bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lymphocyte membrane potential and Ca2+‐sensitive potassium channels described by oxonol dye fluorescence measurements
Authors: Wilson H. Alexander , Chused Thomas M. 
Published: February 2005

AbstractA method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (Ψ) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of Ψ than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte Ψ was calculated to be −70 mV. T and B lymphocytes maintain a constant Ψ as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (Ek) is shown to result from Na+ permeability. At [K+]o values greater than 10 mM, lymphocytes behave as K+ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca2+‐sensitive K+ channel by pharmacologic manipulations. Hence, T cells depolarized by 4‐aminopyridine (4‐AP, 10 mM) were observed to return to resting Ψ by A23187‐induced elevation of [Ca2+]i. Cells depolarized by quinine (100 μM) were unaffected by A23187. The Ca2+‐activated channel does not contribute to resting Ψ in T cells since it may be selectively blocked by quinine (20 μM) or modulated by calmodulin antagonists (5 μM trifluperazine) without affecting resting Ψ.
Induction of metallothionein synthesis in cultured cells derived from rabbit kidney
Authors: Koizumi Shinji , Sone Toshio , Kimura Masami 
Published: February 2005

AbstractMetallothionein (MT) synthesis in rabbit kidney‐derived RK‐13 cells was studied. In response to Cd2+, RK‐13 cells synthesized proteins closely similar in chromatographic and electrophoretic behaviors to the liver MTs induced in Cd2+ ‐injected rabbit. These proteins were specifically immunoprecipitated by anti‐mouse liver MT‐II serum. The rate of RK‐13 thionein (apoprotein of Mt) synthesis rapidly increased after exposure to 1μg/ml of Cd2+, and reached the maximum in 7 h. The dose‐response curve for the synthesis was biphasic; a sharp increase up to 0.5 μg/ml Cd2+ and a slower increase at higher concentrations. RK‐13 cells retained kidney‐specific properties in terms of responsiveness of thionein synthesis to inducers; The MTs were inducible also by Zn2+ and probably by Hg2+, but not by dexamethasone. This system would therefore be a useful model in vitro for studying the regulation of MT synthesis in kidney cells.
Adenosine uptake, transport, and metabolism in human erythrocytes
Authors: Plagemann Peter G. W. , Wohlhueter Robert M. , Kraupp Martin 
Published: February 2005

AbstractUsing rapid kinetic techniques, we have determined the kinetics of zero‐trans influx and equilibrium exchange of adenosine, and its uptake and in situ phosphorylation at 25°C in human erythrocytes which were pretreated with 2′‐deoxycoformycin to inhibit deamination of adenosine. Both the Km and Vmax for adenosine transport were about 300 times higher than those for the in situ phosphorylation of adenosine (Km about 0.2 μM), so that the first order rate constants for both processes were about the same. In contrast, the first order rate constant for adenosine deamination by untreated, intact cells was about 20% of that of adenosine transport or phosphorylation. These kinetic properties of the various steps, in combination with substrate inhibition of adenosine phosphorylation above 1 μM adenosine, assure that, at extracellular concentrations of physiological relevance ( < 1 μM), adenosine is very rapidly and efficiently salvaged by the erythrocytes and converted to ATP, whereas at extracellular concentrations of 10 μM or higher, practically all adenosine transported into the cells is deaminated. When the concentration of adenosine was 0.1 μM, a 10% (v/v) suspension of erythrocytes depleted the extracellular fluid of adenosine within 1 min of incubation at 25°C.
Growth requirements and characterization of rat cervical epithelial cells in culture
Authors: Wright Thomas C. , Nahabedian Christine 
Published: February 2005

AbstractThe extended culture of rat cervical epithelial cells can be achieved in the absence of a fibroblast feeder layer by utilizing collagen gels and a complex growth medium. The medium contains a 1:1 mixture of RPMI‐1640 and Ham's F12 supplemented with 7.5% porcine serum and epidermal growth factor, cholera toxin, transferrin, insulin, and hydrocortisone, Under these culture conditions the cells show rapid log‐phase growth and high saturation densities while retaining the ultrastructural characteristics of immature squamous metaplastic cells of the rat uterine cervix even after extended passage. In a manner similar to epithelial cells from a variety of sources, rat cervical epithelial cells form hemicysts at confluence in vitro when cultured on impermeable substrates. The development of these methods for culturing cervical epithelial cells provides an experimental system for the study of factors important in regulating the growth and differentiation of metaplastic squamous epithelial cells.
Isolation and characterization of hexose transport mutants in L6 rat myoblasts
Authors: D'Amore Tony , Duronio Vincent , Cheung Matthias O. , Lo Theodore C. Y. 
Published: February 2005

AbstractA method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2‐deoxy‐D‐glucose (2‐DOG)‐and 2‐deoxy‐2‐fluoro‐D‐glucose (2FG)‐resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2‐DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2‐DOG, 2FG, or 3‐O‐methyl‐D‐glucose (3‐OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four‐ to five‐fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for α‐aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2‐DOG and 3‐OMG transport in mutant F72 demonstrated that the Vmax for 2‐DOG uptake was significantly reduced, whereas the Vmax for 3‐OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high‐affinity hexose transport system.
Proteoglycans synthesized by gingival fibroblasts derived from human donors of different ages
Authors: Bartold P. Mark , Boyd Robin R. , Page Roy C. 
Published: February 2005

AbstractThe proteoglycans synthesized by fibroblasts derived from human donors of ages ranging from 12 years to 68 years have been studied. In addition, the in vitro proliferation rates of the various cell strains were studied and demonstrated that increasing donor age correlated with a decrease in proliferative activity. The incorporation of [35S]‐sulfate into proteoglycans decreased with increasing donor age with cells from the oldest donor demonstrating a 50% reduction compared with cells from the youngest donor. Analysis on Sepharose CL‐4B of isolated [35S]‐labeled proteoglycans for molecular size distribution revealed few differences between the cell‐layer‐associated proteoglycans of all cell strains studied. However, analysis of the medium‐associated [35S]‐labeled proteoglycans demonstrated an increase in the amount of small molecular size proteoglycans with increasing age. More specific analysis of the glycosaminoglycan composition revealed an increase in heparan sulfate from 52% to 73% in the cell‐layer‐associated proteoglycans of cells from the youngest and oldest donors, respectively. Accompanying this increase was a relative decrease in dermatan and chondroitin sulfate content from 24% to 13% and 25% to 16%, respectively, with increasing donor age. Additionally, the degree of N‐sulfation of cell layer heparan sulfate increased with age. Heparan sulfate levels increased in the medium as well with increasing age, with a concomitant decrease in chondroitin sulfate. The quantity of medium‐derived dermatan sulfate remained relatively evenly distributed throughout the various ages studied. The various differences noted are considered to reflect the general metabolic changes associated with aging. In particular the increase in heparan sulfate content with age is considered to be related to the decreased proliferative activity of the fibroblasts with increasing age.
Studies of DNA polymerases alpha and beta from cultured human cells in various replicative states
Authors: Krauss Sharon Wald , Linn Stuart 
Published: February 2005

AbstractDNA polymerase activities from HeLa cells and from cultured diploid human fibroblasts in various growth states were compared. α‐Polymerase activities from log phase fibroblasts treated with sodium butyrate and from stationary phase HeLa cells had DEAE‐cellulose elution patterns that differed from those of polymerases from dividing cells. Moreover, α‐ and β‐polymerases from nondividing cells replicated synthetic polymers less faithfully. Although similar changes were observed previously for polymerases from late‐passage and postconfluent early passage fibroblasts, amounts of α‐polymerase activity recovered from nondividing cells in this study did not dramatically decline as they had in the former cases. The α‐polymerase activities from HeLa cells and fibroblasts in various growth states sedimented near 7.5S in 0.4 M KCl and could be inhibited by a monoclonal lgG fraction prepared against KB cell α‐polymerase. By several criteria, there was no significant differences in levels of UV‐stimulated repair synthesis observed in early or late‐passage postconfluent fibroblasts or in log phase fibroblasts treated with sodium butyrate. In summary, levels of α‐polymerase do not necessarily correlate either with replicative activity or with apparent levels of repair synthesis. However, cells with decreased replicative activity always yielded enzyme with decreased fidelity in vitro and altered chromatographic behavior. It appears, therefore, that the alterations observed for α‐polymerase from late‐passage cells may be attributed more generally to the nondividing nature of these cells.
Gangliosides stimulate astroglial cell proliferation in the absence of serum
Authors: Katoh‐Semba Ritsuko , Facci Laura , Skaper Stephen D. , Varon Silvio 
Published: February 2005

AbstractPrevious studies with microcultures of astroglial (AG) cells from newborn rat cerebrum had shown an ability of gangliosides to interact with AG cells cultured under defined conditions. We have now investigated the capability of gangliosides to stimulate DNA synthesis and cell number increases in similar secondary microcultures of newborn rat cerebrum AG cells. At a concentration of 6 × 10−5M, GM1 ganglioside stimulated DNA synthesis and increased cell numbers, with DNA synthesis leading cell increases by 12–24 hr. The ganglioside‐induced AG cell proliferative response occurred with GD1a, GD1b and GT1b, GT1b being the most potent at 10−5M—while asialo GM1 and sialic acid were without effect. In the standard test cultures, DNA synthesis declined very steeply after the first day, with cell numbers stabilizing at the level reached after 2 days. Ganglioside was not itself responsible for the restricted proliferative response, as serum produced the same behaviors.
Effects of hypoxia and hyperoxia on proteoglycan production by bovine pulmonary artery endothelial cells
Authors: Humphries Donald E. , Lee Sheu‐Ling , Fanburg Barry L. , Silbert Jeremiah E. 
Published: February 2005

AbstractBovine pulmonary artery endothelial cells in culture were used to assess the influence of oxygen tension on proteoglycan synthesis. Cells exposed to 3% O2 (hypoxia) for 72 h and then labeled with [35S]sulfate for 5 h accumulated significantly less [35S]proteoglycan in medium than cells exposed to 20% O2 (control). This decrease was due primarily to a reduction in heparan sulfate. Cells exposed to 80% O2 (hyperoxia) for 72 h secreted slightly more [35S]proteoglycan into medium than controls. Greater accumulation of chondroitin sulfate was responsible for the increase. The amount of cell‐associated proteoglycan did not change significantly in cells cultured in 3% or 80% O2 as compared with control cells cultured in 20% O2. Proteoglycans produced by hypoxia‐ or hyperoxia‐treated cells were found to be similar in size to proteoglycans produced by cells cultured at 20% O2. Glycosaminoglycan sulfation, as measured by ion‐exchange chromatography, did not appear to change with varying oxygen tensions. Our results demonstrate that production of proteoglycans secreted by endothelial cells in culture is sensitive to oxygen tension.
Markedly altered membrane transport and intracellular binding of vincristine in multidrug‐resistant Chinese hamster cells selected for resistance to vinca alkaloids
Authors: Sirotnak F. M. , Yang C.‐H. , Mines L. S. , Oribé E. , Biedler J. L. 
Published: February 2005

AbstractStudies of a multidrug‐resistant variant (DC‐3F/VCRd‐5L) of Chinese hamster lung cells selected for resistance to vinca alkaloids revealed marked alterations in transport and intracellular binding of [3H]vincristine compared to parental DC‐3F cells. Influx of [3H]vincristine in DC‐3F cells appears to be an equilibrating, but mediated, process. Although saturation kinetics for [3H]vincristine influx were not demonstrated, an extremely high temperature‐dependence (Q1027–37°C = 5–6) and trans‐inhibition of influx following preloading of cells with nonradioactive vincristine argue in favor of a carrier‐mediated process. Efflux of [3H]vincristine from parental cells conformed to first‐order kinetics (t1/2 37° = 3.6 ± 0.4) and exhibited a lower temperature‐dependence (Q10 27–37°C = 3–3.5) than influx. In variant vs. parental cells, influx of [3H]vincristine was reduced 24‐fold and efflux was increased twofold, accounting for the large (approximately 48‐fold) reduction in steady‐state level of exchangeable drug accumulating in variant cells. Otherwise, transport of [3H] vincristine in these cells showed characteristics similar to parental DC 3F cells. Also, the rate and amount of intracellular binding of [3H]vincristine in variant cells was almost 40‐fold lower than in parental cells. These alterations in influx and efflux of [3H]vincristine and its intracellular binding appear to account, at least to a major extent, for the high level of resistance (2,750‐fold) of this variant to vinca alkaloids. In contrast, cross‐resistance of this variant to daunomycin (178‐fold) could be explained only minimally by a transport alteration. Only a two‐fold increase in efflux of [3H]daunomycin was demonstrated in variant vs. parental cells along with some decrease in intra‐cellular binding. Influx of [3 H]daunomycin was unaltered. In view of these results, we conclude that these two agents most likely do not share the same route for entry in these cells but might share the same efflux route.
Alterations in plasma membrane lipid organization during lymphocyte differentiation
Authors: Del Buono Brian J. , Williamson Patrick L. , Schlegel Robert A. 
Published: February 2005

AbstractThe fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen‐induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial system.
Norepinephrine decreases EGF binding in primary rat hepatocyte cultures
Authors: Cruise Jennifer L. , Cotecchia Susanna , Michalopoulos George 
Published: February 2005

AbstractNorepinephrine (NE) produced a dose‐dependent inhibition of 125I‐epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an α1‐specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.
Transforming growth factor type β regulation of actin mRNA
Authors: Leof Edward B. , Proper Jacqueline A. , Getz Michael J. , Moses Harold L. 
Published: February 2005

AbstractStimulation of quiescent cultures of AKR‐2B cells with transforming growth factor type β (TGFβ) resulted in a transitory increase in actin cytoplasmic poly(A) + RNA. Levels of actin mRNA peaked approximately 4–8 hours subsequent to TGFβ addition and approached basal levels by 24 hours. The accumulation of actin transcripts was dose dependent and insensitive to inhibitors of protein synthesis; 1–3 ng/ml TGFβ induced maximal actin gene expression. Actin isotype‐specific probes demonstrated that both β‐ and γ‐cytoplasmic actins are induced by TGFβ.
Altered polyamine metabolism in chinese hamster cells growing in a defined medium
Authors: Sertich Gary J. , Glass James R. , Fuller David J. M. , Gerner Eugene W. 
Published: February 2005

AbstractChinese hamster cells (line CHO) maintained in McCoy's 5A medium (modified) supplemented with insulin (10 μg/ml), transferrin (5 μg/ml), and ferrous sulfate (1.1 μg/ml) proliferate at rates similar to cultures growing in the McCoy's medium supplemented with 10% fetal bovine serum. Colony‐forming ability is similar in cultures supplemented with either serum or the combination of growth factors. By 6 hours after replacement of serum with growth factors, ornithine decarboxylase (ODCase) activity increases, reaching a maximum value by 24 hours after serum replacement. This maximum is cell density dependent and can exceed a 30‐fold increase over enzyme activity in cultures supplemented with serum. The increased enzyme activity is due to a decrease in the turnover rate of the enzyme, based on protein synthesis inhibition studies, and an accumulation of active enzyme molecules rather than an activation of existing molecules, since the catalytic activity of ODCase, determined using the radiolabeled form of α‐difluoromethylornithine (an enzyme‐activated, irreversible inhibitor of ODCase) in concert with immuno‐chemical techniques, is unchanged by the different growth medium supplements. Intracellular putrescine and spermidine levels are substantially decreased when cultures are maintained in medium supplemented with insulin, transferrin, and ferrous sulfate, rather than serum, which is the sole source of exogenous ornithine. Titration of cultures growing in the defined medium with ornithine leads to a decrease in ODCase activity and an increase in intracellular putrescine and spermidine levels. Putrescine‐ and spermidine‐dependent S‐adenosyl‐L‐methionine decarboxylase activities are similar in cultures maintained in either medium. These data demonstrate that some, but not all, aspects of polyamine biosynthesis are affected by the availability of ornithine, the first substrate in the pathway.
Isolation of a human stromal cell strain secreting hemopoietic growth factors
Authors: Tsai Schickwann , Emerson Stephen G. , Sieff Colin A. , Nathan David G. 
Published: February 2005

AbstractA diploid fibroblastoid cell strain, termed “ST‐1,” has been established from a long‐term liquid culture of human fetal liver cells. ST‐1 cells are nonphagocytic, nonspecific esterase negative and do not possess factor VIII‐related antigen but stain with antibodies specific for fibronectin and type I collagen. The ST‐1 cells produce nondialyzable hemopoietic growth factors capable of stimulating the development of erythroid bursts, mixed granulocyte‐macrophage colonies, pure granulocyte colonies, and pure macrophage colonies. These factors are active on both human fetal liver and human adult bone marrow progenitors. When liquid cultures of human fetal liver hemopoietic progenitors are established with a preformed monolayer of ST‐1 cells, the yields of nonadherent cells, erythroid progenitors, and myeloid progenitors are greatly increased. These studies demonstrate that the fibroblastoid ST‐1 cells support hemopoiesis in vitro and may be a critical element in the stromal microenvironment in vivo.
A cartilage‐derived growth factor enhances hyaluronate synthesis and diminishes sulfated glycosaminoglycan synthesis in chondrocytes
Authors: Hamerman David , Sasse Joachim , Klagsbrun Michael 
Published: February 2005

AbstractCartilage‐derived growth factor purified to homogeneity by affinity chromatography on columns of heparin‐Sepharose was mitogenic for early passage bovine fetal chondrocytes. Hyaluronate and sulfated glycosaminoglycan synthesis in these cells was analyzed by differential enzymatic digestion of the glycosaminoglycans labeled with [14C] glucosamine or [35S]. It was found that chondrocyte proliferation was accompanied by about a four‐fold increase in hyaluronate synthesis over a two‐day period, while the synthesis of sulfated glycosaminoglycans decreased by about 2‐fold. Chromatographic analysis of the sulfated glycosaminoglycans showed decreases in chondroitin 4 and 6 sulfates. It was concluded from these results that cartilage‐derived growth factor was a proliferative factor for chondrocytes and differed from the somatomedins.