Rate Of Decrease In Fluorescence Research Articles

Corticosterone treatment results in enhanced release of peptidergic vesicles in astrocytes via cytoskeletal rearrangements

While the effect of stress on neuronal physiology is widely studied, its effect on the functionality of astrocytes is not well understood. We studied the effect of high doses of stress hormone corticosterone, on two physiological properties of astrocytes, i.e., gliotransmission and interastrocytic calcium waves. To study the release of peptidergic vesicles from astrocytes, hippocampal astrocyte cultures were transfected with a plasmid to express pro-atrial natriuretic peptide (ANP) fused with the emerald green fluorescent protein (ANP.emd). The rate of decrease in fluorescence of ANP.emd on application of ionomycin, a calcium ionophore was monitored. Significant increase in the rate of calcium-dependent exocytosis of ANP.emd was observed with the 100 nM and 1 μM corticosterone treatments for 3 h, which depended on the activation of the glucocorticoid receptor. ANP.emd tagged vesicles exhibited increased mobility in astrocyte culture upon corticosterone treatment. Increasing corticosterone concentrations also resulted in concomitant increase in the calcium wave propagation velocity, initiated by focal ATP application. Corticosterone treatment also resulted in increased GFAP expression and F-actin rearrangements. FITC-Phalloidin immunostaining revealed increased formation of cross linked F-actin networks with the 100 nM and 1 μM corticosterone treatment. Alternatively, blockade of actin polymerization and disruption of microtubules prevented the corticosterone-mediated increase in ANP.emd release kinetics. This study reports for the first time the effect of corticosterone on gliotransmission via modulation of cytoskeletal elements. As ANP acts on both neurons and blood vessels, modulation of its release could have functional implications in neurovascular coupling under pathophysiological conditions of stress.

Deletion of a Conserved, Central Ribosomal Intersubunit RNA Bridge

Elucidation of the structure of the ribosome has stimulated numerous proposals for the roles of specific rRNA elements, including the universally conserved helix 69 (H69) of 23S rRNA, which forms intersubunit bridge B2a and contacts the D stems of A- and P-site tRNAs. H69 has been proposed to be involved not only in subunit association and tRNA binding but also in initiation, translocation, translational accuracy, the peptidyl transferase reaction, and ribosome recycling. Consistent with such proposals, deletion of H69 confers a dominant lethal phenotype. Remarkably, in vitro assays show that affinity-purified Deltah69 ribosomes have normal translational accuracy, synthesize a full-length protein from a natural mRNA template, and support EF-G-dependent translocation at wild-type rates. However, Deltah69 50S subunits are unable to associate with 30S subunits in the absence of tRNA, are defective in RF1-catalyzed peptide release, and can be recycled in the absence of RRF.

Pyridine nucleotide flux and glutathione oxidation in the cultured rat conceptus

It is proposed that protection of the developing embryo from chemical and environmental insults that produces oxidative stress requires a proper glutathione (GSH) and pyridine nucleotide status in both the embryo and extra-embryonic membranes. Modulation of pyridine nucleotide flux {NAD(H) and NAD(P)H} in the visceral yolk sac (VYS) by the thiol oxidants diamide and tert-butyl hydroperoxide (tBH) was studied in real time using microfiberoptic sensors in GD 10 rat conceptuses. Consecutive 5-min exposures to 125- and 250-μM diamide resulted in a fluorescence decrease of 14 and 32 Arbitrary Fluorescence Units (AFU). An additional consecutive exposure to 500-μM diamide caused an attenuated decrease followed by a rebound increase of 22 AFU. Consecutive 5-min exposures to tBH at 250 and 500 μM produced fluorescence decreases similar to that of 500 μM diamide, but the decreases were attenuated at 1000 μM. However, there was variability in the rebound increase. A 5-min exposure to tBH (500 μM) alone caused a fluorescence decrease of 14 AFU followed by a rebound increase of 8 AFU. The rate of fluorescence decrease was attenuated by 50% with pretreatment with the glutathione reductase (GSSG-Rd) inhibitor, BCNU (1,3, bis(2 chloroethyl)-1-nitrosourea), indicating that the decrease in surface fluorescence was probably attributable to a decrease in NADPH. Decreases in fluorescence, observed from the surface of the VYS, correlated with decreases in GSH/GSSG ratios in the embryos and the VYS. After exposure to tBH, GSH levels in conceptuses decreased at the end of 5 and 15 min, with a corresponding increase in oxidized glutathione (GSSG) at the end of 3, 5, and 15 min. Our results demonstrate that the increased production of GSSG on exposure to thiol oxidants correlates with a decrease in the reduced pyridine nucleotide, implying the presence of an active GSSG-Rd pathway in the conceptus during organogenesis, and implicating an important role of the pyridine nucleotides in the restoration of GSH homeostasis in the developing rat conceptus during organogenesis.

A continuous fluorescence displacement assay for phospholipase D.

Phospholipases D (PLD) are receiving increasing attention as important enzymes in lipid signalling pathways. Their biochemical function is to remove the headgroup (e.g. choline) from phospholipids by hydrolysis thus producing phosphatidic acid (PA). Primary alcohols can displace the active site water resulting in transphosphatidylation to produce phosphatidylalcohols [ 11. Whilst dioleoyl phosphatidylcholine (DOPC) is a poor substrate for human nonpancreatic secretory phospholipase 4 (hnpsPLAJ [2] the presence of either phosphatidic acid or phosphatidyl-alcohols can promote activity (papers in preparation). We report here on a novel adaptation of a fluorescence displacement assay, used to monitor fatty acid release, which makes use of activation of hnpsPLA, to indirectly monitor PLD activity. Phospholipases act by the process of interfacial catalysis which can be summarised by the following scheme: E*E* + S + E*S 4 E*P+ E* + P in which E* represents the activated form of the enzyme bound to the interface. The consequence of this mechanism is that if the enzyme does not bind productively to the interface (E*) it remains in the E-form and interfacial catalysis cannot proceed. Thii is the case for hnpsPLA, in the presence of vesicles composed of longchain phosphatidylcholines. The ability of membrane components to promote the E + E* step can therefore have significant effects on the apparent activity of hnpsPLA,. We have examined the effects of phosphatidylmethanol and phosphatidic acid, produced in situ by PLD from Sireptomyces chromofuscus, on the activity of hnpsPLA,. This enhancement of PLA, activity forms the basis of the assay described here. The assay system described does not require the separation of products h m substrates, as is the case with radiometric assays. This fluorescence displacement assay is continuous and sensitive, adaptable to mictrotitre plate format and may be suitable for high throughput screening systems. In the current format pure unlabelled DOPC vesicles were used as substrate. There is no requirement for detergents, indeed these may be deleterious due to activating effects on hnpsPL4. The indirect nature of the assay means that it is susceptible to factors that might alter the hnpsPLA, activity independent of the PLD activity, e.g. calcium concentrations. Assays performed here were carried out in 1 ml aliquots of assay cocktail containing 0.1 M Tris-HC1,O.l M NaCl, 1 mM CaCl,, pH 8.0, DOPC (50 pg/ml) and 1 pM dansyl undecanoic acid (DAUDA, Molecular Probes, USA). The phospholipid was present as small unilammelar vesicles produced either by alcohol injection or by sonication. Liver fatty acid binding protein (FABP) [3] was added to give a 1 : 1 ratio with the fluorescent probe. The complex so formed yields a high fluorescent signal which was monitored over approximately 1 minute. A second trace was recorded immediately on addition of 1.1 pg hnpsPLA, and the rate of decrease of fluorescence assessed. This rate is attributable to a very slow hydrolysis of DOPC by hnpsPLA, (see Figure. 1: blank) which has negligable effect on the composition of the vesicles. Having established the background rate PLD could then be added.The fluorescence signal is monitored immediately on addition of enzyme. Typical traces are depicted in Figure 1. The enhancement in the rate of decrease in fluorescence is proportional to the amount of PLD present (see Figure 2). The addition of PLD alone (in the absence of hnpsPLAJ does not result in a decrease in the fluorescence signal (result not shown). The assay can be performed in a variety of buffer systems including Hank's balanced salt solution. A variety of substrates including whole cells (results not shown) can be used in place of DOPC so long as hnpsPL4 remains predominantly in the E form and thereby expresses a negligible background rate. From Figure 2 it can be seen that the transphosphatidylation assay (i.e. in the presence of 1% v/v methanol) is more sensitive to PLD activity. This is in agreement with observations for the effect of phosphatidyl alcohols and phosphatidic acid on hnpsPL4 activity.

Mechanism of microtubule kinesin ATPase.

A six-step mechanism is derived for the activation of kinesin K379 ATPase by microtubules. The data are fitted by the kinetic scheme [Formula see text] where T, D, and P refer to nucleotide triphosphate, nucleotide diphosphate, and inorganic phosphate, respectively; MtK refers to the complex of a K379 unit with the microtubule binding site. The initial binding and release steps, 1 and 6, are treated as rapid equilibria: k2 = 200 s-1, k3 = 100 s-1, k5 = 35-40 s-1, maximum steady-state rate = 25 s-1 (50 mM NaCl, 20 degrees C). k2 was obtained from the maximum rate of fluorescence enhancement with mant-ATP as substrate, k3 was obtained from the hydrolysis transient phase for ATP or mant-ATP, and k5 was obtained from the rate of decrease in fluorescence of mant-ADP in the reaction [Formula see text]. A large excess of ATP was present with the Mt to block rebinding of mant-ADP. The rate was measured as a function of microtubule concentration and extrapolated to give the maximum rate k5. The same method was used to obtain k5 for ADP by mixing K.ADP with microtubules plus excess mant-ATP. The enhancement of fluorescence for the binding of mant-ATP is followed by a decrease in fluorescence with a rate constant of 35-40 s-1. Since the decrease must occur after hydrolysis, it may be correlated with a step or steps leading to the low fluorescence MtK.D state. In the kinetic scheme, steps 4 and 5 both contribute to determining the maximum turnover rate. At higher ionic strengths or lower protein concentrations, the MtK complex is dissociated by ATP. The maximum rate is 12 +/- 2 s-1 in 50 mM NaCl; consequently, hydrolysis occurs before dissociation. The dissociation constant of MtK in the presence of ADP is twice as large as the dissociation constant in the presence of ATP and four times larger than the KM for microtubule activation. The proposed kinetic scheme, which treats the K379 units of a dimer as independent, provides a satisfactory description of the transient and steady-state properties of the system with the possible exception of results at very low substrate concentrations.

A continuous fluorescence-displacement assay for triacylglycerol lipase and phospholipase C that also allows the measurement of acylglycerols

A new continuous fluorescence-displacement assay for enzymes that release long-chain fatty acids [Wilton (1990) Biochem. J. 266, 435-439] is described in detail for pig pancreatic triacylglycerol lipase. The assay involves the displacement of the highly fluorescent fatty acid probe 11-(dansylamino)undecanoic acid from rat liver fatty acid-binding protein by long-chain fatty acids released as a result of enzyme activity. The assay is surprisingly effective for triacylglycerol lipase, allowing the expression of full activity with low concentrations of substrates in the absence of detergents. The initial rate of decrease in fluorescence is linearly related to enzyme concentration, and activity can be detected in the assay down to concentrations of 10 pg of pure enzyme/ml. The assays demonstrated the quantitative conversion of limiting amounts of substrate into the monacylglycerol. This observation allowed the assay to be used to measure substrates such as triacylglycerols and particularly 1,2-diacylglycerols at concentrations down to about 0.1 microM. Because phospholipase C releases 1,2-diacylglycerols, the coupling of this enzyme to excess lipase allowed the measurement of pure phospholipase C from Bacillus cereus at concentrations down to about 2 ng/ml, and the initial rate of fall in fluorescence in the assay was linearly related to enzyme activity.

The liver cell plasma membrane Ca2+ inflow systems exhibit a broad specificity for divalent metal ions

1. The inflow of Mn2+ across the plasma membranes of isolated hepatocytes was monitored by measuring the quenching of the fluorescence of intracellular quin2, by atomic absorption spectroscopy and by the uptake of 54Mn2+. The inflow of other divalent metal ions was measured using quin2. 2. Under ionic conditions which resembled those present in the cytoplasmic space, Mn2+, Zn2+, Co2+, Ni2+ and Cd2+ each quenched the fluorescence of a solution of Ca2(+)-quin2. 3. The addition of Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ to cells loaded with quin2 caused a time-dependent decrease in the fluorescence of intracellular quin2. Plots of the rate of decrease in fluorescence as a function of the concentration of Mn2+ reached a plateau at 100 microM-Mn2+. 4. The rate of decrease in fluorescence induced by Mn2+ was stimulated by 20% in the presence of vasopressin. The effect of vasopressin was completely inhibited by 200 microM-verapamil. Adrenaline, angiotensin II and glucagon also stimulated the rate of decrease in the fluorescence of intracellular quin2 induced by Mn2+. 5. The rate of decrease in fluorescence induced by Zn2+, Co2+, Ni2+ or Cd2+ was stimulated by between 20 and 190% in the presence of vasopressin or angiotensin II. 6. The rates of uptake of Mn2+ measured by atomic absorption spectroscopy or by using 54Mn2+ were inhibited by about 20% by 1.3 mM-Ca2+o and stimulated by 30% by vasopressin. 7. Plots of Mn2+ uptake, measured by atomic absorption spectroscopy or with 54Mn2+, as a function of the extracellular concentration of Mn2+ were biphasic over the range 0.05-1.0 mM added Mn2+ and did not reach a plateau at 1.0 mM-Mn2+. 8. It is concluded that (i) hepatocytes possess both a basal and a receptor-activated divalent cation inflow system, each of which has a broad specificity for metal ions, and (ii) the receptor-activated divalent cation inflow system is the receptor-operated Ca2+ channel.

A continuous fluorescence displacement assay for the measurement of phospholipase A2 and other lipases that release long-chain fatty acids

1. A new continuous fluorescence assay for phospholipase A2 is described which involves the displacement of the highly fluorescent fatty-acid probe 11-(dansylamino)undecanoic acid from rat liver fatty-acid-binding protein by long-chain fatty acids released as a result of phospholipase A2-catalysed hydrolysis of phospholipids. The initial rate of decrease in fluorescence is linearly related to enzyme activity. 2. The assay will detect enzyme activity down to about 10 pmol/min per ml and gives a linear response up to about 10 nmol/min per ml. 3. The assay will work with all phospholipids that have been tested including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol and phosphatidylglycerol. Substrates carrying a net negative charge showed the highest rates of hydrolysis. 4. The assay will work, in principle, with an enzyme catalysing the release of long-chain fatty acids from a fatty-acylated substrate. This has been confirmed with pancreatic lipase and cholesterol esterase.

Acceleration of the rate of fluorescence decrease by high concentrations of ATP under the condition of accumulation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase.

Addition of up to 300 microM ATP in the presence of 2 M NaCl with MgCl2 to pig kidney Na+,K+-ATPase treated with N-[p-(2-benzimidazolyl)phenyl]maleimide seemed to be insufficient to saturate the rate of the fluorescence decrease. However, both the extent of the decrease and the amount of phosphoenzyme at a steady state were saturated below 20 microM ATP. Addition of Mg2+ with Na+ to the enzyme preincubated with 20 to 600 microM ATP gave nearly the same rate constant, which was below 50% of that obtained by adding 300 microM ATP to the Na+-form enzyme in the presence of Mg2+. High concentrations of ATP affected neither the rate of light-scattering change (Taniguchi, K. et al. (1986) J. Biol. Chem. 261, 3272-3281) after ADP-sensitive phosphoenzyme formation (E1P) nor that of the breakdown of E1P. A stoichiometric amount of [32P]Pi was liberated from [32P]E1P. The data suggested that ATP did not bind to E1P in such a way as to increase the extent of phosphorylation further or to accelerate dephosphorylation. The data also suggested that the reason for the large difference in the apparent affinity of ATP as evaluated from the rate and the extent of fluorescence change is the large dissociation constant for ATP of a Michaelis complex.

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes.

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F-actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.

Kinetic-fluorimetric determination of copper(II), based on its catalytic effect on the oxidation of morin with hydrogen peroxide

A kinetic method is described for the determination of trace amounts of copper(II) based on its catalytic effect on the oxidation of 3,5,7,2′,4′-pentahydroxyflavone (morin) by hydrogen peroxide. The reaction is followed by measuring the rate of decrease in fluorescence (λex.= 463 nm, λem.= 548 nm). The optimum conditions are as follows: pH 10.5 (ammonium chloride-ammonia buffer), 0.005% morin, 20% ethanol, 0.9% hydrogen peroxide and 25 °C. The calibration is linear for concentrations up to 50 ng ml–1 with a precision of 1.7% and the detection limit is 0.4 ng ml–1 of Cu(II). The main interferences are due to Be(II), Mn(II), Zr(IV), Fe(III) and Mn(VII).In the absence of H2O2, higher amounts of Cu(II) catalyse the air oxidation of morin. In this instance, the calibration graph is linear between 150 and 500 ng ml–1.

Anion transport heterogeneity detected by flow cytometric measurement of NBD-taurine efflux kinetics.

NBD-taurine [N-(7-nitrobenzofuran-4-yl) taurine], a fluorescent substrate for the human erythrocyte anion exchange system, has been used to test the feasibility of making flow cytometric measurements of anion transport in K562 erythroleukemic cells. Cells were preloaded by incubation with 20 microM-2mM NBD-taurine, then diluted 10-30-fold, and efflux was monitored by measuring fluorescence intensity (FL) as a function of time using excitation at 488 nm. The observed rate of decrease in fluorescence was sensitive to temperature and also to phloretin, a compound known to inhibit anion transport and other carrier-mediated transport processes. The coefficient of variation (CV) of the fluorescence distribution increased markedly over the efflux period, suggesting heterogeneity of the K562 population with respect to the rate constant for NBD-taurine efflux. This heterogeneity was also reflected in the upward curvature of a first order plot of log (FLt - FL infinity) versus time. Half-times calculated from initial linear portions of the first-order plots were found to decrease as the loading concentration of NBD-taurine was decreased, as predicted for a saturable transport system. NBD-taurine is not an ideal anion transport substrate for flow cytometric studies. It appears to bind to high-affinity sites within the cells with consequent fluorescence quenching, complicating interpretation of kinetic curves at low concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)