Luciferase-based GloSensor™ cAMP assay: Temperature optimization and application to cell-based kinetic studies

Methods ◽  
2021 ◽  
Author(s):  
Fang I. Wang ◽  
Gucci Ding ◽  
Garmen S. Ng ◽  
S. Jeffrey Dixon ◽  
Peter Chidiac
2016 ◽  
Vol 17 (2) ◽  
pp. 480-492 ◽  
Author(s):  
T. Ngulube ◽  
M. W. Gitari ◽  
H. Tutu

Excess fluoride in drinking water is harmful to human health and therefore it needs to be removed from water before consumption. The potential of locally available mixed Mukondeni clay soils (MMCS) as a cheap adsorbent for the removal of fluoride from aqueous solution was investigated. Characterization of MMCS was done by X-ray fluorescence, X-ray diffraction, scanning electron microscopy, Fourier transform infrared and Brunauer Emmett Teller. Cation exchange capacity and point of zero charge of the clays were determined using standard methods. Parameters optimized included: contact time, adsorbent dosage, initial fluoride concentration, pH and temperature. Optimization experiments were done in batch procedures. The results showed that the optimum conditions for the defluoridation of water using MMCS are 60 min, 1.5 g, 9 mg/L, 1.5/100 S/L ratios, pH of 2 and a temperature of 25 °C. The equilibrium isotherm regression parameter (R2 = 0.95) showed that the Freundlich isotherm gave a better fit than the Langmuir isotherm (R2 = 0.52) which indicates multilayer adsorption. Kinetic studies revealed that the adsorption followed pseudo second order kinetics. This study indicated that locally available MMCS are good in the defluoridation of groundwater but modification through blending with metal oxide modified clays can enhance their adsorption capacity.


2004 ◽  
Vol 71 ◽  
pp. 1-14
Author(s):  
David Leys ◽  
Jaswir Basran ◽  
François Talfournier ◽  
Kamaldeep K. Chohan ◽  
Andrew W. Munro ◽  
...  

TMADH (trimethylamine dehydrogenase) is a complex iron-sulphur flavoprotein that forms a soluble electron-transfer complex with ETF (electron-transferring flavoprotein). The mechanism of electron transfer between TMADH and ETF has been studied using stopped-flow kinetic and mutagenesis methods, and more recently by X-ray crystallography. Potentiometric methods have also been used to identify key residues involved in the stabilization of the flavin radical semiquinone species in ETF. These studies have demonstrated a key role for 'conformational sampling' in the electron-transfer complex, facilitated by two-site contact of ETF with TMADH. Exploration of three-dimensional space in the complex allows the FAD of ETF to find conformations compatible with enhanced electronic coupling with the 4Fe-4S centre of TMADH. This mechanism of electron transfer provides for a more robust and accessible design principle for interprotein electron transfer compared with simpler models that invoke the collision of redox partners followed by electron transfer. The structure of the TMADH-ETF complex confirms the role of key residues in electron transfer and molecular assembly, originally suggested from detailed kinetic studies in wild-type and mutant complexes, and from molecular modelling.


1975 ◽  
Vol 14 (04) ◽  
pp. 330-338
Author(s):  
L. G. Colombetti ◽  
J. S. Arnold ◽  
W. E. Barnes

SummaryTc-99m pyridoxylidene glutamate has proven to be an excellent biliary scanning agent, far superior in many respect to the commonly used 1-131 rose bengal. The preparation of the compound as previously reported by Baker et al is too time consuming and requires the use of an autoclave which is not available in most nuclear medicine departments. In our facility, we have been preparing similar compounds using several aldehydes and monosodium glutamate to make labeled complexes having the same pharmacological characteristics. The mixture of monosodium glutamate, aldehyde, and Tc-99m pertechnetate is made slightly alkaline, purged with helium, and placed in a sealed vial. The vial, which is protected by a wire basket, is then heated in a laboratory oven at 130° C for a period of 15 to 20 minutes. During this time, the technetium is reduced to a lower valence state and bound to the complex formed. Chromatographic data show that these compounds are chemically similar to that previously reported. The compounds prepared concentrate in the gall bladder of the rabbit in less than 10 minutes. Kinetic studies have been performed on dogs with a scintillation camera and small digital computer to measure rates of blood clearance, liver and gall bladder uptake, and excretion into the intestine. The aldehyde — glutamate complex promises to be a useful scanning agent for the diagnosis of biliary and hepatocellular diseases.


1961 ◽  
Vol 05 (01) ◽  
pp. 001-020
Author(s):  
Douglas M. Surgenor ◽  
Nancy A. Wilson ◽  
Anne S. Henry

SummaryA method is described for the partial purification of a human plasma factor which accelerates the conversion of prothrombin to thrombin in the presence of tissue thromboplastin. This factor may be dried from the frozen state, and may be kept in stable dry form for long periods of time. The quantitative assay of this activity is done in a classical two-stage prothrombin system using tissue thromboplastin and calcium. From its properties, it is concluded that this activity corresponds to factor V, labile factor and plasma Ac-globulin.Chemical and kinetic studies reveal that human factor V is active in plasma and is destroyed by thrombin. Human serum has little or no factor V activity.These results thus fail to support the postulated activation of factor V during clotting. All of the kinetic data are consistent with an enzymatic role for factor V in the formation of tissue prothrombin activator (thromboplastin).


1979 ◽  
Vol 42 (05) ◽  
pp. 1473-1482 ◽  
Author(s):  
A Dup Heyns ◽  
P N Badenhorst ◽  
H Pieters ◽  
M G Lötter ◽  
P C Minnaar ◽  
...  

SummaryFactors influencing labelling of human platelets with 111Indium-8-hydroxyquinoline ([111In]-oxine) in a physiological saline medium were investigated. The efficiency of labelling is influenced by time of incubation, concentration of oxine, and pH of the incubating medium. It was found that a viable platelet population could be labelled under the following conditions: (1) centrifugation of platelet rich plasma in polystyrene conical tubes at 800 g for 15 min; (2) resuspension of the platelet pellet in saline, pH 5.5; (3) incubating for 30 min at 22°C with [111In]-oxine at a concentration of 6.25 mg oxine/litre platelet suspension; (4) washing once with platelet poor autologous plasma (PPP); and (5) finally resuspending the platelets in PPP. The labelled platelets aggregated normally with collagen and ADP. Electron microscopy, done immediately after labelling, showed internal organelle reorganization characteristic of activated platelets. These ultrastructural features were reversible on incubation in PPP at 37°C for 30 min. The 111In is not released from aggregated platelets and the label does not elute from incubated platelets for at least five hr. We conclude that human platelets thus labelled are suitable for in vivo kinetic studies.


Diabetes ◽  
1975 ◽  
Vol 24 (12) ◽  
pp. 1094-1100 ◽  
Author(s):  
A. Rabinovitch ◽  
A. Gutzeit ◽  
A. E. Renold ◽  
E. Cerasi

Author(s):  
Umamaheswara G. ◽  
Anudeep D.

Fluvastatin sodium is a novel compound used as cholesterol lowering agent which acts through the inhibition of 3- hydroxyl-3- methyl glutaryl- coenzyme A (HMG-Co A) reductase. It has short biological half life (1-3h) in humans required a dosing frequency of 20 to 40mg twice a day. Due to its short variable biological half life it has been developed to a sustained gastroretentive system with a natural and synthetic polymer and to study how far the natural mucilage improves the sustained activity. Floating tablets were prepared by direct compression method using in combination of natural mucilage and synthetic polymer. Prior to the preparation of tablets the physical mixtures were subjected to FT IR studies and pre compression parameters. After preparation of tablets they were subjected to various tests like swollen index, drug content, In vitro dissolution and release kinetics with pcp disso software etc. The tablets prepared by direct compression shown good in thickness, hardness and uniformity in drug content, the prepared tablets floated more than 12h except FS1 and FS2 shows 9 and 11h. Swollen index studies shows with increase in concentration of polymer the swelling increases the diffusion path length by which the drug molecule may have to travel and cause lag time. In vitro results shows that on increasing the amount of hibiscus polymer the sustain activity is increased because of its integrity and forms a thick swollen mass and reduces the erosion property of the HypromelloseK100M, kinetic studies shows that FS 1, FS2, FS3 followed the Korsmeyer peppas model and the rest FS 4, FS 5, FS6 follows the zero order respectively. Based on n value indicating that the drug release followed super case II transport mechanism due to the erosion of the polymer.


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