Adaptation of a continuous, calorimetric kinetic assay to study the agmatinase-catalyzed hydrolytic reaction

2020 ◽  
Vol 595 ◽  
pp. 113618
Author(s):  
Liam A. Wilson ◽  
David Garcia ◽  
Marcelo Monteiro Pedroso ◽  
Benjamin L. Schulz ◽  
Luke W. Guddat ◽  
...  
Keyword(s):  
Kinetic Assay ◽  
Hydrolytic Reaction

Spectrocoulometry – a new spectro-electrochemical technique

1987 ◽  
Vol 52 (6) ◽  
pp. 1386-1396 ◽  
Author(s):  
Ján Mocák ◽  
Michal Németh ◽  
Mieczyslaw Lapkowski ◽  
Jerzy W. Strojek
Keyword(s):  
Optical Probe ◽  
Optical Signal ◽  
Open Circuit ◽  
Electrochemical Technique ◽  
Kinetics Of Oxidation ◽  
Hydrolytic Reaction ◽  
Experimental Errors ◽  
Mechanism And Kinetics ◽  
Kinetics Of

A spectrocoulometric macrocell with a direct-view optical probe was designed and constructed, where the optical signal is transferred by light-conducting glass or quartz fibres permitting to work at wavelengths above 410 or 300 nm. The method of measurement on the proposed equipment is described; it was tested in the study of the mechanism and kinetics of oxidation of Fe(bipy)32+ ions (bipy = 2,2'-bipyridyl) with the use of potentiostatic coulometric electrolysis with open-circuit relaxation at a suitable time. The primary product of electrolysis, Fe(bipy)33+, undergoes a follow-up hydrolytic reaction with the formation of a binuclear complex. The rate constant of the reaction of the first order involves the contributions, kBi, from all bases present in solution; the corresponding values for H2O, OH-, bipy, and CH3COO- ions at a ionic strength 0·5 mol dm-3 and 25 °C were determined as kOH = (5·0 ± 0·6) . 105 mol-1 dm3 s-1, kbipy = (1·3 ± 0·2) . 10-1 mol-1 dm3 s-1, kAc = (5·8 ± 1·0) . 10-2 mol-1 dm3 s-1, and kH2O is not significant with respect to experimental errors.

Eritadenines - Novel type of potent inhibitors of S-adenosyl-L-homocysteine hydrolase

1982 ◽  
Vol 47 (1) ◽  
pp. 167-172 ◽  
Author(s):  
Ivan Votruba ◽  
Antonín Holý
Keyword(s):  
Catalytic Activity ◽  
Rat Liver ◽  
Hydrolytic Reaction

Rat liver SAH-hydrolase is strongly inhibited by four stereoisomeric 4-(adenin-9-yl)-2,3-dihydroxybutyric acids (eritadenines). D-Eritadenine, which is the most effective of the four, inactivates the catalytic activity of SAH-hydrolase at IC50 = 1.2 .10-8 mol l-1 in the hydrolytic reaction. The enzyme is irreversibly inhibited (τ/2 = 1.6 min). The inactivation activity decreases in the order D-erythro(2R, 3R) L-erythro(2S, 3S) threo(2S, 3R) threo(2R, 3S) configuration.

Immunoassay or LC-MS/MS for the measurement of salivary cortisol in children?

2016 ◽  
Vol 54 (5) ◽  
Author(s):  
Yoon Ju Bae ◽  
Alexander Gaudl ◽  
Sonia Jaeger ◽  
Stephanie Stadelmann ◽  
Andreas Hiemisch ◽  
...  
Keyword(s):  
Salivary Cortisol ◽  
Social Stress ◽  
Statistical Power ◽  
Stress Test ◽  
Externalizing Disorders ◽  
Cross Reactivity ◽  
Trier Social Stress Test ◽  
Healthy Children ◽  
Kinetic Assay ◽  
The Difference

AbstractBackground:Dysregulation of the adrenal cortex has been assessed with measurement of salivary cortisol. So far salivary cortisol is routinely measured with immunoassay (IA). However, liquid chromatography-tandem mass spectrometry (MS) is known to offer better specificity. We compared the concentrations of salivary cortisol measured by MS and IA at basal and stress induced conditions and evaluated reasons for the difference in method-dependent cortisol results.Methods:Saliva samples (n=2703) were collected from 169 children (age range: 8–14 years; 81 healthy children; 55 with internalizing and 33 with externalizing disorders) under circadian conditions and during the Trier Social Stress Test for Children (TSST-C). Biochemical analyses were performed with MS for cortisol and cortisone, IA (IBL, RE62011) for cortisol, and enzyme kinetic assay for α-amylase.Results:MS and IA showed mostly comparable results for circadian activity and TSST-C response with similar statistical power. However, IA measured cortisol concentrations about 2.39-fold higher than MS. We found that this difference in measured values between MS and IA was mainly due to different standardization of IA compared to MS. In addition, at cortisol IA concentration below 5 nmol/L, cross-reactivity with cortisone was found to contribute to the lower concordance between MS and IA.Conclusions:Immunoassay and LC-MS/MS were largely comparable in the interpretation of salivary cortisol dynamics in stress research. But the IA method revealed a restricted accuracy in the measuring range below 5 nmol/L.

scholarly journals Kinetics of tau aggregation reveals patient specific tau characteristics among Alzheimer's cases

2021 ◽  
Author(s):  
Tarun V Kamath ◽  
Naomi Klickstein ◽  
Caitlin Commins ◽  
Analiese R Fernandes ◽  
Derek H Oakley ◽  
...  
Keyword(s):  
Growth Phase ◽  
Tau Proteins ◽  
Patient Specific ◽  
Lag Phase ◽  
Plateau Phase ◽  
Kinetic Assay ◽  
Cellular Processes ◽  
Morphological Differences ◽  
Tau Aggregation ◽  
Kinetics Of

Abstract The accumulation of tau aggregates throughout the human brain is the hallmark of a number of neurodegenerative conditions classified as tauopathies. Increasing evidence shows that tau aggregation occurs in a “prion-like” manner, in which a small amount of misfolded tau protein can induce other, naïve tau proteins to aggregate. Tau aggregates have been found to differ structurally among different tauopathies. Recently, however, we have suggested that tau oligomeric species may differ biochemically among individual patients with sporadic Alzheimer disease, and have also showed that the bioactivity of the tau species, measured using a cell-based bioassay, also varied among individuals. Here, we adopted a live-cell imaging approach to the standard cell-based bioassay to explore further whether the kinetics of aggregation also differentiated these patients. We found that aggregation can be observed to follow a consistent pattern in all cases, with a lag phase, a growth phase, and a plateau phase, which each provide quantitative parameters by which we characterize aggregation kinetics. The length of the lag phase and magnitude of the plateau phase are both dependent upon the concentration of seeding-competent tau, the relative enrichment of which differs among patients. The slope of the growth phase correlates with morphological differences in the tau aggregates, which may be reflective of underlying structural differences. This kinetic assay confirms and refines the concept of heterogeneity in the characteristics of tau proteopathic seeds among individuals with Alzheimer’s disease and is a method by which future studies may characterize longitudinal changes in tau aggregation and the cellular processes which may influence these changes.

Quantitative alkaline phosphatase isoenzyme determination by electrophoresis on cellulose acetate membranes.

1977 ◽  
Vol 23 (1) ◽  
pp. 28-34 ◽  
Author(s):  
W H Siede ◽  
U B Seiffert
Keyword(s):  
Alkaline Phosphatase ◽  
Cellulose Acetate ◽  
Clinical Laboratory ◽  
Kinetic Assay ◽  
Specific Staining ◽  
Cellulose Acetate Membranes ◽  
Alkaline Phosphatase Isoenzymes ◽  
Elution Method ◽  
Routine Clinical Laboratory ◽  
Alkaline Phosphatase Isoenzyme

Abstract We present a new method for quantitative determination of alkaline phosphatase isoenzymes. This method consists of electrophoretic separation on cellulose acetate membranes, special fixation technique to avoid elution and diffusion of enzyme protein during incubation, specific staining, and quantitative evaluation by densitometric measurement. We highly recommend the precedure for routine clinical laboratory use. In all normal individuals we observe two isoenzymes of hepatic origin and one isoenzyme each of osseous, intestinal, and biliary origin. Quantitative normal values are presented. Precision of the method is calculated, the CV being less than 10%. The exactness of densitometric quantification is proved by comparison with kinetic assay of alkaline phosphatase isoenzymes by use of an elution method. Clinical implications of alkaline phosphatase isoenzymograms are reported and discussed in detail.

scholarly journals Alanine Aminopeptidase, γ-Glutamyl Transferase and β2-microglobulin as Diagnostic Markers in Patients with Rheumatoid Arthritis

2008 ◽  
Vol 27 (1) ◽  
pp. 59-63
Author(s):  
Dejan Spasovski ◽  
Todor Gruev ◽  
Nada Marina ◽  
Jordan Calovski ◽  
Ljubinka Rajčevska ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Diagnostic Markers ◽  
Border Region ◽  
Healthy Individuals ◽  
Kinetic Assay ◽  
Renal Lesions ◽  
Alanine Aminopeptidase ◽  
Glutamyl Transferase ◽  
Higher Sensitivity

Alanine Aminopeptidase, γ-Glutamyl Transferase and β2-microglobulin as Diagnostic Markers in Patients with Rheumatoid Arthritis The purpose of this research is to evaluate the values of alanine aminopeptidase (AAP), γ-glutamyl transferase (γ-GT), and β2-Microglobulin in urine (β2-M), in untreated rheumatoid arthritis (RA) and to define the effect of untreated rheumatoid arthritis on the tubular function and brush border region. We used a kinetic assay for AAP, standard methods by the IFCC for γ-glutamyl transferase and MEIA for the determination of β2-Microglobulin in urine in 70 participants (35 untreated RA patients, 35 healthy individuals). From the total of 35 RA patients, 24 patients had AAP (sensitivity of the test 68.57%), 16 patients had γ-GT enzymuria (sensitivity of the test 45.71%), while the presence of β2-Microglobulin in urine was found in a very low percentage. Out of 18 RF negative patients, 14 patients are AAP positive, 10 patients were γ-GT positive, while the presence of β2-Microglobulin in urine was not detected. Among 17 RF positive RA patients, the presence of AAP was noticed in 10, the presence of γ-GT in 6 patients, while the presence of β2-Microglobulin in urine was not detected. AAP has higher sensitivity than γ-GT and β2-Microglobulin in the detection of asymptomatic renal lesions in untreated RA.

Kinetic Assay for High-Throughput Screening of In Vitro Transthyretin Amyloid Fibrillogenesis Inhibitors

2005 ◽  
Vol 7 (2) ◽  
pp. 246-252 ◽  
Author(s):  
Ignacio Dolado ◽  
Joan Nieto ◽  
Maria João M. Saraiva ◽  
Gemma Arsequell ◽  
Gregori Valencia ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Kinetic Assay

Fabrication of α-TCP, HAp Functionally Graded Porous Beads

2008 ◽  
Vol 57 ◽  
pp. 135-138
Author(s):  
Yuji Kajihata ◽  
Teruo Asaoka ◽  
Katsuko S. Furukawa ◽  
Takashi Ushida ◽  
T. Tateishi
Keyword(s):  
Surface Layer ◽  
High Strength ◽  
Functionally Graded ◽  
The Body ◽  
Acid Solutions ◽  
Bioactive Material ◽  
Hydrolytic Reaction ◽  
Rate Of Absorption ◽  
Porous Beads ◽  
Good Cell

HAp (Hydroxyapatite) and α-TCP (alpha tribasic calcium phosphate) are non-toxic to human cells and, thus, have been studied for applications as biomaterials. HAp is a bioactive material that is not readily absorbed by the body; it offers both high strength and better tissueadhesive properties than α-TCP. In contrast, α-TCP is highly bioabsorbable; it is quickly absorbed by the body, and, therefore, for example, disappears before bone is completely replaced. If porous beads could be fabricated that would take advantage of the useful properties of α-TCP and HAp, they could be used as excellent scaffolds for cultivating cells. In the present study, ceramic beads with α-TCP at the center were fabricated and coated with a functionally graded film of HAp. A scaffold based on this configuration would be expected to have the following characteristics: good cell adhesion; strong beads; and a rate of absorption into the body that would be easy to control. In addition, to accelerate the formation of porous structure, some acid solutions were used to dissolve the beads surface layer and to penetrate pores toward inside of the bead. HAp formation through hydrolytic reaction seemed to be promoted by these acid solutions.

scholarly journals Development of a Kinetic Assay for Late Endosome Movement

2014 ◽  
Vol 19 (7) ◽  
pp. 1070-1078 ◽  
Author(s):  
Milan Esner ◽  
Felix Meyenhofer ◽  
Michael Kuhn ◽  
Melissa Thomas ◽  
Yannis Kalaidzidis ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Membrane Integrity ◽  
Living Cells ◽  
Mechanisms Of Action ◽  
Added Value ◽  
Kinetic Assay ◽  
Late Endosomes ◽  
Automated Imaging ◽  
Phenotypic Clustering ◽  
Large Compound

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.

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