Rate Of Fluorescence Increase Research Articles

Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

Use of molecular beacons for the rapid analysis of DNA damage induced by exposure to an atmospheric pressure plasma jet

A rapid method for evaluating the damage caused to DNA molecules upon exposure to plasma is demonstrated. Here, we propose the use of a molecular beacon for rapid detection of DNA strand breaks induced by atmospheric pressure plasma jet (APPJ) irradiation. Scission of the molecular beacon by APPJ irradiation leads to separation of the fluorophore-quencher pair, resulting in an increase in fluorescence that directly correlates with the DNA strand breaks. The results show that the increase in fluorescence intensity is proportional to the exposure time and the rate of fluorescence increase is proportional to the discharge power. This simple and rapid method allows the estimation of DNA damage induced by exposure to a non-thermal plasma.

Abstract 199: Hydrogen Peroxide Mediates the pH Conditioned Chloride Conductance in Nodose Ganglia Neurons

pH sensitivity has been rarely studied in vagal afferents of nodose ganglia (NG) neurons. Using whole-cell patch-clamp technique in isolated NG neurons, we recently identified a pH-conditioned Cl - current (pH-I) that was evoked following 2 or 3 brief (10s) exposures to low extracellular pH (7.0-6.0) in 16 of 22 (70%) cells (FASEB J, 2012). The current is large (904.3±159.9pA) and prolonged, lasting 10∼15 minutes, and causes significant depolarization (Δ35.2±4.4mV). In the present study, we tested the hypothesis that reactive oxygen species (ROS) mediates this pH-conditioned Cl - conductance. We found that the rate of increase in fluorescence [(F-F 0 )/F 0 ] of NG neurons loaded with dihydroethidine (ROS dye) rose dramatically following the brief exposures to pH 6.0 from a control of 0.04±0.01 to 0.12±0.02 units/min over 10∼15 minutes (n=31 neurons, p<0.01). Moreover superfusion of neurons with H 2 O 2 induced currents that mimicked the pH conditioned currents. Because of similarities between the pH-conditioned Cl - conductance and the previously described “swell-activated” Cl - current induced with hypoosmotic solutions, we superfused the NG neurons with the H 2 O 2 scavenger PEG-catalase (1000 units/ml). PEG-catalase blocked significantly (p<0.01) the “swell” response to 210 mOsm from 23.3±6.3pA/pF to 0.57±0.39 pA/pF (n=4) as well as the pH-conditioned response from 25.7±6.5pA/pF (n=6) to 6.9±1.4 pA/pF (n=12). The superoxide scavenger PEG-SOD did not affect the current. These results indicate that both pH-conditioned and swell-induced responses are mediated by H 2 O 2 . Opening of these outward Cl - conductances that cause sustained depolarization of vagal afferents may induce a beneficial reflex sympathoinhibition during myocardial ischemia/acidosis or initiate a gastro-intestinal post-prandial satiety reflex.

长江口锋面附近咸淡水混合对浮游植物生长影响的现场培养

通过2009年6月调查航次,获得了营养盐等参数断面分布,表明咸淡水混合是控制营养盐分布的主要因素。为了解不同盐度梯度下浮游植物生长与营养盐吸收的关系,采集两个站位水样分别代表长江冲淡水(C1站)和外海水(I10站),按C1站水样比例,分100%、75%、50%、25%、0%不同比例混合进行现场模拟咸淡水混合培养,有以下认识:(1)平行结果表明培养过程中活体荧光极大值在100%混合组,且淡水比例越低,指数生长期0-48 h内生长速率越低,100%、75%、50%、25%组分别为1.18/d、1.12/d、1.14/d、0.77/d;(2)低于26盐度的水体中PO₄^3-在48 h内可被迅速消耗而产生限制作用,是控制浮游植物生长的潜在限制因子;(3)除0%组外,各混合组DIN/P(DIN:溶解无机氮,Dissolved Inorganic Nitrogen,DIN=NO₃^-+NO₂^-+NH₄⁺)比值在浮游植物指数生长期有升高趋势,100%组DIN/P比值增加了一倍。各组培养48 h后DIN/Si比值逐渐降低至原来的0.7左右,初始DIN/Si小于一定时间内硅藻吸收的(ΔDIN/ΔSi)比,是造成各组DIN/Si比值减小的原因。以上结果表明咸淡水混合过程中形成的营养盐梯度可造成浮游植物生长程度和速率差异,且可因局部浮游植物旺发而改变海水营养结构。;The distribution of nutrients and salinity recorded during the June 2009 cruise in the Changjiang Estuary indicated that dilution by Changjiang River water and seawater mixing were the main factors controlling nutrient behavior. To better understand the implications of this variation, phytoplankton growth and nutrient uptake along a salinity gradient representing dilution by the Changjiang River and seawater respectively were recorded. Two water samples were collected from the sampling stations C1 and I10 (as used in the June 2009 cruise) which were representative of the freshwater and saline water end-member. After diluting portions of the two samples by 100%, 75%, 50%, 25% and 0% with freshwater to simulate different levels of freshwater-saline water mixing, the samples were incubated for 3 days. The results were as follows: (1) the higher the percentage of freshwater, the faster was initial growth and the higher the <em>in vivo</em> fluorescence concentration. During the 3 day incubation period, the rate of increase in fluorescence and maximum <em>in vivo</em> fluorescence of the 100% dilution treatment were 2.9 μg/L/d and 9.6 μg/L, respectively,whilst the rate of increase in fluorescence and maximum fluorescence of the 25% dilution treatment were 0.54 μg/L/d and 2.0 μg/L respectively. The <em>in vivo</em> fluorescence of the 0% dilution treatment was low,probably due to the low nutrient levels in the offshore seawater. The lower the percentage of freshwater, the lower the growth rate of phytoplankton during the exponential growth period, e.g. 1.18/d, 1.12/d, 1.14/d and 0.77/d for the 100%, 75%, 50% and 25% dilution treatments respectively. (2) NO₃^-, NO₂^-, PO₄^3- and SiO₃^2- were apparently consumed but not NH₄⁺. The extent and rate of consumption of NO₃^- and SiO₃^2- were very similar during the first 48 h; the extent of consumption was lower with lower initial nutrient concentration. PO₄^3- levels in the 100%, 75% and 50% dilution treatments were depleted within 48 h. This suggested that PO₄^3- was the likely limiting factor for phytoplankton growth at salinities below 26. Meanwhile, the initial concentrations of NO₃^-, SiO₃^2- and PO₄^3- in different treatments had significant positive correlations with their uptake rates during the exponential growth period. For NO₃^- the uptake rates for the 100%, 75%, 50%, 25% and 0% dilution treatments were 25.39, 19.24, 12.84, 6.04 and 0.21 μmol/d,respectively. For SiO₃^2-, the uptake rate decreased from 14.34 μmol/d for the 100% dilution treatment to 3.73 μmol/d for the 25% dilution treatment. For PO₄^3-, uptake rates decreased from 0.46 μmol/d for the 100% dilution treatment to 0.02 μmol/d for the 0% dilution treatment. For the same dilution treatment uptake rates of the nutrients could be sequenced as follows: NO₃^->SiO₃^2->PO₄^3-. (3) The DIN/P(DIN:Dissolved Inorganic Nitrogen,DIN=NO₃^-+NO₂^-+NH₄⁺) ratio for all treatments, except for the 0% treatment, increased during the phytoplankton exponential growth period. For the 100% dilution treatment the ratio doubled as PO₄^3- was consumed very rapidly, while DIN decreased slowly. Similarly, the ratio DIN to PO₄^3- consumed in 100%, 75% and 50% dilution treatments was higher between 48 h and 96 h compared to consumption within the first 48 h. This indicated that under sufficient nutrient conditions, phytoplankton absorb N and P with an increasingly greater ratio during the exponential growth period. The DIN/Si ratio decreased to about 0.7 times the original level during the first 48 h of incubation. This reflected the low initial DIN/Si value compared to the diatom uptake ratio (ΔDIN/ΔSi) during the incubation period. The results demonstrated variation in the extent and rate of phytoplankton growth for different freshwater-saline water mixtures, and the resultant nutrient gradient. Such mixing processes may cause local blooms which change nutrient structure, and could result in phytoplankton regime shifts.

Kinetic Analysis of β-Galactosidase and β-Glucuronidase Tetramerization Coupled with Protein Translation

Both β-galactosidase (GAL) and β-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.

Fluorescence imaging and spectroscopy for real-time, in-situ characterization of interactions of free radicals with oil-in-water emulsions

The overall goal of this study was to develop a rapid method based on optical imaging and spectroscopy for real time characterization of interactions of free radicals with oil in water emulsions. The method was based on encapsulation of a lipid soluble, free radical sensitive dye that transforms from a non-fluorescent to a fluorescent state upon reaction with hydroxyl radicals. Using this approach, interaction of hydroxyl radicals generated using the Fenton's reagents in aqueous phase with free radical sensitive dye encapsulated in the oil phase of whey protein isolate (WPI) stabilized emulsion was characterized. The results based on fluorescence imaging and spectroscopy showed a linear increase in fluorescence of free radical sensitive dye during the initial time period (~30min) of incubation with the Fenton's reagents. The results showed a high degree of correlation between spectroscopy and quantitative fluorescence imaging measurements. Increase in fluorescence of free radical sensitive dye was a function of concentration of ferrous ions and hydrogen peroxide. Incorporation of EDTA reduced the rate of increase in fluorescence of the dye due to the chelation of metal ions (p<0.05). The addition of α-tocopherol in the lipid phase also significantly reduced the rate of increase in fluorescence (p<0.05). Lipid oxidation was measured to validate propagation of radicals in the oil phase of the emulsion. The results showed a positive correlation between an increase in fluorescence of the free radical sensitive dye and the level of lipid peroxidation. In summary, the method developed in this study is sensitive for real time quantitative measurement of interactions of free radicals with emulsions.

ATP mediates flow‐induced nitric oxide production in thick ascending limbs

Mechanical stimulation caused by increasing flow induces the release of ATP and other nucleotides. Both luminal flow and extracellular ATP stimulate production of nitric oxide (NO) in the thick ascending limb (TAL). We hypothesized that luminal flow‐induced NO production in TALs is mediated by ATP. We measured NO production in isolated, perfused rat TALs using the fluorescent dye DAF FM. The rate of increase in fluorescence reflects NO accumulation. Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 17±15 to 130±34 arbitrary units (AU)/min (p &lt; 0.04; n=7). When an ATP scavenging system (hexokinase; 10 U/ml plus 5.5 mM glucose) was added to the luminal perfusate and basolateral bath, luminal flow did not increase NO production (control: 32±5; after ATP scavenging: 19±4 AU/min; n=6). When luminal flow was increased with the ATPase apyrase (5 U/ml) in the luminal perfusate and basolateral bath, NO levels did not change significantly (control: 41±12; after apyrase: 59±11; n=6). The P2 receptor antagonist suramin (300 umol/L) in the lumen and bath reduced flow‐induced NO production by 73% (control: 56±11; with suramin: 13±12; n=5). We concluded that ATP mediates flow‐induced NO production in thick ascending limbs via P2 receptors.Support: PO1 HL 090550, RO1 HL 070985

Kinetics of ADP Release From Cycling Cross Bridges In Contracting Skinned Cardiac Muscle Monitored With A Fluorescent Probe

Kinetics of ADP release from cycling cross bridges were studied in Ca activated skinned papillary muscle by displacement of fluorescent ADP bound to the cross bridges in AM*ADP state(s) by non-fluorescent ADP photogenerated from caged ADP. A strip of glycerinated papillary muscle (100um, 2mm) from guinea pig left ventricular in Ca-free solution was loaded with a mixture of non-fluorescent ATP (1mM), fluorescent 3′-amino-deoxy ATP (aminoATP) (50μM) and 5 mM caged ADP in the presence of an ATP back-up system. At the plateau of force at pCa5.8 the muscle was rapidly transferred into the photolysis trough filled with silicone oil and irradiated by a 437 nm laser pulse. Alternatively, a muscle loaded with only fluorescent amino ATP (50μM) was allowed to contract at pCa4.5 in oil and develop rigor with amino ADP bound to the cross bridges. Following photolysis of caged ADP the kinetics of force and fluorescent transients were found markedly different in contracting and rigor muscles. In contracting muscle the force and fluorescence both increased following caged ADP photolysis, while in rigor muscle the photolysis induced an increase in fluorescence, but decrease in force. Kinetics of ADP release estimated by the rate of fluorescence increase was significantly slower in contracting muscle than that in rigor: 2-4 s-1 vs 18-20s-1, suggesting that at least two different AM*ADP states exist during ATP hydrolysis by cycling cross bridge in contracting papillary muscle. Supported by NIH grant R03 AR05 2885 for A.K.

The technique of measuring thrombin generation with fluorogenic substrates: 1. Necessity of adequate calibration

In fluorogenic thrombin generation (TG) measurement the concentrations of thrombin are obtained from the course of fluorescence intensity. Because of fluorescence quenching, in one series of normal plasmas (n = 60), the rate of fluorescence increase at fixed thrombin activity was 70 +/- 13% of that in buffer, in another (n = 139) 75 +/- 8%. Using a calibration factor (CF) measured in buffer therefore underestimates thrombin concentrations in plasma and introduces a source of error. A fixed CF also neglects the 25-35% increase of CF during the experiment and thus distorts the form of the TG curve so that the ETP cannot be determined. Continuous individual calibration (CIC), in which CF is determined continuously in a parallel sample, avoids such systematic errors but adds random error because the thrombin course is calculated from two different measurements. We determined the intra-individual coefficients of variation (CV) of the peak-height and ETP as obtained with CIC to those obtained with a fixed CF measured in buffer. With the fixed CF, the CVs varied between 18% and 49%; with CIC they lowered to 4-7% (n = 5 x 12), i.e. in a range allowing clinical application. It is shown that CIC can be discarded for the measurement of peak thrombin values and replaced by comparison to a reference plasma only if quenching is not a systematic confounder. This was shown to be the case in the set of 139 normal plasmas but not in the set used for determining the intraindividual CVs, a difference that may depend upon preanalytical conditions.

Fluorescence-Based Adenylyl Cyclase Assay Adaptable to High Throughput Screening

The second messenger cAMP has been implicated in numerous cellular processes such as glycogen metabolism, muscle contraction, learning and memory, and differentiation and development. Genetic evidence suggests that the enzyme that produces cAMP, adenylyl cyclase (AC), may be involved in pathogenesis in many of these cellular processes. In addition, these data suggest that membrane-bound ACs may be valuable targets for therapeutics to treat pathogenesis of these processes. The development of a robust real-time adenylyl cyclase assay that can be scalable to high-throughput screening could help in the development of novel therapeutics. Here we report a novel fluorescence-based cyclase assay using Bodipy FL GTPgammaS (BGTPgammaS). The fluorescence of the Bodipy moiety of BGTPgammaS was dramatically enhanced by incubation with the minimal catalytic core of wild-type-AC (wt-AC) and a mutant with decreased purine selectivity (mut-AC), in an AC activation-dependent manner. No increase in fluorescence was observed using Bodipy FL ATPgammaS (BATPgammaS) as substrate for either wt-AC or mut-AC. Using BGTPgammaS, forskolin, Gsalpha.GTPgammaS and the divalent cation Mn(2+) potently enhanced the rate of fluorescence increase in a concentration-dependent manner. The fluorescence enhancement of the Bodipy moiety was inhibited by known inhibitors of AC such as 2'deoxy,3'AMP and 2',5'-dideoxy-3'ATP. Furthermore, the fluorescence assay is adaptable to 96-well and 384-well multiplate format and is thus applicable to high throughput screening methodologies.

Ca 2+-Dependent Excitation-Contraction Coupling Triggered by the Heterologous Cardiac/Brain DHPR β2a-Subunit in Skeletal Myotubes

Molecular determinants essential for skeletal-type excitation-contraction (EC) coupling have been described in the cytosolic loops of the dihydropyridine receptor (DHPR) α1S pore subunit and in the carboxyl terminus of the skeletal-specific DHPR β1a-subunit. It is unknown whether EC coupling domains present in the β-subunit influence those present in the pore subunit or if they act independent of each other. To address this question, we investigated the EC coupling signal that is generated when the endogenous DHPR pore subunit α1S is paired with the heterologous heart/brain DHPR β2a-subunit. Studies were conducted in primary cultured myotubes from β1 knockout (KO), ryanodine receptor type 1 (RyR1) KO, ryanodine receptor type 3 (RyR3) KO, and double RyR1/RyR3 KO mice under voltage clamp with simultaneous monitoring of confocal fluo-4 fluorescence. The β2a-mediated Ca 2+ current recovered in β1 KO myotubes lacking the endogenous DHPR β1a-subunit verified formation of the α1S/ β1a pair. In myotube genotypes which express no or low-density L-type Ca 2+ currents, namely β1 KO and RyR1 KO, β2a overexpression recovered a wild-type density of nifedipine-sensitive Ca 2+ currents with a slow activation kinetics typical of skeletal myotubes. Concurrent with Ca 2+ current recovery, there was a drastic reduction of voltage–dependent, skeletal-type EC coupling and emergence of Ca 2+ transients triggered by the Ca 2+ current. A comparison of β2a overexpression in RyR3 KO, RyR1 KO, and double RyR1/RyR3 KO myotubes concluded that both RyR1 and RyR3 isoforms participated in Ca 2+-dependent Ca 2+ release triggered by the β2a-subunit. In β1 KO and RyR1 KO myotubes, the Ca 2+-dependent EC coupling promoted by β2a overexpression had the following characteristics: 1), L-type Ca 2+ currents had a wild-type density; 2), Ca 2+ transients activated much slower than controls overexpressing β1a, and the rate of fluorescence increase was consistent with the activation kinetics of the Ca 2+ current; 3), the voltage dependence of the Ca 2+ transient was bell-shaped and the maximum was centered at ∼+30 mV, consistent with the voltage dependence of the Ca 2+ current; and 4), Ca 2+ currents and Ca 2+ transients were fully blocked by nifedipine. The loss in voltage-dependent EC coupling promoted by β2a was inferred by the drastic reduction in maximal Ca 2+ fluorescence at large positive potentials (Δ F/ F max) in double dysgenic/ β1 KO myotubes overexpressing the pore mutant α1S (E1014K) and β2a. The data indicate that β2a, upon interaction with the skeletal pore subunit α1S, overrides critical EC coupling determinants present in α1S. We propose that the α1S/ β pair, and not the α1S-subunit alone, controls the EC coupling signal in skeletal muscle.

Improving the accuracy of genetic diagnosis from single cells through analysis of real-time PCR signal kinetics

Improving the accuracy of genetic diagnosis from single cells through analysis of real-time PCR signal kinetics

Rate Determination in Phosphorylation of Shark Rectal Na,K-ATPase by ATP: Temperature Sensitivity and Effects of ADP

Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na + was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E 2-P, which follows formation of E 1-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k obs ∼90 s −1 and a minor slow phase with a k obs of ∼9 s −1 (20°C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of ∼3.1 × 10 7 M −1 and 1.8 × 10 5 M −1, respectively. Increasing [ADP] decreased k obs for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E 1-P followed by a conformational change to E 2-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K + the highly fluorescent E 2-P is slowly transformed into the “K +-insensitive” E′ 2-P subconformation forming a side branch of the main cycle.

A Rapid Homogeneous Fluorescence Assay for Subtilisin

Abstract A rapid and sensitive homogeneous assay method has been developed for the determination of subtilisin. The method employs a protein substrate labelled with two fluorescent dyes with fluorescence energy transfer (FET) characteristics. The doubly-labelled substrate was prepared by chemically coupling bovine serum albumin with lucifer yellow and rhodamine dyes. The fluorescence emission from the lucifer labels was initially quenched due to the FET to the adjacent rhodamine labels. However, upon the addition of subtilisin into the labelled substrate solution, increased fluorescence was observed as the enzyme hydrolyzed the substrate and reduced the FET effect. The rate of increase in fluorescence due to substrate hydrolysis was used to calibrate the subtilisin assay. It was linear over the range 0–150 ng of the enzyme (n=8, r2=0.985). The assay was fast with a time of 30 sec to exceed the limit of detection (LOD) signal for 60 ng of subtilisin in 600 μl. In this volume, the LOD for the enzyme was 4.2...

Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5′[γ-thio]triphosphate

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631-642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]0) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]0. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 microM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]1 and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281-309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5'-[beta-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes: (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.

Dynamics of Lactose Permease of Escherichia coli Determined by Site-Directed Fluorescence Labeling

Recently we described the use of site-directed pyrene labeling of engineered lactose permease containing paired Cys residues to obtain proximity relationships between helices in the C-terminal half of the molecule [Jung, K., Jung, H., Wu, J., Privé, G. G., & Kaback, H.R. (1993) Biochemistry 32, 12273]. Pyrene excimer fluorescence was detected for the double Cys mutants His322-->Cys/Glu325-->Cys, Arg302-->Cys/Glu325-->Cys, and Glu269-->Cys/His322-->Cys, indicating that helix X (His322-->Cys/Glu325-->Cys) is in an alpha-helical conformation and that helices VIII (Glu269-->Cys) and IX (Arg302-->Cys) are close to helix X (His322-->Cys and Glu325-->Cys). In this report, these interactions are used to study dynamic aspects of the permease. Excimer fluorescence between helices VIII and X or helices IX and X is markedly diminished by sodium dodecyl sulfate, while the excimer observed within helix X is unaffected, suggesting that tertiary interactions are disrupted by the denaturant with little effect on secondary structure. Furthermore, excimer fluorescence observed between helices VIII (Glu269-->Cys) and helix X (His322-->Cys) is quenched by Tl+, and the effect is markedly and specifically attenuated by ligands of the permease, suggesting that the pyrene becomes less accessible to the aqueous phase. The reactivity of single Cys residues at positions 269 or 322 was also examined by studying the rate of increase in fluorescence with N-(l-pyrenyl)maleimide. With both mutants, ligands of the permease cause a dramatic increase in reactivity which is consistent with the notion that these positions are transferred into a more hydrophobic environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu 2+ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu 2+, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu 2+, i.e., at relatively lowver oxidative stress. At 5 μM Cu 2+, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.

Mg 2+ and ATP or adenosine 5′-[γ-thio]-triphosphate (ATPγS) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase

ATP and Mg 2+ caused a transient increase in the intrisinc fluorescence of Rubisco activase which was inhibited by the presence of ADP. Only minor changes in fluorescence were observed with ATP or Mg 2+ alone. The fluorescence increase was stabilized by addition of an ATP regenerating system or by substitution of ATP with a non-hydrolyzable analog, adenosine 5′-[γ-thio]-triphosphate (ATPγS). The initial rate of increase in fluorescence also depended on the concentration of protein in a manner consistent with second-order kinetics. The concentration dependence for the effect of ATPγS was sigmoidal, although at pH 8 the half-saturation requirements for both ATPγS (12 μM) and Mg 2+ (1.5 mM) were not too dissimilar to the binding affinities (6 μM and 2 mM, respectively) determined indirectly with the fluorescent probe, 1-anilinonapthalene-8-sulfonate. However, the concentration dependence of ATP was about 5-fold higher than its binding affinity, also sigmoidal and quite similar to the concentration responses of ATP hydrolysis and activation of Rubisco by the protein. These characteristics of the intrinsic fluorescence indicate that it monitors a conformational change in the protein occurring after binding of the nucleotide and associated with increased aggregation. Direct evidence of increased aggregation in the presence of Mg 2+ and ATP or ATPγS was obtained by gel-filtration chromatography. However, the apparent molecular mass was heterogenous and also varied with temperature. The increased aggregation of the protein resulted in altered kinetic properties. The ATP hydrolysis activity of the protein increased and the half-maximal ATP concentration decreased as the protein concentration was increased in the assay. Also, a brief pretreatment of the protein with ATP and Mg 2+ to increase aggregation eliminated the otherwise observed time delays in the Rubisco activation and ATP hydrolysis kinetics.

Fluorometric Analysis of Alkaline Phosphatase in Fluid Dairy Products

A new quantitative assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products including whole milk, low fat and skim milks, chocolate milk, and creams. ALP in the test sample hydrolyzes a nonfluorescent substrate, FluorophosR, to a highly fluorescent product. Product formation is monitored continuously during a short incubation period and enzyme activity is calculated from the rate of fluorescence increase. Total test time is 3 min. Reaction rates are linear up to 0.5% raw milk (equivalent to 5 μg phenol/ml/15 min) with a detection limit of 0.006% raw milk. Within and between run precision of the fluorometric method was assessed by repeated analysis of a pasteurized milk sample containing added mixed herd raw milk. The within run (N=10) mean was 190.4 mU/L, standard deviation (SD) 3.2, and a coefficient of variance (CV) of 1.7%. The procedure provides a rapid, sensitive, precise, and easy-to-use ALP assay, applicable to a wide variety of dairy products.

Amylase substrate based on fluorescence energy transfer

A fluorigenic substrate for measuring α-amylase (E.C. 3.2.1.1) activity was prepared by double labeling soluble starch with 5-(4,6-dichlorotrizain-2-yl)aminofluorescein and Procion Red MX8B. Because the absorption spectrum of Procion Red MX8B overlaps the fluorescein emission spectrum, Procion Red efficiently quenches fluorescein emission when it is closer than the critical radius for fluorescence energy transfer. When amylase catalyzes cleavage of a starch molecule between a fluorescein and a Procion Red MX8B, the distance between the two labels increases and the degree of quenching decreases. The rate at which the fluorescence intensity increases is proportional to amylase activity. To maximize the sensitivity it is critical to maximize the amount of Procion Red MX 8B coupled to the starch and to use a high-precision spectrofluorimeter which can measure a small rate of increase in fluorescence above a large constant background.