmr Studies ◽  
Extracellular Medium ◽  
Mean Values ◽  
Cell Calcium ◽  
Novel Approach ◽  
Sickle Erythrocytes

Abstract In this study, we used a recently developed nuclear magnetic resonance (NMR) technique to measure ionized calcium in sickle erythrocytes. The NMR technique, which involves 19F NMR studies of a fluorinated calcium chelator quinMF, [2-(2-amino-4-methyl-5-fluorophenoxy)methyl-8- aminoquinoline-N,N,N′,N′- tetraacetic acid] provides a novel approach to the study of ionized calcium in erythrocytes since the presence of hemoglobin precludes the use of fluorescent calcium indicators. The mean value for ionized calcium in oxygenated sickle erythrocytes was 18 +/- 2 nmol/L (SE). Experiments with normal RBCs gave a mean value of 21 +/- 2 nmol/L (SE). After 1 hour of deoxygenation, mean values for ionized calcium in sickle erythrocytes did not increase as compared with values obtained under oxygen. To investigate whether deoxygenation stimulated endocytosis, sickle erythrocytes were deoxygenated for 1 hour in the presence of impermeant FBAPTA (1,2 bis-(2-amino-5- fluorophenoxy) ethane N,N,N′,N′-tetraacetic acid). Cells were then separated from the extracellular medium and assayed for the presence of FBAPTA; they had incorporated significant quantities of the extracellular FBAPTA. This incorporation was not observed with normal erythrocytes. These data are consistent with at least a portion of the elevation in total cell calcium in sickle erythrocytes arising as a consequence of an endocytotic process in which extracellular calcium ions are incorporated into vesicles. Additional experiments show that these intracellular vesicles accumulate Ca2+ on further deoxygenation, consistent with a transient increase in ionized cell calcium. These studies represent the first use of NMR spectroscopy to evaluate endocytotic processes.

scholarly journals "Structural characterization of aminoglycoside modifying enzyme ANT(2"")-Ia"

2014 ◽  
Vol 70 (a1) ◽  
pp. C702-C702
Author(s):  
Angelia Bassenden ◽  
Dmitry Rodionov ◽  
Nilu Sabet-Kassouf ◽  
Tahereh Haji ◽  
Kun Shi ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Specific Activity ◽  
Ribosomal Subunit ◽  
Bactericidal Effect ◽  
Nmr Studies ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
16S Rna

Aminoglycosides are a class of broad-spectrum antibiotics used in the treatment of serious Gram-negative bacterial infections, they target the 16S RNA subunit and upon binding cause errors in translation, eventually inducing a bactericidal effect [1]. Aminoglycoside nucleotidyltransferase (2")-Ia (ANT(2")-Ia) is an aminoglycoside modifying enzyme that prevents aminoglycosides from binding to the ribosomal subunit, making this enzyme a principle candidate structure-based drug design [2]. Characterization of ANT(2")-Ia has been proven to be difficult due to the low stability and solubility of overexpressed protein, where 95% of the protein being expressed is in the form of inclusion bodies [3]. We describe a protocol that has lead to successful expression and purification of ANT(2")-Ia. A successful enzymatic assay has also been adapted and the protein is active and stable under these conditions with a specific activity of 0.14 U/mg. Furthermore, nuclear magnetic resonance (NMR) studies have allowed for the assignment of 144 of the 176 non-proline backbone residues. Substrate binding NMR experiments have shown unique global chemical shift perturbations upon binding ATP and tobramycin, suggesting unique binding sites for each substrate. Structural determination of ANT(2")-Ia using NMR in conjunction with x-ray crystallography can be utilized in order to develop small molecules that will act as more effective aminoglycosides in order to inhibit ANT(2")-Ia from binding and modifying these antibiotics.

Physiochemical Characterization of Herbo-Metallic NanoMedicine-Abhrak Bhasma by Ayurvedic and Novel Techniques

2018 ◽  
Vol 24 (8) ◽  
pp. 5907-5914
Author(s):  
G Harish ◽  
M Shivashankar
Keyword(s):  
Physical Nature ◽  
Arsenic Content ◽  
Nmr Studies ◽  
Chemical Evaluation ◽  
Potential Development ◽  
Qualitative Level ◽  
Human Need ◽  
Standardization Technique

The Ideal Physicochemical nature of the Bhasma preparation in the Indian System of medicine (ISM) is not as clear required as per the regulatory conditions, hence the analytical studies are one of the significant parts of drug standardization in ISM, such as Siddha, Ayurveda and Unani. The Metallic medicine used in the treatment of disease in human need scientific community for its Authentication, potential development and regulatory aspects. The Present investigation deals with the Physio-chemical evaluation of the ayurvedic medicine Abhrak bhasma by various traditional and novel techniques. In ayurvedic procedure the nature of the bhasmahad been studied by various techniques to determine the quantitative and qualitative level for standardization purpose. In the Present study the Bhasma scrutinized for Physical nature by Varna (Colour), Varitara (Floating), Rekhapurva (furrow filling), Anjana sannibha (softness), Nischandra (Lusterless), Sukshmatva (fineness) and novel techniques such as Nature, Hardness, Solubility, Ash (Acid insoluble), LOD, Assay for determination of percentage silicon dioxide, Heavy metals and arsenic content. The chemical nature is determined by Amla pareeksha (bitter taste), Gatarasatva (absence of taste), Aksharatwa (absence of alkaline taste), Nirdhuma (absence of smoke) and by novel technique such as SEM, Raman spectroscopy, NMR and EDAX. The Abhrak bhasma fulfilled the traditional and novel standardization technique and confirm the presence of Metal oxides of Si, Ca, Fe, Al. NMR studies show the presence of characteristic peak. Therefore the quality of the Abhrak bhasma was customary by both traditional and novel standardization technique.

13C and 15N NMR studies of 2,3,4-pentanetrione 3-phenylhydrazone, dimethyl 2-phenylhydrazonopropanedioate and ethyl 2-phenylhydrazono-3-oxobutanoate

1980 ◽  
Vol 45 (12) ◽  
pp. 3354-3359 ◽  
Author(s):  
Antonín Lyčka
Keyword(s):  
Nmr Spectra ◽  
Chemical Shifts ◽  
Coupling Constants ◽  
15N Nmr ◽  
Nmr Studies ◽  
Carbon And Nitrogen

The 13C AND 15N NMR spectra of 2,3,4-pentanetrione 3-phenylhydrazone, dimethyl 2-phenylhydrazonopropanedioate and ethyl 2-phenylhydrazono-3-oxobutanoate have been measured in deuteriochloroform at 30 °C. The carbon and nitrogen signals have been assigned, and the chemical shifts and coupling constants 1J(CH) have been determined. Ethyl 2-phenylhydrazono-3-oxobutanoate has been measured first as the (E) isomer and then as a mixture of the (E) and (Z) isomers. This compound has also been prepared with one or two 15N atoms enabling determination of absolute values of the coupling constants nJ(15N13C) and 1J(15N15N), respectively.

Sign in / Sign up

Export Citation Format

Share Document