bluuebunny/crossref_metadata_2025_split huggingface dataset:
Automatic detection of fragile X chromosomes using an X centromere probe
Authors: Piper Jim , Fantes Judith , Gosden John , Ji Liang 
Published: August 2005

AbstractIn order to score for the fragile X syndrome, blood samples are prepared with absorption stain labeling by in situ hybridisation of the X chromosome centromeres. Metaphases are located, digitised at high resolution, and segmented fully automatically. A three Stage adaptive classification scheme for labeled X chromosomes is then applied. This consists of a simple box classifier to identify plausible X and false positive X chromosomes, followed by a quadratic discriminant classifier that is re‐trained for each sample. The modal number of X chromosomes is then determined for each sample and used to refine the classification. A simple fragile site detector is applied to the distal portion of the detected X chromosome long arms. From the results we estimate computer and operator time requirements for a screening system in which the operator reviews only the apparently fragile X chromosomes detected by the computer.
Flow cytometric detection of β‐globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization
Authors: Bayer Jan A. , Bauman Jan G. J. 
Published: August 2005

AbstractThe novel method for flow cytometric detection of cellular RNA species in suspended cells by fluorescent in situ hybridization (FC‐FISH) was applied in the evaluation of β‐globin expression in murine haemopoietic tissues. Normal murine bone marrow cells and regenerating bone marrow cells obtained after lethal irradiation and bone marrow transplantation as well as murine 15 d fetal liver were examined. Furthermore, spleens and bone marrow of phenylhydrazine‐induced anaemic mice were studied. Biotinylated sense‐ and antisense single strand RNA probes, obtained by transcription of a 510 nucleotides murine β‐globin cDNA sequence subcloned into the pGEM1 plasmid were used as hybridization probes. For detection of the hybrids formed, avidin‐FITC was used.Only the antisense β‐globin probe gave strongly positive fluorescence signals in a defined population of cells in each of the tissues examined, whereas the sense probe did not give signals higher than control samples. Melting characteristics of the hybrids showed the specificity of the in situ hybridization reaction. Forward light scatter distributions, reflecting cell size of the positive cells were as expected from erythroid cells. Within the erythrocyte subpopulation both β‐globin‐negative and ‐positive cells were detected. The percentages of positive cells determined flow cytometrically correlated with the percentages observed in May‐Grünwald/Giemsa stained preparations. Differences observed in fluorescence intensity between positive cells of different organs were no larger than about a factor of two, indicating a rather constant β‐globin mRNA content over the entire differentiation range. An exception was 15 d fetal liver, which was shown biochemically to contain about eight times more β‐globin RNA and which had a 2.4 times higher fluorescence intensity. We estimate that the sensitivity of the present method is such that as little as 500 copies per cell of a specific mRNA of 1 kb length would be detectable.
Quantification of the DNA content of structurally abnormal X chromosomes and X chromosome aneuploidy using high resolution bivariate flow karyotyping
Authors: Trask Barbara , van den Engh Ger , Nussbaum Robert , Schwartz Charles , Gray Joe 
Published: August 2005

AbstractQuantification of the Hoechst and chromomycin A3 fluorescence intensities of mitotic human chromosomes isolated from karyotypically normal and abnormal cells was performed with a dual beam flow cytometer. The resultant flow karyotypes contain information about the relative DNA content and base composition of chromosomes and their relative frequencies in the mitotic cell sample. The relative copy number of X and Y chromosomes was determined for 38 normal males and females and 6 cell lines with X or Y chromosome aneuploidy. Flow karyotype diagnoses corresponded with conventional cytogenetic results in all cases. We show that chromosome DNA content can be derived from peak position in Hoechst vs. chromomycin flow karyotypes. These values are linearly related to propidium iodide staining intensity as measured with flow cytometry and to the binding of gallocyanin chrome alum to phosphate groups as measured with slide‐based scanning photometry. Cell lines with deleted or dicentric X chromosomes ranging in length from 0.53 to 1.95 times normal were analyzed by using flow cytometry. The measured difference in DNA content between a normal X and each of the structurally abnormal chromosomes was linearly correlated to the difference predicted from cytogenetics and/or probe analyses. Deletions of 3–5 Mb, which were at and below the detection limits of conventional cytogenetics, could be quantified by flow karyotyping in individuals with X‐linked diseases such as Duchenne muscular dystrophy, choroideremia, and ocular albinism/ichthyosis. The results show that the use of flow karyotyping to quantify the size of restricted regions of the genome can complement conventional cytogenetics and other physical mapping techniques in the study of genetic disorders.
Construction of gene libraries for each human chromosome
Authors: Van Dilla Marvin A. , Deaven Larry L. 
Published: August 2005

AbstractWe describe the construction of two complete sets of small insert, complete digest DNA libraries for each of the 24 human chromosomal types by the National Laboratory Gene Library Project. Flow sorting was used to purify the chromosomes which provided the DNA for cloning. One set of libraries was cloned into the HindIII site of the lambda vector Charon 21A, and the other set was cloned into the EcoRI site of the same vector. Characterization information from both in‐house experiments and user feedback is presented. These chromosome‐specific libraries are available to the general scientific community from a repository at the American Type Culture Collection, Rockville, MD. The second phase of the project, the construction of large insert, partial digest libraries in both lambda and cosmid vectors, is underway.
Cell kinetics in mouse epidermis studied by bivariate DNA/bromodeoxyuridine and DNA/keratin flow cytometry
Authors: Kirkhus Bente , Clausen Ole Petter Fraas 
Published: August 2005

AbstractHairless mice were injected intraperitoneally with bromodeoxyuridine (BrdUrd). Basal cells were isolated from epidermis, fixed in 70% ethanol, and prepared for bivariate BrdUrd/DNA flow cytometric (FCM) analysis. Optimum detection of incorporated BrdUrd in DNA was obtained by combining pepsin digestion and acid denaturation. The cell loss was reduced to a minimum by using phosphate‐buffered saline containing Ca2+ and Mg2+ to neutralize the acid. The percentage of cells in S phase and the average uptake of BrdUrd per labelled cell in eight consecutive windows throughout the S phase were measured after pulse labelling at intervals during a 24 h period. Furthermore, the cell cyle progression of a pulse‐labeled cohort of cells was followed up to 96 h after BrdUrd injection. In general the results from both experiments were in good agreement with previous data from 3H‐thymidine labelling studies. The percentage of cells in S phase was highest at night and‐lowest in the afternoon, whereas the average uptake of BrdUrd per labelled cell showed only minor circadian variations. There were no indiications that BrdUrd significantly perturbed normal epidermal growth kinetics. A cell cycle time of about 36 h was observed for the labelled cohort. Indications of heterogeneity in traverse through G1 phase were found, and the existence of slowly cycling or temporarily resting cells in G2 phase was confirmed. There was, however, no evidence of a significant population of temporarily resting cells in the S phase Bivariate DNA/keratin FCM analysis revealed a high purity of basal cells in the suspensions and indicated that the synthesis of the differentiation‐keratin K10 was turned on only in G1 phase and after the last division.
Flow cytometric analysis of erythrocyte populations in tn syndrome blood using monoclonal antibodies to glycophorin A and the Tn antigen
Authors: Bigbee William L. , Langlois Richard G. , Stanker Larry H. , Vanderlaan Martin , Jensen Ronald H. 
Published: August 2005

AbstractFlow cytometric analysis employing monoclonal antibodies to the Tn antigen and glycophorin A was used to characterize the erythrocyte populations present in blood samples from individuals with Tn syndrome. Four monoclonal antibodies specific for the Tn antigen, GalNAc monosaccharide, on human erythrocytes were obtained from a fusion of splenocytes from a Biozzi mouse immunized with red cells from a Tn individual. These monoclonal antibodies specifically recognize GalNAc monosaccharide sites located on the erythrocyte cell surface sialoglycoproteins, glycophorin A and glycophorin B, and do not bind to fixed normal red cells presenting the Neu‐NAcα2‐3Galβ1‐3(NeuNAcα2‐6)GalNAcα1‐O‐Ser(Thr) tetrasaccharide or to fixed neuraminidase‐digested cells presenting the Gal‐GalNAc disaccharide. The percentages of Tn‐positive red cells in samples from six unrelated Tn donors ranged from 28 to 99%. Binding of the glycophorin A‐specific monoclonal antibodies showed that the erythrocytes composing the Tn‐negative fraction presented normal amounts of the M and N epitopes on glycophorin A. The presumed somatic mutational origin of Tn‐positive cells was tested in blood samples from five normal donors; three possible Tn cells were observed after analysis of a total of 1.1 X 107 erythrocytes, suggesting that the frequency of such cells in normal individuals is > 1 X 10−6.
Changes in neutrophil right‐angle light scatter can occur independently of alterations in cytoskeletal actin
Authors: Kraus Eliyahu , Niederman Richard 
Published: August 2005

AbstractForward‐angle light scatter (FALS) and right‐angle light scatter (RALS) are commonly employed to discriminate between leukocyte subclasses. Recently the application of RALS has expanded, and it is now also used as an indicator of neutrophil actin polymerization. In this communication we critically examine the relationship of RALS to changes in cytoskeletal actin. The data indicate that agonists which stimulate an increase, a decrease, or no change in F‐actin content can all stimulate a biphasic change in RALS. We therefore conclude that changes in RALS can occur independently of changes in F‐actin content. This leads us to suggest that caution must be taken when interpreting RALS data in relation to changes in F‐actin. Furthermore, the data also support the idea originally proposed by Yuli and Snyderman (J Clin Invest 73:1408–1417, 1984), that RALS may be an exceptionally sensitive indicator of cell activation.
Partition of protein and DNA during cytokinesis in human breast cancer cell lines
Authors: Sennerstam Roland , Auer Gert 
Published: August 2005

AbstractThree human breast cancer cell lines (HTB‐126, MDA‐231, and HTB‐122) with DNA index (DI) values between 1.26 and 1.72 were analysed together with a diploid mouse embryonal carcinoma cell line (PCC3) by a TV‐video time‐lapse technique (pedigree analysis). Cytochemical parameters (DNA and proteins) were studied in individual cells in a rapid scanning microspectrophotometer. Postmitotic sister cell pairs were analysed after Feulgen‐naphthol‐yellow staining. The DI values of the cell lines were selected to reflect various well‐known clinical ploidy entities differing in malignancy potentials. A mitotic disturbance of the partition of DNA and protein to daughter cells was found in particular in MDA‐231 closest to the triploid DNA modal value (DI = 1.37). Duration of mitosis was considerably longer in the near triploid line compared to the other lines. The MDA‐231 line was also least sensitive to suboptimal growth conditions.This report calls attention to a possible causality between mitotic error and intraclonal genotype and cell mass heterogeneity.
Interfacing a flow cytometer computer with a PC‐based patient information system
Authors: Blume Philip 
Published: August 2005

AbstractWe have established an interface between our flow cytometer's computer and the personal computer (PC) which supports our patient database system. The PC has been equipped with a commercially available IEEE‐488 bus interface board which is connected to the interface bus of the cytometer's Hewlett‐Packard 9000/300 computer (HP). The PC is set as a bus device with the same address as that of the HP's printer. It is programmed to examine the stream of data sent to the printer and extract from it and store in an MS‐DOS text file selected information which subsequently may be transferred to the database system.
Flow cytometric competitive binding assay for determination of actinomycin‐D concentrations
Authors: Saunders George C. , Martin John C. , Jett James H. , Perkins Ann 
Published: August 2005

AbstractA single step, separation free competitive binding reaction between the fluorescent antibiotic mithramycin and actinomycin‐D for common binding sites on DNA coated 10 μm diameter microspheres is described. The fluorescence of the microspheres is measured with a flowcytometer. In the presence of a constant amount of mithramycin, the microsphere fluorescence is inversely proportional to actinomycin‐D concentration.
Improved method for computing potential doubling time from flow cytometric data
Authors: White R. Allen , Terry Nicholas H. A. , Meistrich Marvin L. , Calkins Dennise P. 
Published: August 2005

AbstractRelative movement methods use the timed progression of the mean fluorescence of cells which have heen labeled with monoclonal antibodies against bromodeoxyuridine and displayed with bivariate flow cytometry according to DNA and label content to compute duration of DNA synthesis, Ts. The relative movement is the difference of the mean DNA fluorescence of the labeled undivided cells from the G1 channel relative to the difference between the G1 and G2M channels. In this communication, we show how to extend this method to compute the potential doubling time, Tpot, the time required for a population of cells to double, given quiescent cells but no cell loss. A quantity vis introduced that is a function of the fraction of labeled divided cells and the fraction of labeled undivided cells. We show that v is independent of time and is equal to In(2)T8/Tpot so that Tpot (equal to In(2)T8/v) can be directly found from the information available in computing the relative movement. The method is applied to Chinese hamster ovary cells to demonstrate its utility.
Cytofluorometric determination of protein‐bound thiols and DNA in cell nuclei
Authors: Schabronath J. , Streffer C. , Zamboglou N. 
Published: August 2005

AbstractThe aim of the present study was to establish a cytofluorometric method for the simultaneous determination of protein‐bound sulfhydryl‐groups (PSH) and DNA in isolated cell nuclei. DNA was stained with ethidiumbromide and PSH with N‐iodoacetyl‐N(5‐sulfo‐1‐naphthyl) ethylendiamine (AEDANS). Disulfide groups of nuclear proteins were determined by the same method after reduction with sodium borohydride or thioglycollic acid. The method was established by using nuclei of human lymphocytes, which then served as a biological standard for further investigations of the nuclei of different mammalian cell types: nuclei from mouse liver cells and nuclei from the cells of two human melanoma cell lines. For non‐proliferating lymphocytes distinct DNA‐ and PSH‐values could be measured. The PSH‐values detected in the nuclei of the other cell types were higher by comparison and varied within the cell cycle; i.e., PSH increased during the S‐phase and was almost doubled during the cell generation cycle from G1‐ to G2‐phase. Cell line and cell cycle‐dependent variations of nuclear disulfides could also be detected. These results are discussed with respect to their radiobiological implications. In conclusion, thiol groups may represent one factor determining the radiosensitivity of cells, but they are not the only decisive one.
Intracellular pH, esterase activity, and DNA measurements of human lung carcinomas by flow cytometry
Authors: Liewald F. , Demmel N. , Wirsching R. , Kahle H. , Valet G. 
Published: August 2005

AbstractAn important intention of flow cytometric investigations is to obtain biochemical and biophysical information about cells which is suitable for automated tumor diagnosis. In this study, the ploidy status, the intracellular pH value, the intracellular esterase activity, and the cell volume of vital cells and the DNA and cell volume of dead cells were measured in cancerous tisse and normal lung tissue of 30 patients by flow cytometry. The cell samples were simultaneously stained with the pH and esterase indicator dye 1.4‐diacetoxy‐2,3‐dicyano‐benzene (ADB) and propidium iodide (PI). The flow cytometric measurements were performed in three‐parameter list mode. The data were evaluated on an AT‐compatible personal computer with the DIAGNOS1 program system for automated diagnosis of flow cytometric list mode data.Significant differences were found between normal and malignant tissue in DNA ploidy, in the intracellular esterase activity, in the cell, volume and in the percentage of inflammatory cells and parameters of necrosis. DNA‐aneuploidy was observed in 38% of the lung carcinomas. The simultaneous detection of DNA‐aneuploidy and tumor‐associated properties in a multifactorial analysis led to correct automatic tumor diagnosis in 85% of cases. DNA‐aneuploidy was found at a significant higher frequency in advanced tumors. Adenocarcinomas displayed DNA‐aneuploidy more often (80%) than squamous cell carcinomas (33%).
Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry
Authors: Johnson N. F. , Wilson J. S. , Habbersett R. , Thomassen D. G. , Shopp G. M. , Smith D. M. 
Published: August 2005

AbstractBasal and secretory cells have been separated as highly enriched viable populations from single‐cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2° and 90° light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony‐forming efficiency than secretory cells (3.4%). Significant differences in blue auto‐fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.
Relationship of DNA ploidy to chemoresistance of tumors as measured by in vitro tests
Authors: Volm Manfred , Efferth Thomas 
Published: August 2005

AbstractTo examine whether patients with aneuploid tumors might derive more benefit from chemotherapy than would patients with diploid tumors, predictive tests for determining resistance in human tumors were carried out and the test results compared with the DNA ploidy of the corresponding tumors. Multidrug‐resistance in 15 kidney carcinomas grown as primary cultures was determined by immunofluorescence by Mab C219, which is specific for the plasma membrane glycoprotein P‐170, and by the use of tritiated nucleotide incorporation after addition of doxorubicin. Aneuploid tumors had a higher tendency to be more sensitive than diploid tumors, but the correlation was not significant. This was confirmed by reanalyzing our earlier data on ovarian and lung cancers. In conclusion, DNA measurement using flow cytometry does not appear to be a suitable tool for prediction of resistance of human tumors to chemotherapy.
Cyanine dye labeling reagents—carboxymethylindocyanine succinimidyl esters
Authors: Southwick Philip L. , Ernst Lauren A. , Tauriello Erica W. , Parker Stephen R. , Mujumdar Ratnakar B. , Mujumdar Swati R. , Clever Hester A. , Waggoner Alan S. 
Published: August 2005

AbstractTen carboxymethylindocyanine dyes which from the basis of a new series of fluorescent probes have been synthesized and converted into succinimidyl active esters for fluorescent labeling of proteins or other amino‐containing substances. Fluorescence emission maxima for members of the series range from 575 to 780 nm. Hydrophilic, water‐soluble reagents have been obtained which yield labeled antibodies with little tendency to form precipitates. The fluorescence intensities achieved are higher than those produced by labeling with the cyanine isothiocyanates described previosly (Mujumdar et al.: Cytometry 10:11–19, 1989). The utility of these reagents has been demonstrated in antibody labeling for two‐color immunofluorescent imaging of internal structures in a mammalian cell and for two‐color flow‐cytometry experiments. The use of values of chromophore‐equivalent weight (W/Ceq), calculated from quantitative absorption data on dye samples, is proposed as an aid in formulating labeling procedures.
Cytolytic activity of human natural killer cell subpopulations isolated by four‐color immunofluorescence flow cytometric cell sorting
Authors: Srour Edward F. , Leemhuis Tom , Jenski Laura , Redmond Robert , Jansen Jan 
Published: August 2005

AbstractThe natural killer activity and phenotypic properties of six different subpopulations of normal human peripheral blood lymphocytes obtained by four‐color immunofluorescence cell sorting were examined. Phycoerythrin‐conjugated CD16 and CD56 were used simultaneously to identify (CD16+CD56+) NK cells. The cells most effective in mediating NK cytolysis against K562 target cells were CD3−(16+56+)57‐8+. Although most of the K562 killing was found in the CD3−(16+56+) groups of cells, a substantial degree of NK activity was detected in the CD3+(16+56+) subpopulations of some individuals. The level of expression of CD57 and CD8 was significantly higher on CD3+(16+56+) than on CD3−(16+56+) cells.
Logarithmic amplifiers
Authors: Gandler William , Shapiro Howard 
Published: August 2005

AbstractLogarithmic amplifiers (log amps), which produce an output signal proportional to the logarithm of the input signal, are widely used in cytometry for measurements of parameters that vary over a wide dynamic range, e.g., cell surface immunofluorescence. Existing log amp circuits all deviate to some extent from ideal performance with respect to dynamic range and fidelity to the logarithmic curve; accuracy in quantitative analysis using log amps therefore requires that log amps be individually calibrated. However, accuracy and precision may be limited by photon statistic and system noise when very low level input signals are encountered.
Effect of focus on cell detection and recognition by the cell analyzer
Authors: Spadinger Ingrid , Poon Steven S. S. , Palcic Branko 
Published: August 2005

AbstractThe effect of defocusing on the quality of signals from live cells detected by an automated image cytometry device, the Cell Analyzer, was examined. The influence of these effects on the ability of this device to automatically locate cells plated into a tissue culture flask was then determined by measuring the performance of cell detection and recognition procedures as a function of focus setting. Acceptable limits for deviation from the optimal focus setting (as determined by microscope objective position) were found to be similar for both these procedures, ranging from 40 μm below to 25 μm above the optimal focus position. These limits were asymmetrical about ideal focus due to a pronounced asymmetry in the effects of positive and negative defocusing on the cell signal.