bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth requirements of low‐density rabbit costal chondrocyte cultures maintained in serum‐free medium
Authors: Kato Yukio , Gospodarowicz Denis
Published: February 2005

The factors required for the active proliferation of low‐density rabbit costal chondrocytes exposed to 9:1 (v/v) mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium have been defined. Low‐density primary cultures of rabbit costal chondrocytes proliferated actively when the medium was supplemented with high‐density lipoprotein (300 μg/ml), transferrin (60 μg/ml), fibroblast growth factor (FGF) (1 ng/ml), hydrocortisone (10−6 M), and epidermal growth factor (EGF) (30 ng/ml). Insulin, although it slightly decreased the final cell density, was required for reexpression of the cartilage phenotype at confluence. Optimal proliferation of low‐density chondrocyte cultures was only observed when dishes were coated with an extracellular matrix (ECM) produced by cultured corneal endothelial cells, but not on plastic. Furthermore, serum‐free chondrocyte cultures seeded at low density and maintained on ECM‐coated dishes gave rise to a homogeneous cartilage‐like tissue composed of spherical cells. These chondrocytes therefore seem to provide a good experimental system for analyzing factors involved in supporting proliferation of chondrocytes and their phenotypic expression.
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

The 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

The 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

Platelets 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

Accurate 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

The 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

Experiments 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

The 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

Normal 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

Quiescent 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

Biochemical 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

The 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

The 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

The 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

Previous 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

It 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

Fluorescence 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.
Role of membrane potential in the regulation of lectin‐induced calcium uptake
Authors: Gelfand Erwin W. , Cheung Roy K. , Grinstein Sergio
Published: February 2005

Incubation of lymphocytes with mitogenic lectins triggers Ca2+ uptake. This increase in free cytoplasmic Ca2+ is postulated to be an important signal in the initiation of DNA synthesis. Transmembrane fluxes of monovalent ions and changes in membrane potential are also associated with lectin‐induced activation of lymphocytes. We have examined the relationship between extracellular monovalent ion substitution, the associated electrical potential changes (measured with cyanine dyes), phytohemagglutinin‐induced Ca2+ uptake (measured with Quin‐2) and proliferation in human T cells. The results show that (1) the magnitude of the increase in free cytoplasmic Ca2+ concentration is correlated with the extent of the lymphoproliferative response, (2) lectin‐induced Ca2+ fluxes are sensitive to membrane potential, decreasing with depolarization, and are likely conductive, and (3) the presence of extracellular Na+ during incubation with phytohemagglutinin is not essential to mitogenic triggering.
Fluid‐phase endocytosis by isolated rat adipocytes
Authors: Gibbs E. Michael , Lienhard Gustav E.
Published: February 2005

We have developed an assay, which uses radiolabeled sucrose as the marker, to measure the rate of fluid‐phase endocytosis in isolated rat adipocytes. In addition, the assay was adapted to allow measurement of the release of sucrose from previously loaded cells (fluid‐phase exocytosis). Adipocytes take up sucrose at an approximately linear rate for at least 1.5 hours. A portion of the pinocytosed sucrose is rapidly (half‐time about 20 minutes) returned to the medium. The minimal value for fluid uptake by endocytosis is 57 nl/106 cells‐h at 37°C; this value corresponds to the formation of 110,000 endocytic vesicles of 100‐nm diameter per cell per hour and the internalization of about 20% of the plasma membrane per hour. Insulin caused a small and variable increase in the rate of sucrose uptake. The average increase of 31% from 11 experiments is statistically significant at the level of P < 0.01. A small insulin effect upon the uptake of the calcium complex of [14C]EDTA was also observed. Since this complex was taken up at 2.5 times the rate of sucrose, it probably entered by a combination of fluid‐phase and adsorptive pinocytosis. Insulin did not elicit a significant change in the rate of sucrose release from preloaded cells.
Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity
Authors: Lanfrancone Luisa , Ferrero Dario , Gallo Eugenio , Foa Robin , Tarella Corrado
Published: February 2005

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long‐term cultured T cell growth factor (TCGF)‐dependent human T lymphocytes. Seven cell lines tested produced colony‐stimulating activity (CSA) as well as burst‐promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven Tcell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU‐GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long‐term culture and this property is not related to the helper or suppressor activity of the cultured cells.
Formation of proliferative tetraploid cells after treatment of diploid cells with sodium butyrate in rat 3Y1 fibroblasts
Authors: Yamada Koji , Kimura Genki
Published: February 2005

When randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.
Culture kinetics of glycolytic enzyme induction, glucose utilization, and thymidine incorporation of extended‐exposure phytohemagglutinin‐stimulated human lymphocytes
Authors: Tollefsbol Trygve O. , Cohen Harvey Jay
Published: February 2005

Glycolytic enzyme activity is significantly (P < 0.05) induced between 24 and 48 hours of incubation in phytohemagglutinin‐stimulated human lymphocytes. Nonstimulated cultured cells do not show this induction although these cells have an approximate daily doubling of thymidine incorporation. Maximal glycolytic enzyme activity is reached between 96 and 120 hours of culture in stimulated cells (3.5‐fold increase) and maintained until at least 168 hours. There is no significant induction of the hexosemonophosphate shunt or the TCA cycle during seven‐day transformation. Induction of glucose utilization becomes significantly (P < 0.05) greater in stimulated as compared to nonstimulated cultures between 48 and 72 hours of culture and is significantly elevated for at least an additional 96 hours. There is a 17% increase in total protein in the stimulated cells after 24 hours of culture and higher levels of protein content are then maintained over the control. Thymidine incorporation is significantly greater in stimulated cells from 24–144 hours of culture but is not significantly different from the nonstimulated cells at 168 hours (P = 0.98) although glycolytic enzyme activity remains elevated in the stimulated cells. There is a greater enzyme induction of the latter phase of glycolysis during transformation and this phenomenon continues in extended cultures. Increases in glycolytic enzyme activity during mitogenesis appear to be an intrinsic phenomenon independent of cell proliferation and glucose transport. The mitogen‐induced increase in the activity of the glycolytic enzymes accompanies blastogenesis and the sustained elevated activity of these enzymes appears to be related to the high metabolic rate of transformed cells.
Distribution of insulin receptors between plasmalemma and intracellular compartments: Effect of amino acids
Authors: Galbraith Richard A. , Buse Maria G.
Published: February 2005

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1–2‐hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37°C. Dissociation experiments to quantitate intracellular ligand after 37°C binding assays showed increases in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.
Utilization of iron complexes in an animal cell
Authors: Rasmussen Leif , Toftlund Hans , Suhr‐Jessen Peter
Published: February 2005

The ciliate protozoan Tetrahymena thermophila was grown in synthetic nutrient medium in the absence of the iron chelator citrate. Utilization and toxicity of various iron compounds or complexes in iron‐starved cells were assessed from the number of cell doublings obtained within a standard time. The compounds tested included complexes formed between ortho‐phosphates and two forms of ferric hydroxides, native and cationized ferritin, and tris‐acetylacetonato Fe(III). The ferric hydroxo ortho‐phosphate particles are toxic and can be removed from the medium by Millipore filtration. Uptake of ferritin and tris‐acetylacetonato‐Fe(III) is independent of food vacuole formation and seems to occur by micropinocytosis and by plasma membrane translocation, respectively.
Entry of diphtheria toxin linked to concanavalin A into primate and murine cells
Authors: Guillemot Jean Claude , Sundan Anders , Olsnes Sjur , Sandvig Kirsten
Published: February 2005

Diphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with α‐methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12‐O‐tetradecanoylphorbol‐13‐acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L‐acetamido‐4′‐isothiocyano‐stilbene‐2,2′disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells.
Limb development in chick embryos: Cyclic AMP‐dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis
Authors: Smales William P. , Biddulph David M.
Published: February 2005

The effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20–23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25–28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP‐dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80–85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane‐enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP‐dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP‐protein phosphorylation system in the differentiation of limb tissues.
Adaptive regulatory control of system a transport activity in a kidney epithelial cell line (MDCK) and in a transformed variant (MDCK‐T1)
Authors: Boerner Paula , Saier Milton H.
Published: February 2005

Adaptive regulatory control of system A activity was investigated using MDCK cells and a chemically induced, oncogenic transformant of MDCK cells, MDCK‐T1. Within 7 hours after transfer to an amino‐acid‐deficient medium, A activity of subconfluent MDCK cells had maximally derepressed, but this activity in confluent MDCK cells and in subconfluent transformed cells showed little capacity for derepression. Amino‐acid‐starved, subconfluent MDCK cells were used to study trans‐inhibition and repression of A activity by individual amino acids. Trans‐inhibition and repression were defined as the cycloheximide‐insensitive and cycloheximide‐sensitive components, respectively, of the total inhibition. Trans‐inhibition correlated well with substrate affinity, but repression did not. Trans‐inhibition and repression were further characterized using α‐(methylamino) isobutyric acid (mAIB), a trans‐inhibitor, and glutamate, an effective repressor. The apparent initial T1/2 for inhibition by mAIB in the presence of cycloheximide was 0.5 hours, while that for repression by glutamate was 4.7 hours. Half‐maximal inhibition by mAIB and repression by glutamate occurred at approximately 0.02 mM and 0.07 mM, respectively. Reversal of trans‐inhibition by methionine occurred in the presence of cycloheximide within 1–4 hours after removal of methionine. The A system of the transformed MDCK‐T1 cells showed elevated activity, little capacity for derepression, resistance to repression by amino acids, but retention of sensitivity to trans‐inhibition. Kinetic analysis of mAIB uptake indicated that the A system of MDCK‐T1 cells has become kinetically more complex in a manner which resembled amino‐acid‐starved rather than amino‐acid‐fed MDCK cells. These results suggest that the A system of MDCK‐T1 cells has become resistant to adaptive regulatory control.
Neutral amino acid transport in embryonal carcinoma cells
Authors: Zuzack John S. , Tasca Richard J. , DiZio Stephen M.
Published: February 2005

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems—A, L, and ASC—although one system may make a barely measurable contribution in some cases. The characterization of N‐methyl‐aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single‐component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+‐independent leucine transport, reveals a high‐affinity, single‐component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2‐aminobicyclo‐[2,2,1]heptane‐2‐carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenyialanine is primarily transported by Na+ ‐dependent Systems A and ASC (83% Na+‐dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+‐independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+‐dependency of phenyl‐alanine transport in mouse uterine blastocysts (82% Na+‐dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+‐independent, BCH‐inhibitable mechanism.
Uridine phosphorylase from Novikoff rat hepatoma cells: Purification, kinetic properties, and its role in uracil anabolism
Authors: McIvor R. Scott , Wohlhueter Robert M. , Plagemann Peter P. G.
Published: February 2005

Uridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330‐fold from Novikoff rat hepa‐toma cells by ammonium sulfate precipitation, DEAE‐Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G‐200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 μM, KmRib−1‐P = 88 μM, KmUrd = 16 μM, and KmPi = 130 μM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 μM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose‐1‐phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N‐(phosphona‐cetyl)‐L‐aspartate (“PALA”), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose‐1‐phosphate plays a rate‐limiting role in the uptake of uracil mediated by uridine phosphorylase.
The effects of lysosomotropic amines on protein degradation, migration of nonhistone proteins to the nucleus, and cathepsin D in lymphocytes
Authors: Polet Herman
Published: February 2005

Various lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit the degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit the degradation of short‐and long‐lived proteins equally. Fractionation of the [3H] NHP of the nucleus according to pH 2.5‐6.5 shows that [3H] NHP with a high rate of degradation in untreated cells correspond to [3H] NHP with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit the cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of NHP from the cytoplasm to the nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines.
Requirement of spermidine for induction of both heme synthesis and globin transcription in murine erythroleukemia cells
Authors: Watanabe T. , Shafman T. , Kufe D. W.
Published: February 2005

We have previously demonstrated that spermidine is required for proliferation and differentiation of murine Friend erythroleukemia (MEL) cells. Other studies have indicated that inhibition of MEL differentiation by dexamethasone (DEX) and 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) is reversed by the addition of exogenous polyamines. The present work has thus monitored the requirements of polyamines for induction of MEL hemoglobin synthesis by dimethyl sulfoxide. The results demonstrate that spermidine depletion inhibits induction of both heme synthesis and transcription of α‐ and β‐globin mRNA. In contrast, the results also demonstrate that polyamines are not involved in the inhibition of MEL differentiation by DEX and TPA. Thus, spermidine is required at a transcription level for induction of MEL hemoglobin synthesis, but DEX and TPA act by mechanisms other than polyamine depletion.
Regulation of K562 cell transferrin receptors by exogenous iron
Authors: Rudolph Natalie S. , Ohlsson‐Wilhelm Betsy M. , Leary James F. , Rowley Peter T.
Published: February 2005

Single‐cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 μg/ml/ by the addition of iron in the form of FeCl3, iron‐saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8‐fold increase in external receptors, detected as binding by both transferrin and monoclonal anti‐TfR antibodies, and a 1.5‐fold increase in the pool of internal receptors, as detected by anti‐TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cyclohexionide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.