Determination of Ca2+/Calmodulin-Stimulated Phosphodiesterase Activity in Intact Cells

2005 ◽  
pp. 085-092
Author(s):  
Chen Yan
Keyword(s):  
Phosphodiesterase Activity ◽  
Intact Cells

scholarly journals A commonly used photosynthetic inhibitor fails to block electron flow to photosystem I in intact systems

2019 ◽  
Author(s):  
Duncan Fitzpatrick ◽  
Eva-Mari Aro ◽  
Arjun Tiwari
Keyword(s):  
Electron Flow ◽  
Photosynthetic Apparatus ◽  
Intact Cells ◽  
Membrane Inlet Mass Spectrometry ◽  
Redox Kinetics ◽  
Species Formation ◽  
Intact Leaves ◽  
Transfer Chain ◽  
Photosynthetic Electron Transfer

AbstractIn plant science, 2,4-dinitrophenylether of iodonitrothymol (DNP-INT) is frequently used as an alternative to 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) to examine the capacity of plastoquinol and semiquinone to reduce O2. DNP-INT is considered an effective inhibitor of the photosynthetic electron transfer chain (PETC) through its binding at the Q0 site of Cyt-b6f. The binding and action of DNP-INT has been previously characterized spectroscopically in purified Cyt-b6f complex reconstituted with Plastocyanin, PSII membranes and plastoquinone, as well as in isolated thylakoids based on its property to block MV-mediated O2 consumption. Contrary to the conclusions made from these experiments, we observed clear reduction of P700+ in samples incubated with DNP-INT during our recent investigation into the sites of oxygen consumption in isolated thylakoids. Therefore, we carried out an extensive investigation of DNP-INT’s chemical efficacy in isolated thylakoids and intact leaves. This included examination of its capacity to block the PETC before PSI, and therefore its inhibition of CO2 fixation. P700 redox kinetics were measured using Dual-PAM whilst Membrane Inlet Mass Spectrometry (MIMS) was used for simultaneous determination of the rates of O2 evolution and O2 consumption in isolated thylakoids and CO2 fixation in intact leaves, using two stable isotopes of oxygen (16O2,18O2) and CO2 (12C,13C), respectively. Based on these investigations we confirmed that DNP-INT is unable to completely block the PETC and CO2 fixation, therefore its use may produce artefacts if applied to isolated thylakoids or intact cells, especially when determining the locations of reactive oxygen species formation in the photosynthetic apparatus.

scholarly journals A Novel H+ Conductance in Eosinophils

1999 ◽  
Vol 190 (2) ◽  
pp. 183-194 ◽  
Author(s):  
Botond Bánfi ◽  
Jacques Schrenzel ◽  
Oliver Nüsse ◽  
Daniel P. Lew ◽  
Erzsébet Ligeti ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
The Novel ◽  
Experimental Conditions ◽  
Intact Cells ◽  
Electrophysiological Studies ◽  
Dependent Signal ◽  
Low Threshold ◽  
Nadph Oxidase Activation ◽  
In Cells

Efficient mechanisms of H+ ion extrusion are crucial for normal NADPH oxidase function. However, whether the NADPH oxidase—in analogy with mitochondrial cytochromes—has an inherent H+ channel activity remains uncertain: electrophysiological studies did not find altered H+ currents in cells from patients with chronic granulomatous disease (CGD), challenging earlier reports in intact cells. In this study, we describe the presence of two different types of H+ currents in human eosinophils. The “classical” H+ current had properties similar to previously described H+ conductances and was present in CGD cells. In contrast, the “novel” type of H+ current had not been described previously and displayed unique properties: (a) it was absent in cells from gp91- or p47-deficient CGD patients; (b) it was only observed under experimental conditions that allowed NADPH oxidase activation; (c) because of its low threshold of voltage activation, it allowed proton influx and cytosolic acidification; (d) it activated faster and deactivated with slower and distinct kinetics than the classical H+ currents; and (e) it was ∼20-fold more sensitive to Zn2+ and was blocked by the histidine-reactive agent, diethylpyrocarbonate (DEPC). In summary, our results demonstrate that the NADPH oxidase or a closely associated protein provides a novel type of H+ conductance during phagocyte activation. The unique properties of this conductance suggest that its physiological function is not restricted to H+ extrusion and repolarization, but might include depolarization, pH-dependent signal termination, and determination of the phagosomal pH set point.

scholarly journals Studies on the Biosynthesis of γ–Decalactone in Sporobolomyces odorus

1990 ◽  
Vol 45 (3-4) ◽  
pp. 207-216 ◽  
Author(s):  
Wolfgang Albrecht ◽  
Roland Tressl
Keyword(s):  
Linoleic Acid ◽  
Intact Cells

Abstract Incubation of racemic ethyl [2,2-2H2]-(E)-3,4-epoxydecanoate and the corresponding acid with intact cells of Sporobolomyces odorus led to the formation of deuterium labeled γ-decalaetone. The detection of labeled 2-decen-4-olide and 4-oxodecanoic acid made it possi­ble to propose the biogenetic sequence leading to γ-decalactone. The determination of the en­ antiomeric ratio of the lactone revealed an unspecific metabolism of the precursor. In contrast to the (E)-isomer, after administration of ethyl [5,6-2H2]-(Z)-3,4-epoxydeeanoate no transfor­mation could be detected. These results indicate that (E)-3,4-epoxydecanoic acid, formed from (E)-3-decenoyl-CoA , an intermediate of the β-oxidation of linoleic acid, is the genuine precur­sor in the biosynthesis of γ-decalactone.

Determination of phosphodiesterase activity in rat mast cells using the fluorescent cAMP analogue anthraniloyl cAMP

1995 ◽  
Vol 7 (5) ◽  
pp. 513-518 ◽  
Author(s):  
A. Alfonso ◽  
M. Estévez ◽  
M.C. Louzao ◽  
M.R. Vieytes ◽  
L.M. Botana
Keyword(s):  
Mast Cells ◽  
Phosphodiesterase Activity ◽  
Camp Analogue ◽  
Rat Mast Cells

scholarly journals Assay method for monitoring the inhibitory effects of antimetabolites on the activity of inosinate dehydrogenase in intact human CEM lymphocytes

1992 ◽  
Vol 287 (3) ◽  
pp. 785-790 ◽  
Author(s):  
J Balzarini ◽  
E De Clercq
Keyword(s):  
Mycophenolic Acid ◽  
Marked Effect ◽  
De Novo ◽  
Potent Inhibitor ◽  
Assay Method ◽  
The Novel ◽  
Inhibitory Effects ◽  
Intact Cells ◽  
Imp Dehydrogenase

A rapid and convenient method has been developed to monitor the inhibition of inosinate (IMP) dehydrogenase by antimetabolites in intact human CEM lymphocytes. This method is based on the determination of 3H release from [2,8-3H]hypoxanthine ([2,8-3H]Hx) or [2,8-3H]inosine ([2,8-3H]Ino). The validity of this procedure was assessed by evaluating IMP dehydrogenase inhibition in intact CEM cells by the well-known IMP dehydrogenase inhibitors ribavirin, mycophenolic acid and tiazofurin. As reference materials, several compounds that are targeted at other enzymes in de novo purine nucleotide anabolism (i.e. hadacidine, acivicin) or catabolism (i.e. 8-aminoguanosine, allopurinol) were evaluated. There was a strong correlation between the inhibitory effects of the IMP dehydrogenase inhibitors (ribavirin, mycophenolic acid, tiazofurin) on 3H release from [2,8-3H]Hx and [2,8-3H]Ino in intact CEM cells and their ability to decrease intracellular GTP pool levels. The other compounds (hadacidine, acivicin, 8-aminoguanosine, allopurinol) had no marked effect on 3H release from [2,8-3H]Hx. Using this method, we demonstrated that the novel ribavirin analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide, is a potent inhibitor of IMP dehydrogenase in intact cells.

Determination of pyruvate oxidation rate and citric acid cycle activity in intact human leukocytes and fibroblasts.

1978 ◽  
Vol 24 (2) ◽  
pp. 200-203 ◽  
Author(s):  
H L Willems ◽  
T F de Kort ◽  
F J Trijbels ◽  
L A Monnens ◽  
J H Veerkamp
Keyword(s):  
Citric Acid ◽  
Cell Membrane ◽  
Citric Acid Cycle ◽  
Marker Enzyme ◽  
Intact Cells ◽  
Acid Cycle ◽  
Oxidation Rates ◽  
Pyruvate Oxidation ◽  
14Co2 Production

Abstract We measured pyruvate oxidation in intact leukocytes and fibroblasts by measuring 14C02 production. The optimal pyruvate concentration appeared to be higher than that usually applied. Activities remained constant during the incubation and were proportional to the amount of tissue protein added. Man values (+/-SD) were 2.8 +/- 0.9 nmol/h per 10(6) cells and 37 +/- 14 nmol/h per mg of protein for leukocytes and fibroblasts, respectively, for [1-14C]pyruvate oxidation; and 2.1 +/- 0.8 nmol/h per 10(6) cells and 18 +/- 7 nmol/h per mg of protein, respectively for [2-14C]pyruvate oxidation. We compared oxidation rates of pyruvate and 2-oxoglutarate by intact cells with those of isolated mitochondria. The ratio of 14CO2 production vs. activity of mitochondrial marker enzyme demonstrated that the rate of pyruvate oxidation can adequately be assayed in intact cells, but that the permeability of the cell membrane is rate-limiting in the oxidation of 2-oxoglutarate. No significant oxidation of other intermediates of the citric acid cycle was found, presumably owing to a low rate of transport of these substances across the cell membrane.

scholarly journals Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells.

1985 ◽  
Vol 38 (10) ◽  
pp. 1085-1092 ◽  
Author(s):  
J Burns ◽  
V T Chan ◽  
J A Jonasson ◽  
K A Fleming ◽  
S Taylor ◽  
...  
Keyword(s):  
Sex Determination ◽  
Dna Probes ◽  
Intact Cells
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