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Fluorescence Intensity (fluorescence + intensity)
Kinds of Fluorescence Intensity Selected AbstractsBortezomib as the Sole Post-Renal Transplantation Desensitization Agent Does Not Decrease Donor-Specific Anti-HLA AntibodiesAMERICAN JOURNAL OF TRANSPLANTATION, Issue 3 2010R. Sberro-Soussan Persistence of donor-specific anti-HLA antibodies (DSA) associated with antibody-mediated graft injuries following kidney transplantation predicts evolution toward chronic humoral rejection and reduced graft survival. Targeting plasma cells, the main antibody-producing cells, with the proteasome inhibitor bortezomib may be a promising desensitization strategy. We evaluated the in vivo efficacy of one cycle of bortezomib (1.3 mg/m2× 4 doses), used as the sole desensitization therapy, in four renal transplant recipients experiencing subacute antibody-mediated rejection with persisting DSA (>2000 [Mean Fluorescence Intensity] MFI). Bortezomib treatment did not significantly decrease DSA MFI within the 150-day posttreatment period in any patient. In addition, antivirus (HBV, VZV and HSV) antibody levels remained stable following treatment suggesting a lack of efficacy on long-lived plasma cells. In conclusion, one cycle of bortezomib alone does not decrease DSA levels in sensitized kidney transplant recipients in the time period studied. These results underscore the need to evaluate this new desensitization agent properly in prospective, randomized and well-controlled studies. [source] Simultaneous quantification of cell motility and protein-membrane-association using active contoursCYTOSKELETON, Issue 4 2002Dirk Dormann Abstract We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns. Cell Motil. Cytoskeleton 52:221,230, 2002. © 2002 Wiley-Liss, Inc. [source] The differentiation of biodegradable and non-biodegradable dissolved organic matter in wastewaters using fluorescence spectroscopyJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 8 2002M Reynolds Abstract The chemical and biochemical oxygen demand values of a number of synthetic and wastewater samples were determined using fluorescence spectroscopy. Treated and untreated sewage samples were obtained from a local sewage treatment works while synthetic samples were analysed before, during, and after treatment via a rotating biodisc contactor. Fluorescence intensities were normalised using the water Raman signal as an internal standard and corrections applied to take into account the attenuation effects caused by the sample matrix. The fluorescence emission spectra (,exc,=,280,nm) of synthetic and sewage samples were very similar in that two main fluorescence bands centred around 350,nm and 440,nm were observed in all samples. Normalised fluorescence data, centred at 350,nm, correlate well with corresponding BOD, COD and TOC values (R2 values ranging between 0.93 and 0.98). Using BOD, COD and TOC data the fluorescence at 350,nm and 440,nm can be apportioned to biodegradable and non-biodegradable dissolved organic matter respectively. The findings of this research show that fluorescence data can be used to quantify oxygen demand values (chemical and biochemical) and total organic carbon values. Furthermore, the fluorescence spectral response can be apportioned to biodegradable (BOD) and non-biodegradable (COD,,,BOD) dissolved organic matter. The potential of using fluorescence spectroscopy as a possible tool for real-time monitoring of sewage wastes is discussed. © 2002 Society of Chemical Industry [source] Ethanol Alters Cell Fate of Fetal Human Brain-Derived Stem and Progenitor CellsALCOHOLISM, Issue 9 2010Sharada D. Vangipuram Background:, Prenatal ethanol (ETOH) exposure can lead to fetal alcohol spectrum disorder (FASD). We previously showed that ETOH alters cell adhesion molecule gene expression and increases neurosphere size in fetal brain-derived neural stem cells (NSC). Here, our aim was to determine the effect of ETOH on the cell fate of NSC, premature glial-committed precursor cells (GCP), and premature neuron-committed progenitor cells (NCP). Methods:, NSC, GCP, and NCP were isolated from normal second-trimester fetal human brains (n = 3) by positive selection using magnetic microbeads labeled with antibodies to CD133 (NSC), A2B5 (GCP), or PSA-NCAM (NCP). As a result of the small percentage in each brain, NSC were cultured in mitogenic media for 72 hours to produce neurospheres. The neurospheres from NSC and primary isolates of GCP and NCP were used for all experiments. Equal numbers of the 3 cell types were treated either with mitogenic media or with differentiating media, each containing 0 or 100 mM ETOH, for 120 hours. Expression of Map2a, GFAP, and O4 was determined by immunoflourescence microscopy and western blot analysis. Fluorescence intensities were quantified using Metamorph software by Molecular Devices, and the bands of western blots were quantified using densitometry. Results:, ETOH in mitogenic media promoted formation of neurospheres by NSC, GCP, and NCP. Under control conditions, GCP attached and differentiated, NSC and NCP formed neurospheres that were significantly smaller in size than those in ETOH. Under differentiating conditions, Map2a expression increased significantly in NSC and GCP and reduced significantly in NCP, and GFAP expression reduced significantly in GCP and NCP, and Gal-C expression reduced significantly in all 3 cell types in the presence of ETOH compared to controls. Conclusions:, This study shows that ETOH alters the cell fate of neuronal stem and progenitor cells. These alterations could contribute to the mechanism for the abnormal brain development in FASD. [source] Saponaria officinalis karyology and karyotype by means of image analyzer and atomic force microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2008S. Di Bucchianico Abstract The aim of this work was to offer a contribution to the characterization of taxonomic entity of Saponaria officinalis (2n = 28; an herbaceous perennial species; saporin, a type 1 Ribosome Inactivating Protein, is present in leaves and seeds) by a cytogenetic and karyomorphological approach. We investigated the karyotype's morphometry correlated with Stebbin's symmetric index; the same information has been used for computing the indices resemblance between chromosomes (REC), symmetric indices (SYI), and total form (TF%) which allow the comparison between species and evaluation of karyological evolution. Fluorescence intensities of the stained nuclei were measured by a flow cytometer and, for the first time, values for nuclear DNA content were estimated by comparing nuclei fluorescence intensities of the test population with those of appropriate internal DNA standards. Our study is also aimed to introduce chromosomal volumes, which were determined by atomic force microscopy (AFM), as novel karyomorphological parameter which could allow for chromosome discrimination especially when tiny ones are present. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc. [source] Fluorescence lifetime imaging of activatable target specific molecular probesCONTRAST MEDIA & MOLECULAR IMAGING, Issue 1 2010Raphael Alford Abstract In vivo optical imaging using fluorescently labeled self-quenched monoclonal antibodies, activated through binding and internalization within target cells, results in excellent target-to-background ratios. We hypothesized that these molecular probes could be utilized to accurately report on cellular internalization with fluorescence lifetime imaging (FLI). Two imaging probes were synthesized, consisting of the antibody trastuzumab (targeting HER2/neu) conjugated to Alexa Fluor750 in ratios of either 1:8 or 1:1. Fluorescence intensity and lifetime of each conjugate were initially determined at endosomal pHs. Since the 1:8 conjugate is self-quenched, the fluorescence lifetime of each probe was also determined after exposure to the known dequencher SDS. In vitro imaging experiments were performed using 3T3/HER2+ and BALB/3T3 (HER2,) cell lines. Changes in fluorescence lifetime correlated with temperature- and time-dependent cellular internalization. In vivo imaging studies in mice with dual flank tumors [3T3/HER2+ and BALB/3T3 (HER2,)] detected a minimal difference in FLI. In conclusion, fluorescence lifetime imaging monitors the internalization of target-specific activatable antibody,fluorophore conjugates in vitro. Challenges remain in adapting this methodology to in vivo imaging. Copyright © 2010 John Wiley & Sons, Ltd. [source] A fluorescence study on swelling of hydrogels (PAAm) at various cross-linker contentsADVANCES IN POLYMER TECHNOLOGY, Issue 4 2009Demet Kaya Akta Abstract Disk-shaped acrylamide (AAm) gels were prepared from AAm with various N,N,-methylenebisacrylamide (Bis) contents as cross-linker in the presence of ammonium persulfate as an initiator by free-radical cross-linking copolymerization in water. Polyacrylamide (PAAm) gels were dried before using for swelling experiments. Steady-state fluorescence spectrometer was employed during the swelling of PAAm hydrogels in water. Pyranine was introduced as a fluorescence probe. Fluorescence intensity of pyranine from various Bis content gel samples was measured during in situ swelling process. It was observed that fluorescence intensity decreased as swelling has proceeded. Gravimetric and volumetric experiments were also performed. The Li,Tanaka equation was used to determine the swelling time constants, ,c, and cooperative diffusion coefficients, Dc, from intensity, weight, and volume variations during the swelling processes. It was observed that swelling time constants, ,c, increased and diffusion coefficients, Dc, decreased as the cross-linker content was increased. © 2010 Wiley Periodicals, Inc. Adv Polym Techn 28:215,223, 2009; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/adv.20163 [source] Particle size effect on the film-forming process of PS/PBA composite latexesPOLYMER COMPOSITES, Issue 9 2010aziye U In this work, the effect of hard particle size and blend ratio on the film formation behavior of hard polystyrene (PS) and soft poly(n -butyl acrylate) (PBA) latex blends was studied by means of steady-state fluorescence and UV,visible techniques in conjunction with atomic force microscopy. Three different sets of latexes were synthesized: PBA latex (diameter 97 nm), pyrene (P)-labeled large PS (LgPS; diameter 900 nm), and small PS (SmPS; diameter 320 nm). Two different series of latex blends (LgPS/PBA and SmPS/PBA) were prepared with varying blend composition at room temperature separately. Films were then annealed at elevated temperatures above glass transition (Tg) temperature of PS. Fluorescence intensity (IP) from P and photon transmission intensity (Itr) were measured after each annealing step to monitor the stages of film formation. The results showed that a significant change occurred in IP and Itr at a certain critical weight fraction (Rc) of PBA. Below Rc, two distinct film formation stages, which are named as void closure and interdiffusion, were seen. However, at PBA concentrations nearer to or above Rc, no film formation can be achieved. Comparing to the LgPS/PBA, the sintering process of SmPS/PBA particles occurred at much lower temperatures. Film formation stages for R < Rc were modeled, and related activation energies were calculated. Void closure (,H) and interdiffusion (,E) activation energies for SmPS/PBA were also found smaller in comparing with LgPS/PBA series. However, ,H and ,E values were not changed much with the blend composition for both series. POLYM. COMPOS., 31:1637,1652, 2010. © 2009 Society of Plastics Engineers [source] Determination of bisphenol A in rat brain by microdialysis and column switching high-performance liquid chromatography with fluorescence detectionBIOMEDICAL CHROMATOGRAPHY, Issue 5 2002Yen Sun A sensitive column switching HPLC-fluorescence detection for determination of bisphenol A (BPA) in rat brain by coupling with microdialysis was developed. A microdialysis probe was inserted into the hypothalamus of rat brain and an artificial cerebrospinal fluid was used for perfusion. BPA in brain dialysate was subjected to a fluorescent derivatization with 4-(4,5-diphenyl-1H -imidazol-2-yl)benzoyl chloride (DIB-Cl), and the excess reagent was removed by a column-switching technique. Separation was carried out on two ODS semimicro-columns with the mobile phase of acetonitrile,H2O,methanol,tetrahydrofuran (55:10:35:2.5, v/v) and acetonitrile,0.1,M acetate buffer (pH 3.0),methanol (35:10:55, v/v) at a flow rate of 0.10 and 0.15,mL/min for a precolumn and a separation column, respectively. Fluorescence intensity was monitored at 475,nm with excitation of 350,nm. BPA could be sensitively detected at 0.3 ppb in 60,µL brain microdialysate at a signal-to-noise ratio of 3. By the proposed method, concentrations of BPA in rat brain and plasma were monitored for 8,h after single i.v. or oral administration. It is proved that BPA is capable of penetrating the blood,brain barrier. The ratio of the area under the concentration,time curve of BPA in rat brain to that in blood was estimated to be about 3.0,3.8%. Copyright © 2002 John Wiley & Sons, Ltd. [source] Tyrosine Hydrogen Bond Properties for the Two Binding Sites of ApoovotransferrinCHINESE JOURNAL OF CHEMISTRY, Issue 10 2005Ying-Qi Li Abstract The interaction of gallium(III) with the ligands containing phenolic group(s), such as salicylic acid, 8-hydroxyquinoline, N,N' -bis(2-hydroxybenzyl)ethylenediamine- N,N, -diacetic acid (HBED), N,N, -ethylenebis[2-(o -hydroxyphenyl)glycine (EHPG), and ovotransferrin, was studied, respectively, by means of fluorescence in 0.01 mol/L Hepes at pH 7.4 and room temperature. Fluorescence intensity showed an increase when gallium(III) was bound to 8-hydroxyquinoline and HBED. In contrast, it was decreased with the interaction of gallium(III) with salicylic acid and EHPG. At pH 7.4, there was N···HO type intramolecular hydrogen bond in the former, and the latter existed O···HO type intramolecular hydrogen bond. Fluorescence titration of apoovotransferrin with gallium(III) displayed that the fluorescence intensity was decreased at the N-terminal binding site, while enhanced at the C-terminal binding site. It can account for the O···HO type intramolecular hydrogen bonds for the phenolic groups of Tyr92 and Tyr191 residues at the N-terminal binding site. And there are N···HO type intramolecular hydrogen bonds for Tyr431 and Tyr524 residues at the C-terminal binding site. In addition, under the same conditions, the conditional binding constant of gallium(III) with EHPG or HBED determined by fluorescence method is lg KGa-EHPG=19.18 or lg KGa-HBED=19.08. [source] Simultaneous quantification of cell motility and protein-membrane-association using active contoursCYTOSKELETON, Issue 4 2002Dirk Dormann Abstract We present a new method for the quantification of dynamic changes in fluorescence intensities at the cell membrane of moving cells. It is based on an active contour method for cell-edge detection, which allows tracking of changes in cell shape and position. Fluorescence intensities at specific cortical subregions can be followed in space and time and correlated with cell motility. The translocation of two GFP tagged proteins (CRAC and GRP1) from the cytosol to the membrane in response to stimulation with the chemoattractant cAMP during chemotaxis of Dictyostelium cells and studies of the spatio-temporal dynamics of this process exemplify the method: We show that the translocation can be correlated with motility parameters and that quantitative differences in the rate of association and dissociation from the membrane can be observed for the two PH domain containing proteins. The analysis of periodic CRAC translocation to the leading edge of a cell responding to natural cAMP waves in a mound demonstrates the power of this approach. It is not only capable of tracking the outline of cells within aggregates in front of a noisy background, but furthermore allows the construction of spatio-temporal polar plots, capturing the dynamics of the protein distribution at the cell membrane within the cells' moving co-ordinate system. Compilation of data by means of normalised polar plots is suggested as a future tool, which promises the so-far impossible practicability of extensive statistical studies and automated comparison of complex spatio-temporal protein distribution patterns. Cell Motil. Cytoskeleton 52:221,230, 2002. © 2002 Wiley-Liss, Inc. [source] Variation in gene content among geographically diverse Sulfolobus isolatesENVIRONMENTAL MICROBIOLOGY, Issue 1 2008Dennis W. Grogan Summary The ability of competitive (i.e., comparative) genomic hybridization (CGH) to assess similarity across entire microbial genomes suggests that it should reveal diversification within and between natural populations of free-living prokaryotes. We used CGH to measure relatedness of genomes drawn from Sulfolobus populations that had been shown in a previous study to be diversified along geographical lines. Eight isolates representing a wide range of spatial separation were compared with respect to gene-specific tags based on a closely related reference strain (Sulfolobus solfataricus P2). For the purpose of assessing genetic divergence, 232 loci identified as polymorphic were assigned one of two alleles based on the corresponding fluorescence intensities from the arrays. Clustering of these binary genotypes was stable with respect to changes in the threshold and similarity criteria, and most of the groupings were consistent with an isolation-by-distance model of diversification. These results indicate that increasing spatial separation of geothermal sites correlates not only with minor sequence polymorphisms in conserved genes of Sulfolobus (demonstrated in the previous study), but also with the regions of difference (RDs) that occur between genomes of conspecifics. In view of the abundance of RDs in prokaryotic genomes and the relevance that some RDs may have for ecological adaptation, the results further suggest that CGH on microarrays may have advantages for investigating patterns of diversification in other free-living archaea and bacteria. [source] Enhanced Calcium Influx in Hippocampal CA3 Neurons of Spontaneously Epileptic RatsEPILEPSIA, Issue 3 2001Hiroko Amano Summary: ,Purpose: The spontaneously epileptic rat (SER: tm/tm, zi/zi) shows both absence-like seizures and tonic convulsions. Our previous electrophysiologic studies have demonstrated that SER has abnormal excitability of hippocampal CA3 neurons, which shows a long-lasting depolarization shift by a single stimulation of mossy fibers, probably resulting from the Ca2+ channel abnormalities. The present study was performed to determine whether Ca2+ influx is actually enhanced in the CA3 area of SER. Methods: Hippocampal slices were prepared from normal Wistar rats and SER aged 11,16 weeks old, when the epileptic seizures had been observed, and loaded with fura-2AM. Intracellular Ca2+ concentration ([Ca2+]i) was monitored as the ratio of fluorescence intensities excited at wavelengths of 340 and 380 nm (RF340/F380) with photometric devices. Results: High K+ (10,60 mM) applied to the bath for 2 min increased [Ca2+]i in hippocampal CA1, CA3, and dentate gyrus (DG) areas of both the normal rats and SER in a concentration-dependent manner. However, the high K+,induced increase in [Ca2+]i was significantly more pronounced in the CA3 area of the SER than in that of the normal animals, whereas there were no significant differences in high K+,induced increases of [Ca2+]i in CA1 or DG between the SER and controls. The high K+,induced increases in [Ca2+]i of CA1, CA3, and DG were inhibited by nifedipine (1,10 nM), a Ca2+ channel antagonist in both SER and controls. However, the inhibition of the high K+,induced increase in [Ca2+]i by nifedipine (1 nM) was significantly greater in the CA3 area of SER than that of controls. Conclusions: These findings suggest that Ca2+ influx through the L-type Ca2+ channels is much greater in the CA3 area of SER than in that of normal animals and is involved in the epileptic seizures of the SER. [source] Tuning Specific Biomolecular Interactions Using Electro-Switchable Oligopeptide SurfacesADVANCED FUNCTIONAL MATERIALS, Issue 16 2010Chun L. Yeung Abstract The ability to regulate biomolecular interactions on surfaces driven by an external stimuli is of great theoretical interest and practical impact in the biomedical and biotechnology fields. Herein, a new class of responsive surfaces that rely on electro-switchable peptides to control biomolecular interactions on gold surfaces is presented. This system is based upon the conformational switching of positively charged oligolysine peptides that are tethered to a gold surface, such that bioactive molecular moieties (biotin) incorporated on the oligolysines can be reversibly exposed (bio-active state) or concealed (bio-inactive state) on demand, as a function of surface potential. The dynamics of switching the biological properties is studied by observing the binding events between biotin and fluorescently labeled NeutrAvidin. Fluorescence microscope images and surface plasmon resonance spectral data clearly reveal opposite binding behaviors when +0.3 V or ,0.4 V vs. SCE are applied to the surface. High fluorescence intensities are observed for an applied positive potential, while minimal fluorescence is detected for an applied negative potential. Surface plasmon resonance spectroscopy (SPR) results provided further evidence that NeutrAvidin binding to the surface is controlled by the applied potential. A large SPR response is observed when a positive potential is applied on the surface, while a negative applied potential induces over 90% reduction in NeutrAvidin binding. [source] Controlled Synthesis of CdSe Nanowires by Solution,Liquid,Solid MethodADVANCED FUNCTIONAL MATERIALS, Issue 22 2009Zhen Li Abstract Semiconductor nanowires prepared by wet chemical methods are a relatively new field of 1D electronic systems, where the dimensions can be controlled by changing the reaction parameters using solution chemistry. Here, the solution,liquid,solid approach where the nanowire growth is governed by low-melting-point catalyst particles, such as Bi nanocrystals, is presented. In particular, the focus is on the preparation and characterization of CdSe nanowires, a material which serves a prototype structure for many kinds of low dimensional semiconductor systems. To investigate the influence of different reaction parameters on the structural and optical properties of the nanowires, a comprehensive synthetic study is presented, and the results are compared with those reported in literature. How the interplay between different reaction parameters affects the diameter, length, crystal structure, and the optical properties of the resultant nanowires are demonstrated. The structural properties are mainly determined by competing reaction pathways, such as the growth of Bi nanocatalysts, the formation and catalytic growth of nanowires, and the formation and uncatalytic growth of quantum dots. Systematic variation of the reaction parameters (e.g., molecular precursors, concentration and concentration ratios, organic ligands, or reaction time, and temperature) enables control of the nanowire diameter from 6 to 33,nm, while their length can be adjusted between several tens of nanometers and tens of micrometers. The obtained CdSe nanowires exhibit an admixture of wurtzite (W) and zinc blende (ZB) structure, which is investigated by X-ray diffraction. The diameter-dependent band gaps of these nanowires can be varied between 650 and 700,nm while their fluorescence intensities are mainly governed by the Cd/Se precursor ratio and the ligands used. [source] Construction of a taste-blind medaka fish and quantitative assay of its preference,aversion behaviorGENES, BRAIN AND BEHAVIOR, Issue 8 2008Y. Aihara In vertebrates, the taste system provides information used in the regulation of food ingestion. In mammals, each cell group within the taste buds expresses either the T1R or the T2R taste receptor for preference,aversion discrimination. However, no such information is available regarding fish. We developed a novel system for quantitatively assaying taste preference,aversion in medaka fish. In this study, we prepared fluorescently labeled foods with fine cavities designed to retain tastants until they were bitten by the fish. The subjects were fed food containing a mixture of amino acids and inosine monophosphate (AN food), denatonium benzoate (DN food) or no tastant (NT food), and the amounts of ingested food were measured by fluorescence microscopy. Statistical analysis of the fluorescence intensities yielded quantitative measurements of AN food preference and DN food aversion. We then generated a transgenic fish expressing dominant-negative G,i2 both in T1R-expressing and in T2R-expressing cells. The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food. The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals. [source] Modified Cyclodextrins as Enantioselective Hosts for Amino AcidsHELVETICA CHIMICA ACTA, Issue 4 2008Pavan Kumar, Vydyula Abstract Two modified , -cyclodextrins, H-2 and H-3, having a flexible appended moiety were studied for the chiral discrimination of the enantiomers of various amino acids by means of fluorescence as signaling option. These hosts quenched the fluorescence intensities of amino acids upon binding. The d- enantiomers were better recognized by these hosts. The association constants (Ks) and enantioselectivity factors (,) of the host,guest complexes were calculated. [source] The role of radixin in altered localization of canalicular conjugate export pump Mrp2 in cholestatic rat liverHEPATOLOGY RESEARCH, Issue 2 2008Hideyuki Kojima Aim:, Cholestasis has been associated with the endocytic retrieval of multidrug resistance protein 2 (Mrp2), but its mechanism is still unclear. Recent studies have indicated that radixin, a cross-linker between the actin filaments and membrane proteins, may be activated by phosphorylation and may be required for the canalicular localization of Mrp2. Methods:, We investigated the role of radixin in the altered localization of Mrp2 in rat models of intrahepatic (ethinyl estradiol treatment) and extrahepatic (bile duct ligation) cholestasis using immunofluorescence microscopy. The changes in localization and expression were analyzed using Scion Image for Windows. Results:, In both models, Mrp2 was localized outside as well as inside the ZO-1 staining, indicating partial dislocation from the canalicular membrane. In contrast to the steep elevation of the immunostaining intensity curves for Mrp2 in the controls, the corresponding curves in both models were broadened and flattened, confirming endocytic retrieval into the hepatocytes. Mrp2 and radixin were colocalized at the canalicular domain in the controls, whereas in both cholestatic rats they were dissociated at some canaliculi, indicating the disturbed colocalization of Mrp2 and radixin in cholestasis. The fluorescence of phosphorylated radixin, an active form of radixin, markedly decreased in both cholestatic models, which was supported by the reduced peak fluorescence intensities. Conclusion:, The disturbed colocalization of Mrp2 and radixin may contribute to the endocytic retrieval of Mrp2 in cholestasis due to the failure to anchor Mrp2 in the canalicular membrane, in which the phosphorylated radixin may play a major role. [source] Determination of genomic copy number with quantitative microsphere hybridization,,HUMAN MUTATION, Issue 4 2006Heather L. Newkirk Abstract We developed a novel quantitative microsphere suspension hybridization (QMH) assay for determination of genomic copy number by flow cytometry. Single copy (sc) products ranging in length from 62 to 2,304 nucleotides [Rogan et al., 2001; Knoll and Rogan, 2004] from ABL1 (chromosome 9q34), TEKT3 (17p12), PMP22 (17p12), and HOXB1 (17q21) were conjugated to spectrally distinct polystyrene microspheres. These conjugated probes were used in multiplex hybridization to detect homologous target sequences in biotinylated genomic DNA extracted from fixed cell pellets obtained for cytogenetic studies. Hybridized targets were bound to phycoerythrin-labeled streptavidin; then the spectral emissions of both target and conjugated microsphere were codetected by flow cytometry. Prior amplification of locus-specific target DNA was not required because sc probes provide adequate specificity and sensitivity for accurate copy number determination. Copy number differences were distinguishable by comparing the mean fluorescence intensities (MFI) of test probes with a biallelic reference probe in genomic DNA of patient samples and abnormal cell lines. Concerted 5, ABL1 deletions in patient samples with a chromosome 9;22 translocation and chronic myelogenous leukemia were confirmed by comparison of the mean fluorescence intensities of ABL1 test probes with a HOXB1 reference probe. The relative intensities of the ABL1 probes were reduced to 0.59±0.02 &!ndash;fold in three different deletion patients and increased 1.42±0.01 &!ndash;fold in three trisomic 9 cell lines. TEKT3 and PMP22 probes detected proportionate copy number increases in five patients with Charcot-Marie-Tooth Type 1a disease and chromosome 17p12 duplications. Thus, the assay is capable of distinguishing one allele and three alleles from a biallelic reference sequence, regardless of chromosomal context. Hum Mutat 27(4), 376,386, 2006. © 2006 Wiley-Liss, Inc. [source] The freshwater dissolved organic matter fluorescence,total organic carbon relationshipHYDROLOGICAL PROCESSES, Issue 16 2007Susan A. Cumberland Abstract The fluorescent properties of dissolved organic matter (DOM) enable comparisons of humic-like (H-L) and fulvic-like (F-L) fluorescence intensities with dissolved organic carbon (DOC) in aquatic systems. The fluorescence-DOC relationship differed in gradient, i.e. the fluorescence per gram of carbon, and in the strength of the correlation coefficient. We compare the fluorescence intensity of the F-L and H-L fractions and DOC of freshwater DOM in north Shropshire, England, featuring a river, wetland, spring, pond and sewage DOM sources. Correlations between fluorescence and DOC varied between sample sites. Wetland water samples for the F-L peak gave the best correlation, r = 0·756; the lowest correlation was from final treated sewage effluent, r = 0·167. The relationship between fluorescence and DOC of commercially available International Humic Substances Society standards were also examined and they generally showed a lower fluorescence per gram of carbon for the F-L peak than the natural samples, whereas peat wetland DOM gave a greater fluorescence per gram of carbon than river DOM. Here, we propose the strength of the fluorescence,DOC correlation to be a useful tool when discriminating sources of DOM in fresh water. Copyright © 2007 John Wiley & Sons, Ltd. [source] Saponaria officinalis karyology and karyotype by means of image analyzer and atomic force microscopyMICROSCOPY RESEARCH AND TECHNIQUE, Issue 10 2008S. Di Bucchianico Abstract The aim of this work was to offer a contribution to the characterization of taxonomic entity of Saponaria officinalis (2n = 28; an herbaceous perennial species; saporin, a type 1 Ribosome Inactivating Protein, is present in leaves and seeds) by a cytogenetic and karyomorphological approach. We investigated the karyotype's morphometry correlated with Stebbin's symmetric index; the same information has been used for computing the indices resemblance between chromosomes (REC), symmetric indices (SYI), and total form (TF%) which allow the comparison between species and evaluation of karyological evolution. Fluorescence intensities of the stained nuclei were measured by a flow cytometer and, for the first time, values for nuclear DNA content were estimated by comparing nuclei fluorescence intensities of the test population with those of appropriate internal DNA standards. Our study is also aimed to introduce chromosomal volumes, which were determined by atomic force microscopy (AFM), as novel karyomorphological parameter which could allow for chromosome discrimination especially when tiny ones are present. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc. [source] The Extent of DNA Deformation in DNA Photolyase, Substrate Complexes: A Solution State Fluorescence StudyPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2008Kongsheng Yang Cyclobutylpyrimidine dimers (CPDs) are the major UV photoproduct formed in DNA containing adjacent pyrimidines. These lesions can be repaired by DNA photolyase, a flavoprotein that utilizes blue light in a direct reversal of the cyclobutane ring. Previous studies have shown that the CPD is base flipped into the protein, with concomitant disruption of the substrate around the CPD. In this study, we use a fluorescent cytidine analog, pyrrolo-dC (PC), to probe how far base flipping propagates along the duplex. From these measurements, the degree of base destacking in the two bases flanking the adenines opposing the CPD appears to be minimal, which was consistent with the protein:substrate crystal structure. Fluorescence-detected melting temperatures for duplexes with and without a CPD were obtained, suggesting that a 5,-pyrimidine-PC-purine-3, motif is more stable than the 5,-purine-PC-pyrimidine-3, motif. This stability trend was reflected in the fluorescence intensities of ss-PC oligos but not for duplexes. The melting point depression due to the PC probe was found to be comparable to other popular fluorescent base analogs. [source] Real-time monitoring of fluorescence anisotropy and temperature during processing of biaxially stretched polypropylene film,POLYMER ENGINEERING & SCIENCE, Issue 4 2004Anthony J. Bur An optical sensor based on fiber optics has been developed to measure fluorescence anisotropy and temperature during processing of biaxially stretched polypropylene films. The sensor, containing optical fibers, polarizing elements and lenses, was mounted above the polypropylene film as it was processed in a tenter frame oven stretching machine. Fluorescence observations were made using the fluorescent dye, bis (di-tert butylphenyl) perylenedicarboximide (BTBP), which was doped into the resin at very low concentrations. To monitor biaxial stretching, fluorescence anisotropy measurements were carried out with light polarized in the machine and the transverse directions corresponding to the directions of biaxial stretching. Fluorescence based temperature measurements were obtained from the ratio of fluorescence intensities at 544 nm and 577 nm. A matrix of experiments involving three levels of stretch ratio in both the machine and transverse directions was undertaken. We observed significant differences between anisotropy in the machine and transverse directions that we attributed to the sequential stretching operation, i.e., the film was stretched in the machine direction first, followed by stretching in the transverse direction, and to film temperature and strain rate for each stretching operation. The result was uniformly higher anisotropies in the machine direction. Film temperature obtained from fluorescence corresponded to oven thermocouple measurements within 2°C. Polym. Eng. Sci. 44:805,813, 2004. © 2004 Society of Plastics Engineers. [source] Overexpression of CD7 in classical Hodgkin lymphoma-infiltrating T lymphocytes,CYTOMETRY, Issue 3 2009Adam C. Seegmiller Abstract Background: Diagnosis of Hodgkin lymphoma (HL) is sometimes complicated by the scarcity of neoplastic cells in a reactive inflammatory background. Immunophenotyping by flow cytometry (FC) has not played a significant role in HL diagnosis because of its consistent failure to identify these neoplastic cells. However, HL-infiltrating T cells have been shown to play a role in HL pathogenesis. This study characterizes the FC immunophenotype of these T lymphocytes to determine whether they can be used to assist in the diagnosis of HL. Methods: Cell suspensions from 76 lymph nodes involved by HL and 156 lymph nodes with reactive lymphadenopathy (LAD) were analyzed by flow cytometry to assess the expression of T-cell antigens. Results: The CD4:CD8 ratio and CD7 expression in both CD4(+) and CD8(+) T cells are increased in HL compared with reactive lymph nodes and there are significant differences between these features in different subtypes of HL. However, only the expression of CD7 in CD4(+) T cells distinguishes between HL and reactive LAD. This is especially true for classical HL in younger patients. Using a CD7 mean fluorescence intensity (MFI) cutoff value generated by this data, 37/47 FNA specimens were correctly diagnosed. Conclusions: There are significant differences in the immunophenotypes of HL-infiltrating T cells. Of these, the CD7 expression in CD4(+) T cells discriminates between HL and reactive LAD, suggesting that this could be a useful and practical adjunctive tool in the diagnosis of HL. It may also further our understanding of the pathophysiology of this disease. © 2008 Clinical Cytometry Society [source] Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemiaCYTOMETRY, Issue 6 2007Eva D. Rossmann Abstract Background: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors. Methods: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITEÔ (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC). Results: The ABCs of CD4 and CD20 antigens estimated with QSC (ABCQSC) were higher than those assigned with QB (ABCQB) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABCQ-MESF) were ,15% higher than ABCQSC, whereas ABCQ-MESF was ,49% higher than ABCQB. Statistically significant correlations were found between the values obtained using various standards. The present study is the first to report down-regulation of CD3 antigen on T cells from patients with CLL. Conclusions: This study emphasizes the relevance of quantitative measurement of fluorescence intensity by flow cytometry as a standardized approach to measure and interpret the expression of some CLL markers and reduce variability of results obtained at different sites in multi-center clinical studies. © 2007 Clinical Cytometry Society [source] Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLLCYTOMETRY, Issue 4 2006Mark Shenkin Abstract Background: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. Methods: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. Results: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. Conclusions: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. © 2006 International Society for Analytical Cytology [source] C-Kit receptor (CD117) expression on myeloblasts and white blood cell counts in acute myeloid leukemiaCYTOMETRY, Issue 1 2004Jolanta Wo Abstract Background The c-Kit receptor is considered to play a crucial role in hematopoiesis. Induction of mobilization of hematopoietic cells in the bone marrow requires cooperative signaling through c-Kit and c-Kit ligand pathway, and these interactions are important in the retention of stem cells within the bone marrow. Therefore, we analyzed c-Kit density on the leukemic myeloblasts of patients with acute myeloid leukemia (AML) in relation to white blood cell count (WBC) in the peripheral blood. Methods Bone marrow aspirates collected from patients with AML and bone marrow aspirates and leukapheresis products after granulocyte colony-stimulating factor blood mobilization from adult volunteers were studied. To determine the level of c-Kit receptor expression, we applied quantitative (relative fluorescence intensity and antibody binding per cell) cytometric methods. Results Our data showed negative correlation between the level of c-Kit expression intensity on myeloblasts and the number of leukocytes in blood of AML patients. The c-Kit receptor density on myeloblasts in patients with low WBC was significantly stronger than that on myeloblasts in patients with high WBC. In the latter patient group, the density c-Kit receptor on myeloblasts was similar to that on CD34+ cells in mobilized peripheral blood. Conclusions The obtained data suggest an involvement of c-Kit receptor in the regulation of leukemic myeloblasts egress to the peripheral blood. © 2004 Wiley-Liss, Inc. [source] Multicenter clinical experience with flow cytometric method for fetomaternal hemorrhage detectionCYTOMETRY, Issue 6 2002Jenn C. Chen Abstract BACKGROUND Enumeration of fetal red blood cells (RBCs) is important in the management of fetomaternal hemorrhage (FMH), particularly in situations of Rh incompatibility. METHODS We evaluated results from three institutions using the flow cytometric method (FCM) to detect fetal RBCs based on the anti-hemoglobin F (HbF) monoclonal antibody method. RESULTS During 1997,2001, 69 of 1,248 patients (5.5%) had measurable fetal erythrocytes (RBCs) in maternal blood. Only 21 patients (1.7%) had more than 30 mL of fetal blood detected in maternal blood. Of the 11 patients with large FMH and clinical follow-up, 7 had fetal demise (64%). In positive samples, significant differences were found in the fluorescence intensity (FI) of anti-HbF antibody staining between HbF-negative erythrocytes (HbF-) and adult HbF containing erythrocytes (F cells; 4 ± 0 versus 57 ± 9 linear mean channels [LMC]; P < 0.001) and between HbF-cells and fetal RBCs (4 ± 0 versus 433 ± 136 LMC; P < 0.001). In addition, significant differences were observed in forward light scatter intensity between HbF-cells and fetal RBCs (298 ± 15 versus 355 ± 68 LMC, P = 0.03). The transportability of the test is also addressed by comparing results from two other laboratories. The experience of our three laboratories, as well as the results from the recently reinitiated College of American Pathologists survey, which compares FCM and manual methods, clearly documents the superiority of the FCM test over the manual Kleihauer-Betke (KB) test. CONCLUSIONS The FCM is a simpler, more objective, and more precise alternative to the KB method in clinical testing. The high mortality rate associated with large FMH and therapeutic implications of these results should give laboratories motivation to abandon the KB method with more robust FCM to detect FMH. Cytometry (Clin. Cytometry) 50:285,290, 2002. © 2002 Wiley-Liss, Inc. [source] Sensitive, label-free protein assay using 1-ethyl-3-methylimidazolium tetrafluoroborate-supported microchip electrophoresis with laser-induced fluorescence detectionELECTROPHORESIS, Issue 9 2008Yuanhong Xu Abstract Based on the dimer,monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) instead of PBS was applied as running buffers in microchip electrophoresis. Due to the excellent properties of EMImBF4, not only nonspecific protein adsorption was more efficiently suppressed, but also approximately ten-fold higher fluorescence intensity enhancement was obtained than that using PBS. Under the optimal conditions, detection limits for BSA, bovine hemoglobin, cytochrome c, and trypsin were 1.00×10,6, 2×10,6, 7×10,7, and 5×10,7 mg/mL, respectively. Thus, without covalent modification of the protein, a protein assay method with high sensitivity was achieved on microchips. [source] Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plantsENVIRONMENTAL MICROBIOLOGY, Issue 3 2006Yunhong Kong Summary The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2,6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus -related PAOs and Actinobacteria -related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus -related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed. [source] | ||||||||||||||||||||||||||||||||||