bluuebunny/crossref_metadata_2025_split huggingface dataset:
Time‐related effects of enzymatic disaggregation on model human lung carcinomas
Authors: Howard Randy B. , Shroyer Kenneth R. , Marcell Tere , Swanson Lucille E. , Pagura Marco E. , Mulvin David W. , Cowen Michael E. , Johnston Michael R. 
Published: June 2005

AbstractEnzyme cocktails used to prepare tumor cell suspensions may influence yield, viability, and cytology, thus time‐related cocktail effects on model human lung carcinomas were examined. A549, NCI‐H125, and NCI‐H460 carcinomas were completely disaggregated at 25°C over 2 h with either (mg/ml) collagenase/DNAse (C/D, 1/0.1), collagenase/hyaluronidase/DNAse (C/H/D, 1/0.1/0.1), or polymyxa protease/DNAse (PP/D, 3/0.1). Trypan blue viabilities, total yields, viable yields, and flow cytometric percent tumor cells (TC) were measured every 20–30 min (n = 4–7 per tumor type). The final percentages of TC, mononuclear cells (MN), polymorphonuclear cells (PMN), lymphocytes, and necrotic cells were determined by cytology (n = 4–5 per tumor type). The time‐dependent measurements showed that (1) disaggregation was progressive and complete with all cocktails; (2) viability was stable or increasing with all cocktails; (3) percent TC was stable for all cocktails, but lower for PP/D than C/D in final suspensions; and (4) PP/D gave lower final total yields, higher final viabilities, but the same final viable yields as the C cocktails, suggesting selective elimination of dead cells by PP/D. Final cytology measurements showed that PP/D gave a lower percent MN and a higher percent PMN than C cocktails. Cocktail effects may importantly influence cell suspension properties. © 1995 Wiley‐Liss, Inc.
Luminescence quenching by long range electron transfer: A probe of protein clustering and conformation at the cell surface
Authors: Matko Janos , Jenei Attila , Wei Taiyin , Edidin Michael 
Published: June 2005

AbstractQuenching of luminescence from fluorescent and phosphorescent probes by nitroxide spin labels with a long range electron transfer (LRET) mechanism (44,45) has been tested as a tool to monitor association/clustering and conformational changes of cell surface proteins. The membrane proteins were labeled with monoclonal antibodies or Fab fragments conjugated with luminescent probes or water‐soluble nitroxide spin labels. The method was tested as a probe of 3 different aspects of protein‐protein association involving class I MHC molecules: (1) interaction between the heavy and light chains of the MHC molecules, (2) clustering, self‐association of MHC molecules, (3) proximity of MHC molecules to transferrin receptors of fibroblasts or surface immunoglobulin molecules of B lymphoblasts. The extent of quenching upon increasing the fractional density of the quencher was sensitive for protein association in accordance with earlier immunoprecipitation and flow cytometric Förster‐type energy transfer (FCET) data obtained on the same cells. These data suggest that the LRET quenching can be used as intra‐ or intermolecular ruler in a 0.5–2.5 nm distance range. This approach is simpler (measurements only on donor side) and faster than many other experimental techniques in screening physical association or conformational changes of membrane proteins by means of spectrofluorimetry, flow cytometry, or microscope based imaging. © 1995 Wiley‐Liss, Inc.
Kinetic analysis of glutathione in anchored cells with monochlorobimane
Authors: Barhoumi Rola , Bailey R. Hartford , Burghardt Robert C. 
Published: June 2005

AbstractA method for the measurement of intracellular glutathione content and glutathione S‐transferase activity with monochlorobimane in adherent cells is described. The method involves the kinetic analysis of monochlorobimane conjugation to glutathione over a relatively short period of time. This permits extrapolation over time for determination of equilibrium fluorescence intensity (relative glutathione level) from scan intensity data that follows first‐order kinetics, minimizing problems commonly associated with the use of monochlorobimane. By using measured fluorescence intensity values from glutathione standards, a suspension calibration curve was generated and, subsequently, was used to determine the photomultiplier tube saturation rate. A theoretical intracellular calibration curve was then generated to quantify glutathione content in cells. This method was also applied to study the changes in glutathione in a variety of rodent and human cell lines and in selected cocultures of cells exhibiting similar or different glutathione levels. Comparison of the glutathione levels obtained with monochlorobimane and a standard colorimetric method (GSH‐400) indicated good correlation between the two methods. These studies support the use of laser cytometry for measuring intracellular glutathione with monochlorobimane as well as changes in glutathione occurring in cells that establish physical contacts with other cells. Laser cytometric analysis of glutathione in anchored cells also provides opportunities to monitor individual cellular responses to a variety of experimental manipulations, such as responses to various toxic insults or the protective effects of gap junction‐mediated intercellular communication. © 1995 Wiley‐Liss, Inc.
DNA and RNA content analysis by flow cytometry in the pathobiologic assessment of bone tumors
Authors: El‐Naggar Adel K. , Hurr Kenneth , Tu Z. Nora , Teague Kim , Raymond Kevin A. , Ayala Alberto G. , Murray John 
Published: June 2005

AbstractStudies of simultaneous DNA and RNA contents by flow cytometry in hematologic and some solid neoplasms have been shown to provide information that may be useful in the pathobiological evaluation of these neoplasms. We contend that similar analysis may be equally valuable in assessing bone tumors. Our data revealed significant statistical differences in DNA ploidy and proliferative fraction between benign and malignant bone neoplasms. Benign tumors manifested predominantly DNA diploidy and low proliferative activity, whereas the majority of malignant tumors were DNA aneuploid and showed high proliferation rate. No significant difference in the RNA content between different histopathologic categories was found. We observed, however, a distinct and consistently high RNA content pattern in giant cell tumors, aneurysmal bone cysts, and chondroblastomas that may be useful in their differential diagnosis. Analysis of different prognostic factors in malignant tumors indicated that histologic grade and DNA content are a significant prognostic factors. Further analysis of malignant tumors showed that a correlation between the proliferative activity and the clinical outcome in the low grade category and between RNA content and patients' survival in osteosarcomas. Our study also showed that preoperative treatment significantly impacted on the extent of the proliferative fraction in malignant tumors. We conclude that DNA/RNA analysis of bone tumor may assist in: (1) the differential diagnosis of certain bone tumors, (2) evaluation of treatment response, and (3) the biological assessment of osteosarcomas. © 1995 Wiley‐Liss, Inc.
Flow cytometric measurement of NK cell immunoconjugates by pulse width processing
Authors: Manogaran P. S. , Kausalya S. , Pande Gopal 
Published: June 2005

AbstractPulse width analysis, in flow cytometry, has been widely used for optimal cell size resolution in cell kinetics analysis. Pulses, generated by scattered light or fluorescence of cells, are electronically analyzed for their height and width. The information generated from these two properties of the pulses is utilized to distinguish signals from single cells vs. signals from cell clumps or aggregates. Pulse width, unlike pulse height, is more sensitive to differences in cell diameter, and therefore can discriminate very small differences in it, which pulse height cannot. We have exploited this property of pulse widths to measure immunoconjugates between NK cells and their targets. Discrimination of the free target cells from the conjugated ones is possible by the pulse widths of only light scatter signals, both forward and/or orthogonal. This resolution was not obtained if pulse height of the same signals was visualized. Using this resolution it was possible to distinguish single cells from the aggregates between target and effector cells. We propose that this is a better method for distinguishing conjugates than the method in which prior vital staining of cells is used. © 1995 Wiley‐Liss, Inc.
Chromosome specific DNA hybridization in suspension for flow cytometric detection of chimerism in bone marrow transplantation and leukemia
Authors: Arkesteijn G. J. A. , Erpelinck S. L. A. , Martens A. C. M. , Hagenbeek A. 
Published: June 2005

AbstractFlow cytometry was used to measure the fluorescence intensity of nuclei that were subjected to fluorescent in situ hybridization in suspension with chromosome specific DNA probes. Paraformaldehyde‐fixed nuclei were protein digested with trypsin and hybridized simultaneously with a biotin‐and DIG labeled chromosome specific centromere probe. A number of probes were tested in the suspension hybridizations. The method yielded fluorescent hybridization signals that allow discrimination between Y chromosome positive and negative nuclei when analyzed by flow cytometry. The method is especially suited for analysis of bone marrow cells derived from patients who have received a sex‐mismatched allogeneic bone marrow transplantation. Male leukemia cells with a trisomy for chromosome 8 were mixed with normal female cells and simultaneously hybridized in suspension with a DIG labeled probe specific for chromosome 8 and the biotin labeled Y chromosome probe. Y chromosome positive or negative nuclei were sorted onto microscope slides and subsequently classified as being leukemic or not by fluorescence microscopy, on the basis of the presence of a trisomy for chromosome 8. A 120‐fold enrichment could be achieved when 300 Y positive nuclei were sorted from a mixture originally containing 0.5% leukemia cells. Given the specificity of the flow cytometry and FISH procedure, the combination of the two methods can reach a lower detection level of 1 per 250,000. © 1995 Wiley‐Liss, Inc.
Cell volume and shape oscillations in rat type‐II somatotrophs at hypotonic conditions
Authors: Engström Karl Gunnar , Sävendahl Lars 
Published: June 2005

AbstractThe size and shape of growth hormone (GH)‐producing rat type‐II somatotrophs was studied during osmotic manipulation. When somatotrophs were exposed to large osmotic stress (200 and 225 mOsm), the peak projected cell area (PCA) was 132.9% ± 12.6% and 116.8% ± 2.8% (P < 0.01) and triggered a regulatory volume decrease (RVD) to avoid lysis. At lower osmotic stress (250 mOsm), the rate of swelling was slower, and the volume reached a steady state at 109.4% ± 2.4% (P < 0.05) and was without RVD. At 275 and 287 mOsm, the swelling was delayed [PCA peak at 3–4 min; 105.8% ± 1.5% (P < 0.05) and 104.2% ± 1.7%] and then showed repeated synchronized cycles of swelling and shrinkage (P < 0.05). The data suggest that somatotrophs may have more than one mechanism for volume regulation. One mechanism is for large swelling (classic RVD response), whereas the other represents more physiological mechanisms for regulating cell volume within a more limited geometry range. For low osmotic stress (250–287 mOsm), the somatotrophs became less spherical during swelling and, thus, were without membrane dilation. Therefore, this type of volume regulation must work independently from membrane stress. Related volume regulation mechanisms may underlie the previously observed volume fluctuations in somatotrophs seen during secretory stimulation with GH‐releasing hormone. © 1995 Wiley‐Liss, Inc.
Anti‐CD3‐Induced Apoptosis in T‐Cells From Young and Old Mice
Authors: Chrest Francis J. , Buchholz Meredith A. , Kim Young Ho , Kwon Taeg‐Kyu , Nordin Albert A. 
Published: June 2005

AbstractLight scatter measurements using flow cytometry indicated that T cells from young and old mice undergo apoptosis following activation with immobilized anti‐CD3. The percentage of cells in apoptosis after 20 h activation was significantly greater (p<.001) in cultures containing cells from older animals. The mean percentages of apoptotic T cells from young and old mice after 20 h activation were 19.3% and 33.0%, respectively. The proportion of viable cells after 20 h activation was significantly higher (p<.003) in the young (mean=78.4%) than in the old animals (mean =: 65.8%). Simultaneous measurements of light scatter and fluorescence indicated that apoptotic T cells contained both the CD4+ and the CD8 + T cell phenotypes. The frequency of apoptotic CD8+ T cells was elevated (p<.007) in older animals, where the mean percentage was 15.1%, compared to 5.3% in the young. The most dramatic difference between young and old (p<.0008) was seen in the percentages of viable CD4+ T cells after 20 h activation. The mean viable CD4+ T‐cell percentage was 33.7% in the young and 21.4% in the old. CD4+ cells expressing high levels of CD45RB (CD45RBhi) after activation for 20 h possessed light scatter and bright fluorescence properties characteristic of viable cells, whereas CD4+/CD45RB10 density cells could be identified as apoptotic based on their decreased CD4 fluorescence and scatter characteristics. CD4+ cells from young animals were predominantly CD45RBhi, whereas CD4+ cells from the old had greater levels of CD454RB10 cells.In addition to light scatter changes, measurement of DNA content after 40 h activation revealed the presence of a sub‐G1 DNA apoptotic peak and a viable cell cycle distribution. After 40 h of activation, there was an increase in the percentage of apoptotic cells in both young and old mice, with the greatest increase seen in the cells from older animals. Further evidence supporting the process of apoptosis in 40 h‐activated cells was confirmed by the appearance of DNA strand breaks detected by in situ nick translation. © 1995 Wiley‐Liss, Inc.
A model for evaluation of in situ hybridization spot‐count distributions in tissue sections
Authors: Pahlplatz M. M. M. , de Wilde P. C. M. , Poddighe P. , v. Dekken H. , Vooijs G. P. , Hanselaar A. G. J. M. 
Published: June 2005

AbstractThe interpretation of in situ hybridization (ISH) spot‐count distributions, obtained from evaluation of ISH signals in tissue sections, is complicated by the unknown impact of nuclear truncation and of the localization of ISH spots within the nuclei. In this study, a mathematical model was developed to investigate the effects of nuclear truncation and of the distribution of ISH spots within the nucleus on the ISH spot‐count distribution in tissue sections. In this model, it was assumed that nuclei are spherical and of constant diameter and that ISH spots have negligible size and are distributed randomly within the nucleus (“volume model”) or along the nuclear membrane (“surface model”). A minimal nuclear profile diameter was introduced in order to study the effect of rejecting small nuclear fragments for spot‐count evaluation. Given the section thickness, the nuclear size, the minimal nuclear profile diameter, and the true number of ISH spots per nucleus and their spatial distribution within the nucleus, the model predicts the proportion of nuclei observable in the section with a specific number of ISH spots. A program that performs the model calculations was developed for PC and is available upon request.For section thickness greater than 50% of the nuclear diameter, the main effect of increasing section thickness on spot‐count distributions was the increase of the proportion of nuclei with the true chromosome copy number of spots. For lower section thickness, the total distribution shifted towards lower spot frequencies. The influence of the minimal profile diameter was most notable for values close to the nuclear diameter. The effect of the localization of ISH spots within the nucleus was shown to be prominent, especially for sections with thickness smaller than the nuclear diameter. Good correspondence between model‐predicted distributions and measured distributions was obtained using the volume model and taking into account only large nuclear profiles. © 1995 Wiley‐Liss, Inc.
Dyes providing increased sensitivity in flow‐cytometric dye‐efflux assays for multidrug resistance
Authors: Frey Tom , Yue Stephen , Haugland Richard P. 
Published: June 2005

AbstractIn an effort to improve detection of P‐glycoprotein‐mediated multidrug resistance (mdr), several dyes were compared to rhodamine 123 (R123) in efflux assays. Two dyes (SY‐38 and SY‐3150) were found that provided better sensitivity. These dyes were effluxed by a cell line known to be mdr‐positive (P388/R84) but not by an mdr‐negative cell line (P388). Efflux was blocked by both verapamil and cyclosporine A. Efflux from KG1a cells was less than from P388/R84, just as has been seen with R123 and daunomycin. In further experiments, a model system was used to demonstrate two‐color immunofluorescence plus efflux measurements. This was done using a sequential staining method. A procedure was devised that, at least for this model system, allowed single‐step staining with both dye and antibody. The sensitivity of the efflux measurement was slightly compromised by using this one‐step staining method. © 1995 Wiley‐Liss, Inc.
Occurrence and a possible mechanism of penetration of natural killer cells into k562 target cells during the cytotoxic interaction
Authors: Radoškevića Katarina , van Leeuwen Anne Marie T. , Segers‐Nolten Ine M. J. , Figdor Carl G. , de Grooth Bart G. , Greve Jan 
Published: June 2005

AbstractThe cytotoxic interaction between cloned human Natural Killer (NK) cells and K562 target cells was studied using confocal laser scanning microscopy (CLSM) and conventional fluorescence microscopy. We observed, using fixed as well as living cells, the occurrence of (pseudo)emperipolesis during the interaction. About 30% of conjugated NK cells penetrated, partly or completely, into the target cells (in‐conjugation). Virtually all in‐conjugated target cells exhibited polymerized actin. Killer cells of in‐conjugates were frequently seen approaching the target cell nucleus or aligning along it. If the cytotoxic process was inhibited by the absence of calcium neither actin polymerization nor in‐conjugation were observed. A kinetic study showed that in‐conjugation starts somewhat later than actin polymerization but still within a few minutes after addition of calcium to conjugates previously formed in the absence of calcium. The presence of cytochalasin D (an inhibitor of actin polymerization) completely inhibited in‐conjugation and partly reduced the cytotoxic activity. Zinc ions (endonuclease inhibition) inhibited in‐conjugation and decreased the total number of target cells with polymerized actin in a concentration dependent manner. Cytotoxic activity was also reduced but not as efficiently as in‐conjugation.Our study demonstrates that in‐conjugation represents a significant fraction of the cytotoxic interaction. The results indicate that it may be a consequence of an actin polymerization and endonuclease activity dependent part of a cytotoxic mechanism. © 1995 Wiley‐Liss, Inc.
High affinity binding of 7‐aminoactinomycin D and 4′,6‐diamidino‐2‐phenylindole to human neutrophilic granulocytes and lymphocytes
Authors: Loborg Helena , Lindén Eva , Lönn Anita , Skoglund Per , Rundquist Ingemar 
Published: June 2005

AbstractThe binding behavior of the DNA binding dyes 7‐aminoactinomycin D (7‐AAMD) and 4′,6‐diamidino‐2‐phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7‐AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid extracted cells, supposedly containing nucleosome‐free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7‐AAMD binding sites remained in the unextracted PFA‐fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7‐AAMD compared to lymphocytes. DAPI had a much higher affinity than 7‐AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ. © 1995 Wiley‐Liss, Inc.
Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in p‐glycoprotein‐expressing MCF7 human breast adenocarcinoma cells
Authors: Merlin Jean‐Louis , Marchal Sophie , Ramacci Carole , Dieterlen Alain , Schultz Guy , Lucas Catherine , Poullain Marie‐Gwenaelle , Berlion Maryse 
Published: June 2005

AbstractA triazinoaminopiperidine derivative synthesized as a modulator of multidrug resistance, S9788, was investigated in the human brest adenocarcinoma MCF7DXR cell line expressing P‐glycoprotein. In addition to being less sensitive to daunorubicin, the resistant cell line showed dramatic alterations in the subcellular distribution of daunorubicin, as observed via fluorescence microscopy and quantified via tritiated daunorubicin nuclear distribution analysis. Compared to verapamil and cyclosporin A at 2 and 5 μol/liter, S9788 proved to be more potent in restoring the cellular accumulation and the subcellular distribution of daunorubicin in the resistant cells. Significant activity of S9788 was observed at 2 μol/liter, which is clinically achievable, and S9788 restored the nuclear distribution of the drug to the level observed in the parental sensitive cell line. Consequently, the restoration of the cytotoxicity of daunorubicin by S9788 was nearly complete (>90%) at 2 μol/liter, whereas cyclosporin A reached this level of activity at 5 μol/liter, and verapamil was always less active at both concentrations. These results suggest that the modulation of multidrug resistance by S9788 is not only related to the enhancement of the cellular accumulation but also especially by the restoration of the subcellular distribution of the drugs to their nuclear sites of action. © 1995 Wiley‐Liss, Inc.