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At least 271 records · Page 15Linked to original sources
PubMed1989

Glucose effects on cytoplasmic Ca2+ of individual pancreatic beta-cells recorded by two procedures for dual-wavelength fluorometry.

Two different dual-wavelength microfluorometric systems were used to measure the cytoplasmatic Ca2+ concentration (Cai2+) of individual pancreatic beta-cells loaded with the indicator fura-2. Fluorescence was excited continuously at 340 nm with frequent manual checks at 360 nm. Alternatively, the 340/380 nm fluorescence excitation ratio was recorded based on automated samples at the two wavelengths every 10 msec. Irrespective of the experimental technique a rise of the glucose concentration from 3 to 20 mM resulted in an initial lowering of Cai2+ followed by a rise with a peak and a subsequent sustained intermediary level. The increase of Cai2+ depended on the presence of extracellular Ca2+. In some cells the pattern differed and the increase of the glucose concentration induced large oscillations of Cai2+. Glucose-induced increase of Cai2+ was associated with small rapid fluctuations, which were detectable only with the fast time-sharing approach. The characteristic Cai2+ response to glucose was maintained by some beta-cells after culture for more than 3 weeks. The results indicate that the glucose sensitivity of individual beta-cells varies, and that many electrically coupled cells within an islet collectively determine the characteristic pattern of rhythmic depolarization.

Animals↗
PubMed2006

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry.

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na(+)/P(i) cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na(+), Li(+), and P(i)-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na(+) and Li(+). Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.

Animals↗
PubMed2006

Quantification of algal biofilms colonising building materials: chlorophyll a measured by PAM-fluorometry as a biomass parameter.

The aim of this study was to quantify algal colonisation on anthropogenic surfaces (viz. building facades and roof tiles) using chlorophyll a (chl a) as a specific biomarker. Chl a was estimated as the initial fluorescence F0 of 'dark adapted' algae using a pulse-modulated fluorometer (PAM-2000). Four isolates of aeroterrestrial green algae and one aquatic isolate were included in this study. The chl a concentration and F0 showed an exponential relationship in the tested range between 0 and 400 mg chl a m(-2). The relationship was linear at chl a concentrations <20 mg m(-2). Exponential and linear models are presented for the single isolates with large coefficients of determination (exponential: r2 > 0.94, linear: r2 > 0.92). The specific power of this fluorometric method is the detection of initial algal colonisation on surfaces in thin or young biofilms down to 3.5 mg chl a m(-2), which corresponds to an abundances of the investigated isolates between 0.2 and 1.5 million cells cm(-2).

Biofilms↗
PubMed2004

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry.

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.

Animals↗
PubMed1991

Quantification of nucleic acids on nitrocellulose membranes with time-resolved fluorometry.

We use a streptavidin-based macromolecular complex (SBMC) labelled with the europium chelate of 4,7-bis (chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) as a staining reagent for biotinylated DNA present on nitrocellulose filters. The fluorescent spots or bands obtained can either be observed under UV illumination, photographed by instant camera photography or quantified by using a specially designed instrument working as a high resolution time-resolved fluorometric scanner. The detection limit is approximately 10 pg of target DNA. Various experiments with use of biotinylated DNA probes hybridized to Southern transferred targets have shown that the new procedure is a useful versatile non-isotopic methodology for staining DNA on solid supports.

Bacterial Proteins↗
PubMed2004

New separation-free assay technique for SNPs using two-photon excitation fluorometry.

A new separation-free method for detection of single nucleotide polymorphisms (SNPs) is described. The method is based on the single base extension principle, fluorescently labeled dideoxy nucleotides and two-photon fluorescence excitation technology, known as ArcDia trade mark TPX technology. In this assay technique, template-directed single base extension is carried out for primers which have been immobilized on polymer microparticles. Depending on the sequence of the template DNA, the primers are extended either with a labeled or with a non-labeled nucleotide. The genotype of the sample is determined on the basis of two-photon excited fluorescence of individual microparticles. The effect of various assay condition parameters on the performance of the assay method is studied. The performance of the new assay method is demonstrated by genotyping the SNPs of human individuals using double-stranded PCR amplicons as samples. The results show that the new SNP assay method provides sensitivity and reliability comparable to the state-of-the-art SNaPshot trade mark assay method. Applicability of the new method in routine laboratory use is discussed with respect to alternative assay techniques.

DNA Primers↗
PubMed1994

Comparison of the binding of beta-cyclodextrin and alpha- and gamma-cyclodextrins with pullulanase from Klebsiella pneumoniae as studied by equilibrium and kinetic fluorometry.

The change in fluorescence spectra of crystalline pullulanase from Klebsiella pneumoniae caused by the addition of alpha-, beta-, and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin was investigated at 25 degrees C and pH 5.6. The fluorescence intensity at around 325 nm (excitation at 280 nm) was increased by the addition of all the cyclodextrins studied. The dissociation constant, Kd, of the enzyme-cyclodextrin complex was evaluated by fluorometric titration for each cyclodextrin, and was consistent with the inhibitor constant, Ki, obtained previously [Iwamoto et al. (1993) J. Biochem. 113, 93-96]. The Kd values of beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin were approximately two orders of magnitude smaller than those of alpha- and gamma-cyclodextrins. Fluorescence titration of a cyclodextrin in the presence of another cyclodextrin revealed competition among alpha-, beta-, and gamma-cyclodextrins for binding with the enzyme, which indicates that the binding region of beta-cyclodextrin overlaps those of alpha- and gamma-cyclodextrins. On the other hand, with excitation at 295 nm, a fluorescence spectral change similar to that excited at 280 nm was observed for alpha- and gamma-cyclodextrins and 6-O-alpha-glucosyl-alpha-cyclodextrin, whereas beta-cyclodextrin and 6-O-alpha-glucosyl-beta-cyclodextrin did not show any such change. These results suggest that the binding site or the binding mode of beta-cyclodextrin is slightly different from those of alpha- and gamma-cyclodextrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMedDate not supplied

Tissue viability measurement by in situ fluorometry.

A prototype laser-fiber optic based sensor for in situ monitoring of nicotinamide adenine dinucleotide (NADH) has been developed. This system is based on a compact neodymium-yttrium-aluminum-garnet (Nd:YAG) laser with associated harmonic generators. Light distribution to and from tissue is handled by a fiber optic network, including a long optical fiber to be inserted into the target tissue. Immobilizing the enzyme lactate dehydrogenase on the fiber tip converts the monitoring channel into a lactate sensor. A new dual beam reflection approach for blood volume artifact compensation is tested. Detection sensitivity of free NADH in the micromolar region is achieved. The method and system configuration appear feasible for continuous in situ monitoring of two important indices of ischemia and hypoxia in a clinical setting.

Animals↗
PubMed1983

Evaluation of the burn wound with perfusion fluorometry.

Determination of depth of burn injury using vital dyes has been unsatisfactory. The present study evaluated the ability of the fiberoptic perfusion fluorometer to assess the depth of burn in the early postburn period. Sixty-three burns were examined with the fluorometer after intravenous administration of sodium fluorescein. The fluorescein kinetics were monitored for 1 hour within the first 48 hours and again between the third and sixth days postburn. The rate of fluorescein uptake and burn wound fluorescence was determined and compared to that of normal unburned skin. Depth of burn was confirmed by biopsy and healing characteristics. Fluorometric analysis during both study periods consistently distinguished between partial-thickness and full-thickness burns. Partial-thickness burns uniformly exhibited fluorescence within 10 minutes; full-thickness burns showed nil fluorescence. None of the patients experienced a change in skin color or complications from the small dose of fluorescein given.

Burns↗
PubMed1998

A dual label oligonucleotide ligation assay for detection of the CYP2C19*1, CYP2C19*2, and CYP2C19*3 alleles involving time-resolved fluorometry.

CYP2C19 (S-mephenytoin hydroxylase) is a polymorphically expressed enzyme. Currently, two defective alleles are known--CYP2C19*2 and CYP2C19*3. The authors have developed an oligonucleotide ligation assay to detect these two alleles. This assay combines the hybridization of one common, biotinylated capture probe and two allele-specific probes to the target DNA, with the ability of a DNA ligase to distinguish mismatched nucleotides. The probes are only ligated if they are base paired correctly to the target strand. The biotin is bound to streptavidin, and all DNA not covalently bound to the biotin-labeled capture probe, is removed in a washing procedure. The allele-specific probes are labeled with either europium or samarium, and their emission can be measured simultaneously. The ratio between the emission separates the genotypes. This method was applied on DNA from 19 whites and 21 Vietnamese living in Denmark. All genotypes determined by the assay were consistent with the results from restriction enzyme cleavage. There were 12 poor metabolizers; 10 homozygous CYP2C19*2/CYP2C19*2, one heterozygous CYP2C19*2/CYP2C19*3, and one heterozygous CYP2C19*1/CYP2C19*2. The authors conclude that this assay is well-suited for a high throughput of samples in a routine laboratory. The finding of an apparently heterozygous CYP2C19*1/CYP2C19*2 poor metabolizer, confirms that there are still unknown mutations in CYP2C19.

Alleles↗
PubMed1991

Characterization of yeast strains of the genera candida, Hansenula, Kluyveromyces, Pichia, Rhodotorula, and Saccharomyces by mixed-dye fluorometry.

An analytical method in which we used the selective adsorption of several fluorophores by yeast cells is described. The suitability of using binary mixtures of 1-pyrene butyric acid, 3,6-dimethylamino acridine, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, and rhodamine B isothiocyanate for the characterization and identification of microorganisms was tested with 98 yeast strains belonging to the genera Candida, Hansenula, Kluyveromyces, Pichia, Rhodotorula, and Saccharomyces. The application of multivariate statistical methods and pattern recognition methods to the allocation of the yeast strains into genus-species-strain structures and to a comparison of fluorescence data sets for differentiation and identification purposes showed the usefulness of the method.

Analysis of Variance↗
PubMed2002

Heterogeneity in the structural dynamics of Sulfolobus solfataricus beta-glycosidase revealed by electron paramagnetic resonance and frequency domain fluorometry.

The local and global dynamics of the Sulfolobus solfataricus beta-glycosidase were studied by electron spin resonance and time-resolved fluorescence techniques. For electron paramagnetic resonance (EPR) investigations, the protein was covalently modified by the maleimido nitroxide spin label, which is specific for cysteine -SH groups, at position 344 and at position 101, where Ser-101 was changed into a cysteine by site-directed mutagenesis. The greater reactivity of exposed Cys-101 suggested the exclusive modification of this amino acid compared with Cys-344. The labeled proteins underwent temperature perturbation in the range 290-335 K and the values of the spin-label rotation correlation frequencies (nu(c)) ranged from 6 x 10(7) to 2 x 10(8) sec(-1) for the protein labeled at position C344 and from 5.62 x 10(7) to 1.10 x 10(8) sec(-1) for the protein labeled at C101. These rotation correlation values are related to the local dynamic characteristics of the protein matrix. The temperature dependence of rotation correlation frequencies expressed in terms of Arrhenius coordinates (log (nu(c)) vs. 1/T) for the protein labeled at C344 exhibited a linear dependence but with a change in the slope at 311 K. For the protein labeled at C101, no change in the slope was observed at the same temperature. General dynamic information was deduced from the analysis of the fluorescence emission decay of the tryptophanyl residues that are present in each region of the protein structure. Fluorescence data analysis highlighted a bimodal distribution of fluorescence lifetimes arising from the contribution of two emitting groups: one consisting of closely clustered tryptophans responsible for the long-lived emission component (7.1 nsec) and the other composed of tryptophans nearer to the protein surface, which can be associated to the short-lived component (2.5 nsec). The temperature dependence of lifetime distribution parameters linked to the long-lived and short-lived components, expressed in Arrhenius coordinates, showed two different points in which the change in the slope occurred (i.e., 328 K and 338 K, respectively). The Arrhenius analysis of data provided the activation energy relative to the conformational changes characterizing the local and global movements running through the protein matrix.

Circular Dichroism↗
PubMed1980

Protein mobility inside pyruvate dehydrogenase complexes as reflected by laser-pulse fluorometry. A new approach to multi-enzyme catalysis.

The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme-free complex is compared with complex to which Mg2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 degrees C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipo-amide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.

Azotobacter↗
PubMed1987

Calcium-dependent and -independent release of glutamate from synaptosomes monitored by continuous fluorometry.

An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.

Animals↗
PubMed1969

Identification of group A streptococci by direct fluorometry.

A simple direct fluorometric method for rapid identification of group A streptococci is described. The method permits the detection of the organism in mixed cultures without the aid of a microscope and is amenable to automated processing of specimens. Experience with the indirect fluorometric method revealed that nontrypsinized cells from a 10-fold dilution of overnight broth cultures could be stained with uniform brilliance with fluorescent antibody (1:15 dilution) and that fluorescent antibody dissociated from such cells at 55 C for 20 min gave serologically specific fluorometric values. With this information, it was possible to develop a simpler fluorometric test which gave results comparable to those obtained by conventional cultural-precipitin grouping techniques. In the direct test described, cultures from throat swabs were incubated overnight, and cells from a 10-fold dilution were stained with specific fluorescent antibody (1:50 dilution) and then rinsed. The stained specimens were transferred to a continuous-filter paper strip (Whatman 3 MM) and read serially in a Turner 110 fluorometer with Corning 5840 and Wratten 2A filters in place. The reagents used required careful standardization and testing to assure that fluorometric readings above a specified value would be indicative of the presence of group A streptococci.

Automation↗