bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biochemical effects of 12‐O‐tetradecanoylphorbol‐13‐acetate, retinoic acid, phenobarbital, and 5‐azacytidine on a normal rat liver epithelial cell line
Authors: Tsao Ming‐Sound , Nelson Karen G. , Grisham Joe W. 
Published: February 2005

AbstractThe effects of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), all trans‐retinoic acid (RA), 5‐azacytidine (5‐AC), and phenobarbital (PB) on the activities of seven enzymes and/or isozymes of a diploid rat liver epithelial cell line have been studied. At 0.1 μg/ml, TPA depressed the specific activities of lactate dehydrogenase and gamma‐glutamyl transpeptidase, whereas 2 mM PB depressed gamma‐glutamyl transpeptidase and alkaline phosphatase. At 0.01 μg/ml, RA markedly depressed the activity of NADH‐diaphorase and lactate dehydrogenase but enhanced the activity of alkaline phophatase. Only 2 μM 5‐AC caused the most significant shift of lactate dehydrogenase isozyme toward the “muscle”‐type isozyme. Histochemical studies revealed that PB and 5‐AC induced focal areas of cells with glycogen deposits, but no significant changes in either ultrastructure or alpha‐fetoprotein and albumin immunohistochemical staining pattern were observed to suggest hepatocytic differentiation. Although none of the enzymatic changes could be consistently correlated with the effects of these biological modifiers on the cellular growth rate, the effect of RA on NADH‐diaphorase, lactate dehydrogenase, and alkaline phosphatase activities was the opposite of the changes observed during carcinogenesis of these rat liver epithelial cells by multiple treatments with N‐methyl‐N′‐nitro‐N‐nitrosoguanidine. The depression of gamma‐glutamyl transpeptidase activity by PB is contradictory to that observed histochemically in hepatocytes in vivo, but such discrepancy may be related to the differences in cell type, growth conditions, or duration of exposure.
Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum‐free medium: Clonal analyses, growth kinetics, and cell cycle studies
Authors: Wille John J. , Pittelkow Mark R. , Shipley Gary D. , Scott Robert E. 
Published: February 2005

AbstractThe effects of growth factors, hormones, and calcium on the growth and differentiation of secondary cultures of normal human prokeratinocytes, i.e., proliferative keratinocytes, derived from adult or neonatal skin were determined by culture in serum‐free basal medium, MCDB 153. Clonal growth was achieved when MCDB 153 was supplemented with either epidermal growth factor (EGF) or bovine pituitary extract (BPE), provided insulin was present. In the absence of insulin, however, both EGF and BPE were required for clonal growth. Using this assay, it was established that colony‐forming efficiency is independent of calcium concentrations above 0.03 mM and averages 56%; colony size, however, was influenced by calcium and EGF concentrations. Optimal clonal growth occurred in medium containing 10 ng/ml EGF and 0.3 mM calcium. By contrast, differentiation was enhanced by the combination of low EGF (0.1 ng/ml) and high calcium (2 mM). This suggests that an inverse relationship exists between the growth response (extent of clonal growth) and the differentiation response (extent of differentiation). These results suggest that proliferation and differentiation are regulated in an integrated manner. Detailed kinetic studies and cytofluorimetric and autoradiographic analyses also showed that exponentially growing secondary cultures of adult and neonatal prokeratinocytes have a 24‐hour cell generation time with G1, S, G2, and M phases of 12, 8, 3, and 1 hours, respectively. In addition, the data show that such cells can be growth arrested in medium that does not induce differentiation and that such a procedure significantly limits the cell's subsequent proliferative potential. Furthermore, prolonged culture of adult (> 30 population doublings) and neonatal prokeratinocytes (> 50 population doublings) is associated with senescence and the G1 arrest of noncycling cells.
The organization of microfilaments in spreading platelets: A comparison with fibroblasts and glial cells
Authors: Karlsson Roger , Lassing Ingrid , Höglund Anna‐Stina , Lindberg Uno 
Published: February 2005

AbstractPlatelets respond to stimulatory agents in general by the formation of long spikelike surface projections built up of tightly bundled microfilaments. During contact stimulation this is followed by a second phase when thin membrane lamellae grow out between the projections. This behaviour resembles that seen for instance in fibroblasts and glial cells, sending out microspikes and lamellipodia as a step in their advancement over solid substrata. Conditions, designed earlier for the preservation and visualization of the fragile organization of microfilaments and microtubules in the peripheral, highly motile parts (leading lamellae) of such cells (Höglund et al. (1980) J. Musc. Res. Cell Motility, 1:127–146), were used here to produce high‐resolution images of the ultrastructural organization of platelets spreading on a solid substratum. This revealed an unexpected arrangement of actin filaments running parallel to the advancing edge, and small tufts of microfilaments on the outside of this edge‐bundle. Cytochalasin D caused a regression of the spikelike projections as well as of both types of structures in the advancing platelet lamella and led to the appearance of a dense filamentous mat in juxtaposition to the plasma membrane. Analysis of the actin pools using the DNAase inhibition assay showed that the dramatic reorganizations of actin seen during the two phases of contact stimulation was reflected in a shift in the G/F‐actin ratio only during the early phase.
Kinetics of clonogenic melanoma cell proliferation and the limits on growth within a bilayer agar system
Authors: Thomson Stephen P. , Moon Thomas E. , Meyskens Frank L. 
Published: February 2005

AbstractAccurate descriptions of the kinetics of cell growth in semi‐solid agar clonogenic systems have been difficult because the number of cells in colonies of different sizes is largely unknown. We stained and removed tumor cell colonies from agar, directly counted their cells, and established equations to quantitate the number of cells within colonies of different sizes. We used these equations to quantitate, in terms of cell number and volume, the total amount and kinetics of clonogenic cell proliferation from biopsies of human melanoma and cell lines of several different tumor types. Daily observations of cells in agar and serial photography indicated a 0‐ to 4‐day delay in the onset of proliferation in agar followed by rapid growth and then abrupt cessation of proliferation. We quantified the extent of proliferation of cells from melanoma biopsies of seven patients and 11 cell lines after they were allowed to proliferate in agar until they stopped. Approximately 10% of cells divided one to five times while only 0.01% divided six to nine times. The total number of cells within the colonies at the end of growth was different while the total volume of cells within the colonies per plate was similar; approximately 109 μm3 cellular volume per plate represents an upper limit for proliferation within the closed, nonrefed bilayer agar system. Previous replating studies using the same biopsy cells have shown that clonogenic melanoma cells can self‐renew and have more proliferative capacity than that expressed during primary colony formation. Thus, the clonogenic assay only measured initial proliferative capacities. Furthermore, variable delays in the onset of proliferation may contribute to the heterogenity of colony size within clonogenic assays.
Characterization of monovalent ion transport systems in an insect cell line (Manduca sexta embryonic cell line che)
Authors: English Leigh H. , Cantley Lewis C. 
Published: February 2005

AbstractThe monovalent ion transport systems of an immortalized insect cell line (CHE) have been investigated. These cells are unusual in that unlike most vertebrate cells, their normal extracellular environment consists of high potassium and low sodium concentrations. CHE cells maintained high intracellular [K+] through both a furosemide‐inhibitable and a vanadate‐inhibitable transport system. Intracellular exchangeable [Na+] was slightly lower than the extracellular [Na+] and was maintained at this level through a vanadate‐sensitive transport system. Na+ uptake was also inhibited by furosemide: however, the stoichiometry of furosemide‐sensitive Na+ uptake when compared with furosemide‐sensitive K+ uptake indicated that these cations are not cotransported. 4,4′‐Diisothiocyano‐2,2′‐disulfonic acid stilbene (DIDS) inhibited Na+, K+, and Cl− uptake. Vanadate and furosemide decreased cytoplasmimic pH, while cytoplasmic pH increased in the presence of DIDS. A model is presented explaining how Na+, K+, Cl−, H+ and HCO3 − fluxes are regulated in these cells.
Collagen degradation in human lung fibroblasts: Extent of degradation, role of lysosomal proteases, and evaluation of an alternate hypothesis
Authors: Bienkowski Robert S. 
Published: February 2005

AbstractExperiments were conducted to determine the extent and variability of collagen degradation in human fetal lung fibroblasts. Cells were incubated with [14C]proline, and degradation was measured by determining the hydroxy[14C]proline in a low molecular weight fraction relative to total hydroxy[14C]proline. Average (basal) degradation in stationary phase HFL‐1 cells incubated for 8 h was 16 ± 3%, and substantial alterations in the composition of the labeling medium, e.g., omitting serum and varying pH between 6.8 and 7.8, had no effect. Organic buffers slightly lowered degradation in a manner that was independent of pH. Collagen degradation in two other lung cell lines, Wl‐38 and IMR‐90, did not differ from the level in HFL‐1. Degradation was significantly higher (23 ± 5%) in HFL‐1 cultures labeled for 24 h rather than 8 h, and pulse‐washout experiments showed that the rate of degradation was not uniform: after an 8‐h pulse, 11% of the hydroxy [14C]proline in the medium was in the low molecular weight fraction, but 31% was in this fraction after a 16‐h washout. The lack of effect of either serum deprivation or elevated pH suggests that lysosomal proteases have no direct role in basal degradation; however, NH4Cl decreased the enhanced degradation observed in ascorbate deficiency to basal level, indicating that abnormal molecules synthesized under those conditions are degraded by lysosomal proteases. The appearance of small hydroxy[14C]proline‐containing molecules was inhibited by αα′dipyridyl and cycloheximide in a dose‐dependent and reversible manner, demonstrating that their production depends on enzymatic hydroxylation of proline and protein synthesis.
Factors affecting the generation of mast cells from multipotential colony‐forming cells
Authors: Li C. L. , Johnson G. R. 
Published: February 2005

AbstractThe cells responsible for the long‐term in vitro generation of murine mast cells have been examined. Sequential analysis of all colony types obtained from cultures of spleen or bone marrow cells showed that only colonies derived from multipotential cells (mixed‐erythroid colonies) or mast cell progenitors, contained cells responsible for mast cell generation in liquid cultures. Primary colony growth and subsequent maintenance of mast cells in liquid cultures was dependent upon pokeweed mitogen‐stimulated spleen cell‐conditioned medium (SCM). Mixed‐erythroid colonies from 14‐day cultures of spleen cells had the greatest capacity for mast cell generation. Analysis by clone splitting and transfer to high (20%) and low (2.5%) concentrations of SCM showed that the concentration of SCM used in either the primary colony culture or subsequent liquid culture phase altered both the proliferative capacity of the mast cells generated and the frequency of mast cell progenitors within individual mixed‐erythroid colonies. Thus, mixed‐erythroid colonies stimulated with 2.5% SCM contained the highest proportion of mast cell progenitors (34% of colonies) and when stimulated with 20% SCM, approximately fourfold higher numbers of mast cells were produced at weekly intervals from liquid cultures maintained in 2.5% SCM compared to parallel liquid cultures containing 20% SCM. These studies confirm the hemopoietic origin of mast cells and demonstrate that a factor(s) in SCM is able to modulate their proliferative potential.
Normal embryo fibroblasts release transforming growth factors in a latent form
Authors: Lawrence David A. , Pircher Renée , Krycève‐Martinerie CéCile , Jullien Pierre 
Published: February 2005

AbstractNormal chicken, mouse, and human embryo fibroblasts release into their culture media transforming growth factors (TGFs) in a latent form. Their soft agar colony‐forming activity on two widely used target cells, rat NRK‐49F and mouse AKR‐2B, is essentially revealed only after prior acidification of cell‐conditioned media. These TGFs are EGF‐dependent when assayed on NRK‐49F cells and EGF‐independent on AKR‐2B cells. The TGF activity from the chicken source is released in three (apparent) molecular weight forms of 500 kd, 125 kd, and 20 kd.
Sodium orthovanadate stimulation of DNA synthesis in nakano mouse lens epithelial cells in serum‐free medium
Authors: Jones Trevor R. , Reid Ted W. 
Published: February 2005

AbstractQuiescent cultured Nakano mouse lens cells incubated for 40 hours with sodium orthovanadate incorporated 3H‐thymidine at an accelerated rate; the greatest response occurred at 20 μM vanadate, whereas by 2 μM an incorporation rate equivalent to unstimulated cells was noted. Microscopic examination of the cells revealed that those exposed to concentrations of vanadate greater than 100 μM had lysed by the end of the 40‐hour incubation. Reduction in vanadate exposure time to 1 hour caused the cells to incorporate the greatest amount of 3H‐thymidine at a vanadate concentration of 200 μM to 500 μM. Half‐maximum incorporation of 3H‐thymidine (after a 40‐hour incubation) was induced by a 2‐hour incubation with 20 μM vanadate. Studies with insulin showed that while 20 ng/ml insulin alone did not increase 3H‐thymidine incorporation, 20 ng/ml insulin in combination with 20 μM vanadate resulted in a significant increase in 3H‐thymidine uptake over cells exposed to only vanadate. Insulin alone will increase cell number and insulin with vanadate are synergistic in the stimulation of DNA synthesis, but the two together show no further increase in cell number over that produced by insulin alone. Thus, vanadate can increase progression from G1/G0 to S‐phase, but cannot stimulate cells to divide. Studies designed to detect DNA damage and repair rather than S‐phase DNA synthesis demonstrated that vanadate was not causing increased 3H‐thymidine uptake by damaging DNA. Cell counts revealed that vanadate, while able to induce DNA synthesis, does not induce mitosis. Autoradiography and equilibrium sedimentation experiments demonstrated that gene amplification was not occurring. A known vanadate exchange inhibitor blocked the ability of vanadate to increase 3H‐thymidine incorporation which is consistent with the idea that cellular internalization of vanadate is required for this effect to be seen. 86Rb+ uptake experiments demonstrate that the vanadate concentration inducing 50% inhibition of (Na+, K+)ATPase is nearly two orders of magnitude more concentrated that vanadate concentrations shown capable of inducing 3H‐thymidine uptake. This strongly suggests that (Na+, K+)ATPase inhibition is not the central mechanism by which DNA synthesis is stimulated by vanadate.
Sodium‐potassium exchange in sea urchin egg. I. Kinetic and biochemical characterization at fertilization
Authors: Ciapa B. , de Renzis G. , Girard J. P. , Payan P. 
Published: February 2005

AbstractBiochemical and kinetic characteristics of the Na+‐K+ exchange were studied in Paracentrotus lividus eggs. Measurement of the 86Rb uptake shows that ouabain‐sensitive 86Rb uptake is dramatically stimulated within the first minute following fertilization. The Na+‐K+ pump‐mediated K+ entry presents a maximal rate at 8 min postfertilization and then decreases to reach a plateau within 30 min. We assess that the steep rise in cell K+ occurring at fertilization (J.P. Girard, P. Payan, C. Sardet, Exp. Cell. Res. 142:215–221, 1982) does not originate from a net entry of external K+. Measured 30 min postfertilization, the half‐maximal activation by K+ of the ouabain‐sensitive Na+‐K+ exchange is 5–6 mM and the ouabain lC50 is 5.10−5 M. Egg cortices from unfertilized and fertilized eggs show comparable Na+‐K+ ATPase activity with a 50% ouabain‐sensitive fraction. Vm and Km for Na+ and K+ of the enzyme are of the same order of magnitude in cortices of unfertilized and fertilized eggs. Cortical Na+‐K+ ATPase from unfertilized eggs shows a ten fold increase of activity between pH 6.7 and pH 7.7. The results strongly suggest that the plasma membrane of unfertilized eggs contains a preexisting Na+‐K+ transporting system which is obligatorily stimulated at fertilization.
The role of iron in the growth of human leukemic cell lines
Authors: Titeux Monique , Testa Ugo , Louache Fawzia , Thomopoulos Pierre , Rochant Henri , Breton‐Gorius Jeanine 
Published: February 2005

AbstractThe growth requirements of three human leukemic cell lines (K 562, HEL, U937) have been studied in the absence of serum. For growth in serum‐free medium, the cells require insulin, transferrin, and albumin. Two highly watersoluble iron salts, ferric ammonium citrate and ferric ammonium sulfate, may completely replace transferrin for supporting the growth of these cell lines. Similar results were obtained when mitogen‐stimulated lymphocytes were grown in serum‐free media. Iron containing compounds, such as hemin or hemoglobin, were also able to replace transferrin. Experiments using 42/6 monoclonal antibody strongly suggest that free‐iron salts are taken up by the cells by a mechanism that is completely independent from transferrin receptors.
A cloning assay for 6‐thioguanine resistance provides evidence against certain somatic mutational theories of aging
Authors: Horn Peggy L. , Turker Mitchell S. , Ogburn Charles E. , Disteche Christine M. , Martin George M. 
Published: February 2005

AbstractThe frequencies of 6‐thioguanine‐resistant primary clones from the kidneys and skeletal muscles of aging male cohorts of two F1 hybrid strains of Mus musculus varied from 0.59 to 10.96 × 10−5 and did not increase as a function of donor age (up to 40 months). Resistant clones were shown to be severely deficient in the activity of hypoxanthine‐guanine phosphoribosyltransferase (EC 2.4.2.8). These deficiencies presumably resulted from molecular alterations at this X‐linked locus, including point mutations. No alterations of the X‐chromosome were observed at the level of the light microscope. These results are inconsistent with predictions of the intrinsic mutagenesis and protein synthesis error catastrophe theories of aging. They do not rule out, however, somatic mutational theories that invoke comparatively large‐scale chromosomal lesions, many of which would be likely to be lethal at the cellular level.
Internalization and acidification of insulin by activated human lymphocytes
Authors: Murphy Robert F. , Bisaccia Emil , Cantor Charles R. , Berger Carole , Edelson Richard L. 
Published: February 2005

AbstractThe binding and internalization of fluorescein isothiocyanate‐conjugated insulin by nonactivated and phytohemagglutinin‐activated circulating human lymphocytes was measured by flow cytometry. In confirmation of previous results, negligible binding or internalization was observed for unstimulated cells, while activated lymphocytes showed significant insulin binding. The majority of this insulin was demonstrated to be internalized via receptor‐mediated endocytosis and acidified within 60 min after addition of insulin. Dual‐fluorescence flow cytometry, using antibodies specific for human T cell subsets, was used to show that the expression of insulin binding sites occurs for at least some cells from both the helper/inducer and cytotoxic/suppressor T cell subsets. Insulin internalization is not an artifact of in vitro stimulation, since more than 90% of the unstimulated lymphocytes from a patient with a helper T cell leukemia are positive for insulin internalization. The usefulness of flow cytometric analysis for measuring lymphocyte activation in unstimulated populations and the therapeutic potential of the reported findings for control of lymphocyte proliferation are discussed.
Effects of 2′,3′‐dideoxynucleosides on mammalian cells and viruses
Authors: Waqar M. Anwar , Evans Mary Jo , Manly Kenneth F. , Hughes Robert G. , Huberman Joel A. 
Published: February 2005

AbstractPrevious studies on the biological effects of the 2′,3′‐dideoxynucleosides (ddNs) have shown that while ddAdo is lethal to E. coli, ddThd has minimal effects on the growth of mammalian cell lines and that it inhibits retrovirus infection of some cell lines but not others. Previous studies have also shown that the 5′‐triphosphate of ddThd, ddTTP, selectively inhibits cellular DNA polymerases β and γ and retroviral reverse transcriptases. Cellular DNA polymerase α is relatively resistant to ddTTP. We have extended these findings to show that the 5′‐triphosphates of the other 3 ddNs (ddATP, ddCTP, and ddGTP) affect cellular DNA polymerases α, β, and γ in the same fashion as does ddTTP. We also show that all four ddNs in concentrations up to 100 μM have negligible effects on the growth of NIH Swiss 3T3 cells. These negligible effects may be due to inefficient intracellular phosphorylation of each nucleoside to the triphosphate. We have determined that, in several different cell lines, ddThd is phosphorylated only at a very slow rate to ddTTP, and in the one cell line tested (monkey CV‐1 cells), ddAdo and ddGuo are also poorly phosphorylated. Both ddAdo and ddGuo, and probably ddThd, are converted by CV‐1 cells to additional unknown compounds which may have biological activity. The four ddNs display effects of different magnitudes on certain virus infections. Although 30 μM ddThd inhibits herpes simplex I infection of CV‐1 cells by 50%, 30 μM ddAdo has no effect. Infection of NIH Swiss 3T3 cells by 334C murine leukemia virus is inhibited 70–80% by ddAdo, ddCyd, and ddThd at 50 μM, but inhibition by 50 μM ddGuo is 100%.
Detection of intranuclear clusters of Sm antigens with monoclonal anti‐Sm antibodies by immunoelectron microscopy
Authors: Eliceiri George L. , Ryerse Jan S. 
Published: February 2005

AbstractIt has been shown that small nuclear RNA (snRNA) species U1, U2, U4, U5, and U6 are found in the nucleus in the form of small nuclear ribonucleoprotein particles (snRNPs), and that anti‐Sm antibodies react with snRNP polypeptides, which are associated with all five snRNAs. We report here a novel intranuclear complex, denoted “Sm cluster,” detected by immunostaining with monoclonal anti‐Sm antibodies in HeLa cells.
Lectin receptor proximity on HL‐60 leukemia cells determined by fluorescence energy transfer using flow cytometry
Authors: Jenis Diane M. , Stepanowski Anita L. , Blair Owen C. , Burger Douglas E. , Sartorelli Alan C. 
Published: February 2005

AbstractFluorescence energy transfer using flow cytometric measurements was utilized to determine the proximity of concanavalin A receptors on the surface of HL‐60 promyelocytic leukemia cells before and after induction of differentiation. The HL‐60 cells were induced to differentiate into granulocytes using dimethylsulfoxide and into macrophages using 12‐O‐tetradecanoylphorbol‐13‐acetate. Concanavalin A was labeled with either fluorescein (donor chromophore) or tetramethylrhodamine (acceptor chromophore), and these species were used to determine lectin proximity. With granulocytic differentiation, the amount of concanavalin A bound remained constant, but a decrease in receptor density was observed. During macrophage differentiation, however, both receptor density and receptor number increased. The increase in concanavalin A binding during differentiation appears to be a result of maturation rather than an initiating event.