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At least 595 records · Page 33Linked to original sources
PubMed2002

Cold vapour atomic fluorescence spectrometry and gas chromatography-pyrolysis-atomic fluorescence spectrometry for routine determination of total and organometallic mercury in food samples.

Two procedures have been investigated for the quantification of the different forms of mercury in food. A two-stage procedure has been developed to determine firstly total inorganic and organometallic species, and then the full separation of all organomercury species. The procedure involves solubilisation of the samples using alkaline extractions or enzymolysis, followed by the extraction of organic mercury in an organic solvent, preferably a mixture of dichloromethane and hexane (3:2). For the total organic mercury determination, the organic extract is analysed for "total" mercury after nitric acid/peroxide digestion, evaporation of the solvent and detection by cold vapour-atomic fluorescence spectrometry. Full organomercury speciation requires a clean-up step before analysis of the final extract in dichloromethane by gas chromatography coupled to a pyrolyser and an atomic fluorescence detector (GC-pyro-AFS). A detection limit of 6 ng l-1, and reproducibility of 2% was achieved for the CV-AFS method; GC-pyro-AFS yielded 200 ng l-1 and 5% for detection limit and coefficient of variation, respectively. Both procedures were validated with the use of various certified reference materials over a wide range of mercury concentrations, and by spiking experiments. The validated methods were tested successfully on a wide range of commercially available food samples.

Chromatography, Gas↗
PubMed2000

Involvement of natural killer cells in patients with myelodysplastic syndrome carrying monosomy 7 revealed by the application of fluorescence in situ hybridization to cells collected by means of fluorescence-activated cell sorting.

Monosomy 7 is the most frequent chromosome abnormality among patients with secondary myelodysplastic syndrome (MDS). We used fluorescence in situ hybridization (FISH) and fluorescence-activated cell sorting (FACS) in order to clarify the lineage involvement. Four patients, three with de novo MDS and one with secondary MDS, were enrolled in this study. Monosomy 7 was observed in pluripotent stem cells (CD34(+)Thy-1(+)), and in B (CD34(+)CD19(+)) and T/natural killer (NK) progenitors (CD34(+)CD7(+)). The number of abnormal cells of B (CD19(+)) and T (CD3(+)) cells was below the cut-off value, but approximately 60% of the NK cells (CD3-CD56(+)) contained monosomy 7 in three of the patients.

Aged↗
PubMed1998

Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells.

Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.

Animals↗
PubMed1991

Complex formation between chlorophyll a and cytochrome c: surface properties at the air-water interface. Absorbance, fluorescence and fluorescence-lifetime in Langmuir-Blodgett films.

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (II), surface potential (delta V) and [14C]cytochrome c surface-concentration (gamma) isotherms were measured versus molecular area (sigma) in mixed films. Compared to the successive-addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN.m-1. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression-expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of [14C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir-Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a-cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energy-transfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.

Amino Acids↗
PubMedDate not supplied

Ability of laser fluorescence device associated with fluorescent dyes in detecting and quantifying early smooth surface caries lesions.

A laser fluorescence (LF) device is a portable tool, but it does not measure minor mineral changes. Our in vitro study aim is to propose the association of an LF with two fluorescent dyes and to evaluate the performance in detecting and quantifying early demineralization. Artificial caries lesions are created in 40 primary canine teeth using a demineralizing solution (pH=4.8) for 12, 24, 48, and 96 h. LF measurements are performed with DIAGNOdent after demineralization in these samples and in 20 sound primary teeth. Measurements with LF with 0.2-mM tetrakis(N-methylpyridyl)porphyrin (LF TMPyP) and with 4-mM protoporphyrin IX (LF PPIX) are made. The amount of calcium loss is determined by atomic emission spectrometry. A correlation between LF and LF with dyes and mineral loss and receiver operating characteristics analysis are performed, as well as comparisons of sensitivity, specificity, and accuracy values. Significant correlation is obtained with LF TMPyP and mineral loss of lesions demineralized for 24, 48, and 96 h. Better performance is achieved with LF TMPyP for all parameters than with LF alone. LF PPIX does not present good results. In conclusion, LF TMPyP provides good performance in detecting and quantifying very early enamel caries lesions.

Cuspid↗
PubMed2004

Visualization of differential gene expression by improved cyan fluorescent protein and yellow fluorescent protein production in Bacillus subtilis.

The distinguishable cyan and yellow fluorescent proteins (CFP and YFP) enable the simultaneous in vivo visualization of different promoter activities. Here, we report new cloning vectors for the construction of cfp and yfp fusions in Bacillus subtilis. By extending the N-terminal portions of previously described CFP and YFP variants, 20- to 70-fold-improved fluorescent-protein production was achieved. Probably, the addition of sequences encoding the first eight amino acids of the N-terminal part of ComGA of B. subtilis overcomes the slow translation initiation that is provoked by the eukaryotic codon bias present in the original cfp and yfp genes. Using these new vectors, we demonstrate that, within an isogenic population of sporulating B. subtilis cells, expression of the abrB and spoIIA genes is distinct in individual cells.

Bacillus subtilis↗
PubMed1982

Comparison of an enzyme-linked immunoassay and a quantitative indirect fluorescent-antibody test with the conventional indirect fluorescent-antibody test for detecting antibodies to Toxoplasma gondii.

Two new methods for the detection of antibodies to Toxoplasma gondii, an enzyme-linked immunosorbent assay and a quantitative immunofluorescence assay, were evaluated and compared with the conventional indirect fluorescent-antibody slide test. Each of 100 human sera was assayed twice by the three procedures. Both the enzyme-linked immunosorbent assay and the quantitative immunofluorescence assay correlated well with serologically positive (indirect fluorescent-antibody titer greater than or equal to 1:32) and negative sera. The enzyme-linked immunosorbent assay was more specific, but less sensitive, than the quantitative immunofluorescence assay. However, the quantitative immunofluorescence assay was more reproducible and more rapid than the enzyme-linked immunosorbent assay.

Animals↗
PubMed1984

Retrospective examination of lung tissue specimens for the presence of Legionella organisms: comparison of an indirect fluorescent-antibody system with direct fluorescent-antibody testing.

Although direct fluorescent-antibody (DFA) testing has been used successfully for a number of years to detect legionellae in clinical specimens, the number of known species and serogroups of Legionella has now increased to such an extent that the performance of DFA testing for all serological variants is impractical. Lung homogenates that were submitted to the Centers for Disease Control, Atlanta, Ga., from patients with suspected legionellosis, from November 1977 through May 1982, were originally screened by DFA testing. In our study 498 of these lung homogenates were screened by indirect fluorescent-antibody (IFA) testing, using a panvalent antiserum pool containing antibodies to 25 serological variants of Legionella spp. Fluorescein isothiocyanate-labeled goat anti-rabbit immunoglobulin was used as the second antibody of the sandwich system. For positive homogenates, i.e., those containing Legionella organisms, species and serogroup identification was made by IFA staining with polyvalent serum pools and then with monovalent antiserum. Of the 498 homogenates screened, 39 (7.8%) were positive by IFA testing. Four (0.8% of total; 10.3% of positive homogenates) of these had previously been negative by DFA testing, but subsequent testing showed that they contained Legionella organisms for which DFA reagents were not available at the initial screening. All specimens that were positive by DFA testing were also positive by IFA testing. IFA testing with polyvalent antisera is a simple, efficient method which is at least as sensitive as DFA testing and which can be used by clinical laboratories to cope with the increasing number of known serological variants of Legionella spp.

Antibodies, Bacterial↗
PubMed1977

Comparison of rabies humoral antibody titers in rabbits and humans by indirect radioimmunoassay, rapid-fluorescent-focus-inhibition technique, and indirect fluorescent-antibody assay.

Rabies humoral antibodies were induced in eight New Zealand rabbits by a single intramuscular injection of inactivated suckling mouse brain rabies vaccine. The primary response to immunization was measured in blood samples taken at selected intervals for 6 months. The anamnestic response was measured in blood samples obtained 2 weeks after the rabbits received a booster immunization. The humoral antibody concentrations were measured by the rapid-fluorescent-focus-inhibition technique (RFFIT), indirect fluorescent-antibody assay (IFA), and indirect radioimmunoassay (RIA). The maximal neutralizing antibody titers as measured by RFFIT were attained by the 4th week and persisted into the 24th week. After booster immunization the antibody response was almost 10-fold higher than the highest level attained in the primary response. The antibody levels as measured by IFA and RIA were similar, but the titers as measured by either procedure were almost 10-fold lower than those determined by RFFIT. After booster immunizations the antibody levels, as measured by IFA and RIA, were three- and sixfold higher, respectively, than the maximal levels attained in the primary response. Twenty-two human serum specimens were tested by the same serological procedures, with disparate results. Both RIA and RFFIT effectively differentiated antirabies-positive sera from antirabies-negative sera.

Animals↗
PubMed2005

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats.

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.

Cyclic AMP-Dependent Protein Kinases↗
PubMed2003

Viable cell detection by the combined use of fluorescent glucose and fluorescent glycine.

The combined use of a fluorescent glucose (2NBDG) and a fluorescent glycine (NBD-Gly) was tried for the detection of viable cells of significant foodborne pathogenic strains in addition to several Escherichia coli strains and coliforms. Thirty-five out of 41 strains showed marked uptake of 2NBDG but 6 strains were not able to take in 2NBDG. Five out of these 6 strains showed NBD-Gly uptake.

4-Chloro-7-nitrobenzofurazan↗
PubMed1976

Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).

The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.

Affinity Labels↗
PubMedDate not supplied

The fluorescence mechanism of 5-methyl-2-pyrimidinone: an ab initio study of a fluorescent pyrimidine analog.

The photophysically important potential energy surfaces of the fluorescent pyrimidine analog 5-methyl-2-pyrimidinone have been explored using multireference configuration-interaction ab initio methods at three levels of dynamical correlation, all of which support a fluorescence mechanism. At vertical excitation S1 (dark, n(N)pi*) and S2 (bright, pipi*) are almost degenerate at 4.4 eV, with S3 (dark, n(O)pi*) at 5.1 eV. The excited system can follow the S1-S2 seam of conical intersections, accessible from the Franck-Condon region, to its minimum and then evolve from this conical intersection on the S1 (pipi*) surface to a global minimum. At lower levels of correlation, the S1 surface shows two minima separated by a barrier of up to 0.18 eV. The secondary minimum found at the lower levels of correlation becomes the global minimum with higher correlation. The S1 population at this minimum can be trapped from accessing the lowest energy S0-S1 (pipi*/gs) conical intersection by an energy gap at least 0.3-0.4 eV higher than the S1 minimum. The calculated emission energy from this minimum is 2.80 eV. Gradient pathways connecting important S1 geometries are presented, as well as other excited state conical intersections.

Fluorescence↗
PubMed2005

Determination of the termination efficiency of the transcription terminator using different fluorescent profiles in green fluorescent protein mutants.

An approach in determining the intrinsic termination efficiency (%T) of transcription termination using green fluorescent protein (GFP) mutants was developed. This approach utilizes a cassette vector in which the tested terminator is introduced between two GFP mutant genes: an ultraviolet-optimized mutant (GFPuv: F99S, M153T, V163A) and a blue-shifted mutant (BFP: F64L, S65T, T145F). The ratio of the fluorescence intensity of BFP to GFPuv after transcription and translation represents the termination efficiency of the terminator. E. coli ribosomal RNA operon T1 terminator, phage lambda terminator site R2, E. coli tryptophane attenuater were introduced into the vector, and their transcriptional efficiencies were estimated as 89, 79, and 24%, respectively, showing good agreement with published data.

Base Sequence↗
PubMed2000

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting.

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8. Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.

Cell Lineage↗
PubMed2002

Fluorescence quenching of dimeric and monomeric forms of yeast hexokinase (PII): effect of substrate binding steady-state and time-resolved fluorescence studies.

Fluorescence quenching studies on the PII isoenzyme of yeast hexokinase have been performed using charged as well as polar uncharged quenchers. In both 'open' (i.e. in the absence of glucose) and 'closed' (i.e. in the presence of glucose) forms of the enzyme, bimolecular quenching rate constant (kq) for acrylamide is significantly larger than that of KI, indicating that all the tryptophans are not fully exposed to the solvent. Overall accessibility of tryptophans towards KI was greater in the presence of glucose than in the absence of glucose. At high ionic strength, the value of bimolecular quenching rate constant (kq) for KI did not change suggesting that the average environment of the accessible tryptophan residue(s) is almost neutral. Quenching by KI is dynamic in nature. Accessibility of tryptophans towards acrylamide at concentration > or = 0.2 M was more in the 'open' form of the enzyme than that observed in the 'closed' form whereas at concentration < or = 0.2 M no significant difference in the extent of quenching was observed. It is reasonable to conclude that glucose induced conformational change leads some tryptophan residue(s) to be more exposed and at the same time some tryptophan residue(s) in the hydrophobic region become more buried. Dimeric and monomeric forms of the enzyme behave similarly towards the quenching by acrylamide. In the unfolded state, the accessibility of tryptophans was considerably higher for both the quenchers. Temperature dependent study and the fluorescence lifetime data indicate that the mechanism of quenching by acrylamide is primarily dynamic in nature.

Acrylamide↗
PubMed2004

Determination of protein phosphorylation and the translocation of green fluorescence protein-extracellular signal-regulated kinase 2 by capillary electrophoresis using laser induced fluorescence detection.

In this study, we developed a method to monitor the phosphorylation and translocation of the extracellular signal-regulated kinase (ERK2) proteins after PC12 cells have been stimulated by a mitogen. The method involves the use of green fluorescent protein (GFP), capillary electrophoresis and the measurement of laser-induced fluorescence (CE-LIF). We showed the prescence of the non-phosphorylated GFP-ERK2 and phosphorylated GFP-ERK2 in cell lysates by CE-LIF, and then compared the phosphorylations of GFP-ERK2 and GFP-183A. Phosphorylated GFP-ERK2 was detected at 6.7 min and the non-phosphorylated GFP-ERK2 at 5.3-5.5 min. The results were compared with confocal laser scanning microscope imaging and western blot results, and suggest that the developed method can be used to detect other enzymatic modifications.

Animals↗