bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transepithelial transport in cell culture: Bioenergetics of Na‐, D‐glucose‐coupled transport
Authors: Sanders Martin J. , Simon Lawrence M. , Misfeldt Dayton S. 
Published: February 2005

AbstractThe renal cell line LLC‐PK1 contransports Na and D‐glucose from the apical to the basolateral side of the cell monolayer, and the short‐circuit current (Isc) measures the net amount of Na transported. Under conditions of maximal cotransport, the addition of phlorizin or removal of Na rreversibly decreased oxygen consumption by one‐hal. In the absence of glycolytic substrates, α‐methyl‐D‐glucoside stimulated Isc and oxygen consumption, although the Isc came to a steady state 50% less than when glycolytic substrates were present. The addition of other aerobic substrates did not increase Isc; however, when non‐contransported glycolytic substrates were introduced the Isc returned to a maximum with an associated fall in oxygen consumption and increased lactate production. Thus, in the absence of glycolytic substrates aerobic ATP formation may be rate‐limiting for Na, D‐glucose contansport. For this epithelium glycolysis makes an impotant contribution to the provision of energy or transport. Oxygen consumption does not correlate well with Isc and is not a good measured off the energy used in transport.
Glucose uptake and ornithine decarboxylase activity in tetradecanoyl phorbol acetate non‐proliferative variants
Authors: Butler‐Gralla Edith , Herschman Harvey R. 
Published: February 2005

AbstractThe potent tumor promoter 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA) is alos an excellent mitogen for 3T3 cells. We have previously isolated two independent variants, 3T3‐TNR‐2 and 3T3‐TNR‐9, that are unable to divide in response to TPA (Butler‐Gralla and Herschman, 1981). We have now tested tow components of the pleiotypic response, elevation of 2‐deoxyglucose uptake and ornithine decarboxylase induction, in these cells. Basal levels of 2‐deoxyglucose uptake were nearly tenfold higher in confluent 3T3‐TNR‐2 and 3T3‐TNR‐9 cells than in 3T3 cells. In contrast, basal ornithine decarboxylase levels were five‐ to tenfold lower in the variants. TPA stimulation of 2‐deoxyglucose uptake was as great in absolute terms in the variant cell lines as that of 3T3 cells but was only half that observed with serum. TPA was unable to induce any elevation of ornithine decarboxylase in 3T3‐TNR‐9 cells. treated with TPA, the maximal specific activity in the variant was less than the unstimulated value for 3T3 cells.
Mouse teratocarcinoma cells resistant to aphidicolin and arabinofuranosyl cytosine: Isolation and initial characterization
Authors: Aizawa Shinichi , Loeb Lawrence A. , Martin George M. 
Published: February 2005

AbstractWith the long‐range goals of elucidating the biochemical genetics and the phenotypic expression, both in vitro and in vivo, of mutator strains of mammalian cells, we have isolated and partially characterized a series of drug resistant mutants from a subline of a feeder‐dependent parental mouse teratocarcinoma line (PSA‐1) known to be capable of chimerizing host blastocyts via the injection of inner cell mass. Two series of stable mutants were isolated—one (Aphr) selected on the basis of resistance to aphidicolin (Aph), a specific inhibitor of DNA polymerase‐α, and the other (AraCr) selected by resistance to arabinofuranosyl cytosine, an analogue of cytosine. Irrespective of the method of selection, most of the 32 mutants isolated were resistant to both agents, although to different degrees, and with variations in growth characteristics, deoxynucleoside sensitivities, and sensitivities to mutagens (5‐bromodeoxyuridine, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine and ultraviolet light). A protocol of multi‐step selection provided stable mutants highly resistant to aphidicolin and only mildly resistant to AraC, even in the absence of prior mutagenesis, and is thus recommended as an approach to the isolation of candidate mutator strains of multipotent teratocarcinomas.
Spontaneous and agonist‐induced 86Rb efflux from rabbit aortic smooth muscle cells in culture: A comparison with fresh tissue
Authors: Martin W. , Gordon J. L. 
Published: February 2005

AbstractPotassium efflux was measured in rabbit aortic strips and smooth muscle cells cultured from them by monitoring the release of isotope from preparations preloaded with 86Rb. The basal rate of 86Rb efflux from rabbit subcultured aortic smooth muscle cells was eightfold higher than from freshly isolated strips, but calculations of reuptake of isotope in the tissue indicated that the measured rate constant for efflux from aortic strips underestimated the true rate by about fourfold. The rate constant for efflux from freshly dispersed cells was less than half that of subcultured cells and remained unchanged for 5 days in culture. It then rose and by around day 10 had reached the value for subcultured cells. The increase in efflux coincided with the onset of cell division. The increased rate of efflux was accompanied by an increased rate of uptake so that the internal potassium content of the cells remained constant. Heparin decreased the efflux of 86Rb from subcultured cells to that of freshly isolated cells concomitant with a reduction in the rate of proliferation. The onset of cell division and increased basal efflux of potassium was associated with a loss of responsiveness to noradrenaline and histamine as assessed by monitoring 86Rb efflux, although depolarising solutions of potassium chloride were still able to elicit a response. Responsiveness to noradrenaline had histamine could be restored by the addition of heparin. These results suggest that the lack of responsiveness of subcultured cells is not due to irreversible dedifferentiation but to a reversible loss in proliferating cells of receptors for vasoactive agents or of a coupling mechanism between receptor occupation and ion gating.
Comparison of indirect probes of membrane potential utilized in studies of human neutrophils
Authors: Seligmann Bruce E. , Gallin John I. 
Published: February 2005

AbstractFour indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3′dipentyloxacarbocyanine [di‐O‐C5(3)], 3,3′ dipentylindocarbocyanine [di‐I‐C5(3)], and 3,3′ dipropylthiodicarbocyanine [di‐S‐C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di‐S‐C3(5) not only exhibits a different fluorescence response mechanism from di‐O‐C5(3) [and di‐I‐C5(3)] but also that the fluorescence of di‐S‐C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB−, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB− are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di‐O‐C5(3) and di‐I‐C5(3) but cannot be avoided with di‐S‐C3(5) or TPMP+/TPB−. Using di‐O‐C5(3) with the fluorescence‐activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe‐specific problems or response heterogeneity.
Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts
Authors: Rudick Victoria L. , Rudick Michael J. , Jones Patricia M. 
Published: February 2005

AbstractUsing cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m̈g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP‐xylose, UDP‐galactose, and UDP‐glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP‐galactose and UDP‐glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.
Development of a serum‐free medium which supports the long‐term growth of human and nonhuman primate lymphoid cells
Authors: Brown Ronald L. , Griffith Richard L. , Neubauer Russell H. , Rabin Harvey 
Published: February 2005

AbstractThe ability to grow lymphoid cells in serum‐free media affords the advantage of separately analyzing those components found to be involved in proliferation and differentiation. Iscove's medium (IMDM) supplemented with bovine serum albumin or casein, cholesterol, ferrous chloride, insulin, β‐mercaptoethanol, L‐α‐phosphatidylcholine, and transferrin supported the long‐term proliferation of a gibbon ape lymphoma T‐cell line, MLA144. These cells continue to produce Interleukin 2 (IL‐2, T‐cell growth factor) constitutively in the serum‐free medium. IL‐2‐dependent human T cells initiated and maintained in culture in serum‐free medium containing IL‐2 have continued to replicate for over 3 months with two population doublings every 3 to 4 days. A normal, IL‐2‐dependent marmoset T‐cell line, OH‐1, also proliferated on the serum‐free medium when supplemented with IL‐2. Several established primate B‐cell lines which do not require IL‐2 for growth were able to proliferate in the serum‐free medium. These B‐cell lines included B95‐8, an Epstein‐Barr virus (EBV)‐transformed marmoset cell line, HuCo/R‐H, a human cord B‐lymphocyte line transformed with EBV, and Namalwa, an EBV‐positive B‐cell line established from a Burkitt's lymphoma. B95‐8 cells grown on serum‐free medium showed high levels of EBV antigen‐positive cells after induction with 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA).
Serum‐free culture of PC13 murine embryonal carcinoma cells
Authors: Heath John K. , Deller M. Jane 
Published: February 2005

AbstractThe requirements for the serum‐free culture of PC13 murine embryonal carcinoma cells were determined. Supplementation of a 50:50 mixture of Dulbecco's modified Eagles medium and MCDB104 with transferrin (5 μg/ml), human high‐density lipoprotein (HDL) (100 μg/ml), and human low‐density lipoprotein (LDL) (50 μg/ml) supported growth comparable to that observed with 5% foetal calf serum. Media supplementation with lipoproteins apparently substitutes for the effects of insulin, desoctapeptide insulin (DOP), or multiplication‐stimulating activity (MSA) on EC cell multiplication. Clonal growth of PC13 EC cells in this serum‐free medium could only be achieved in the presence of suitable feeder cell monolayers. These observations demonstrate that PC13 EC cells do not have an absolute requirement for exogenous mitogens to support multiplication.
Induction of reverse transformation and normal cell cycle regulation by dibutyryl cAMP in a chemically transformed cell line
Authors: Wang Yong Chao , Rao Potu N. 
Published: February 2005

AbstractThe objective of this study was to determine whether N6, O2‐dibutyryl 3′,5′‐adenosine monophosphate (db‐cAMP)‐induced reverse transformation in a chemically transformed mouse cell line, AKR‐MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR‐MCA cells were grown to confluency in the presence or absence of db‐cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+ 1‐ +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)‐treated cells exnibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db‐cAMP‐induced reverse transformation in AKR‐MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.
Na+ K+ pump and passive K+ transport in large and small red cell populations of anemic high and low K+ sheep
Authors: Lauf P. K. , Valet G. 
Published: February 2005

AbstractReticulocytes, isolated by centrifugal elutriation from massively bled sheep and identified by cytometric techniques, were analyzed with respect to their cation transport properties. In sheep with genetically high K+ (HK) or low K+ (LK) red cells, two reticulocyte types were distinguished by conventional or fluorescence‐staining techniques 5–6 days after hemorrhage: Large reticulocytes as part of a newly formed macrocytic (M) erythrocyte population, and small reticulocytes present among the adult red cell population (volume population III of normal sheep blood, Valet et al., 1978). Although cellular reticulin disappeared within a few days, the M‐cell population persisted throughout weeks in the peripheral circulation permitting a transport study of in vivo maturation. At all times, M cells of LK sheep had lower K+ and higher Na+ contents than M cells of HK sheep. Regardless of the sheep genotypes, M cells apparently reduced their volume during their first days in circulation; however, throughout the observation period, they did not attain that characteristic for adult red cells. Both ouabain‐sensitive K+ pump and ouabain‐insensitive K+ leak fluxes were elevated in M cells of both HK and LK sheep. The increased K+ pump flux was mainly due to higher K+ pump turnover rather than to the modestly increased number of pumps as measured by [3H]ouabain binding. In contrast, small reticulocytes enriched from separated volume population III cells by a Percoll‐density gradient exhibited transport parameters close to their prospective mature HK or LK red cells. The data support the concept that the M cells derived from emergency reticulocytes while the small reticulocytes represented precursors of normal red cell maturation. The Na+ and K+ composition found in M cells of HK and LK sheep, respectively, suggest development of the LK steady state at or prior to the reticulocyte state, a finding consistent with that of Lee and Kirk (1982) on low K+ dog red cells.
Adhesion of endothelial cells to extracellular matrix proteins
Authors: Macarak E. J. , Howard P. S. 
Published: February 2005

AbstractHuman and bovine endothelial cells were used in a series of studies examining cell adhesion to extracellular matrix macromolecules. Collagen types I and III, an acetic acid extract of bovine lens capsule basement membrane, laminin, and fibronectin were demonstrated to affect, in varying degrees, cell adhesion, cell growth, and “sprout cell” formation in endothelial cell cultures. All the matrix macromolecules tested promoted endothelial cell adhesion, and the adhesion to the basement membrane extract was blocked by antibodies to fibronectin. When human endothelial cells were grown on defined matrices, the level of serum required for optimum growth was decreased, while bovine endothelium grown on defined matrices did not show sprout cell formation. These data suggest that endothelial cells are able to adhere to a wide variety of connective tissue components and that extracellular matrices significantly influence cell behavior.
Growth‐promoting effects of iron‐ and cobalt‐protoporphyrins on cultured embryonic cells
Authors: Verger Claude , Sassa Shigeru , Kappas Attallah 
Published: February 2005

AbstractThe effects of hemin (Fe‐protoporphyrin) and Co‐protoporphyrin on cellular growth have been investigated principally in cultured fibroblasts, but also in myoblasts and hepatocytes from chick embryos. In the presence of horse serum in the culture medium, which by itself did not stimulate cell growth appreciably, Co‐protoporphyrin stimulated cell attachment while hemin stimulated cell proliferation of fibroblasts. When Co‐protoporphyrin and hemin were added together, the most potent stimulation of cell growth, consisting of increases in cell attachment and rapid cell proliferation, was observed. These findings indicate that the two metalloporphyrins have differential and complementary effects on cellular growth in culture, with synthetic Co‐protoporphyrin principally affecting cellular attachment and Fe‐protoporphyrin stimulating cellular proliferation.
Evidence for monovalent phosphate transport in Ehrlich ascites tumor cells
Authors: Bowen Jesse W. , Levinson Charles 
Published: February 2005

AbstractIn an effort to determine whether the Na+‐dependent Pi transport system of Ehrlich ascites tumor cells exhibits specificity for H2PO4− or HPO4−2, Pi fluxes were determined by measuring 32PiPi self‐exchange. Three experimental approaches were employed. First, the effect of pH on steady‐state Pi transport at 0.5 and 5 mM was studied. Second, the relationship between Pi transport and Pi concentration (0.25–9.2 mM) at pH 5.6 and 7.9 was determined. Third, the dependence of Pi transport on [H2PO4−] (0.05–4.2 mM) at constant [HPO4−2] (0.5 mM), and the converse, [HPO4−2] (0.06–4.5 mM) at constant [H2PO4−] (0.5 mM), was evaluated. Ks (apparent half‐saturation constant) and Jmax (maximal transport rate) were calculated by two methods: weighted linear regression (WLR) and a nonparametric procedure. The dependence of Pi flux on pH indicates that optimum transport occurs at pH 6.9. Pi transport decreases as pH is reduced when extracellular Pi is either 0.5 or 5 mM. However, at pH 7.9, Pi flux is reduced only in 0.5 mM Pi. At pH 5.6, H2PO4− comprises 93% of the total Pi present, and the calculated Ks is 0.055 ± 0.026 mM (WLR). This is the same as the Ks determined from the initial phase of the flux vs. [H2PO4−] relationship (0.056 ± 0.020 mM). However, at pH 7.9 (where 94% of Pi is HPO4−2), the measured Ks is 0.58 ± 0.11 mM (WLR), which is ten times higher than at pH 5.6. This value is also five times greater than the Ks calculated from the flux vs. [HPO4−2] curve (0.106 ± 0.16 mM). Kinetic parameters calculated by the nonparametric method, though somewhat different, gave similar relative results. Taken together, these results support two conclusions: (1) H2PO4− is the substrate for the Na+‐dependent Pi transport system of the Ehrlich cell, and (2) H+ can inhibit Pi transport.
Elastin production by cultured calf pulmonary artery endothelial cells
Authors: Mecham Robert P. , Madaras Judy , McDonald John A. , Ryan Una 
Published: February 2005

AbstractCalf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross‐reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS‐PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one‐third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un‐cross‐linked, 70,000‐dalton tropoelastin. The addition of β‐aminopro‐pronitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross‐linking of tropoelastin does not occur in culture.Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web‐like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell‐cell contact. Extracellular matrix remaining after solu‐bilization of cellular material with Triton X‐100 stained positive for fibronectin, but not for elastin.
Renewal inhibition of human mammary cell growth in vitro: Cortisol and the recruitment of cells to terminal differentiation
Authors: McGrath Charles M. , Soule Herbert D. 
Published: February 2005

AbstractCortisol and insulin stimulated exponential growth of normal human mammary epithelium in short‐term monolayer culture. The response of cells depended on their organization into “growth units” on the surface of the culture dish; single cells did not respond. Growth of cells in the units ceased after only 3–4 doublings, ending in terminal differentiation. The 3–4 divisions that occurred in response to insulin and cortisol and that resulted in terminal differentiation, were not inhibited by short‐range signals normally transmitted at population confluence. When growing above confluence density in response to hormones, cells reduced volume to accommodate the “terminal differentiation” divisions while still largely preserving a monolayer. The longevity of populations of normal cells (9–14 divisions), which occurred in 3 or 4 passages, exceeded the average longevity of individual cells in one passage (3–4 divisions). This disparity between real and apparent longevity was due to the inhibition of growth of divisional cells within growth units, which occurred in concert with terminal differentiation of other cells in these units. Inhibited cells could be recruited to undergo terminal differentiation divisions in response to cortisol and insulin, but only when the growth units were disrupted and terminally differentiated cells were eliminated, which occurred at subculture. We refer to the inhibition of growth that occurs in growth units as “renewal inhibition” to distinguish it from population‐wide “confluence inhibition” and to emphasize three other aspects: (1) it occurred in terminally differentiating growth units; (2) it occurred in the continued presence of an inductive hormonal stimulus for differentiative growth; and (3) it conserved less differentiated cells for recruitment to terminal differentiation. There are parallels between renewal inhibition in vitro and the signals that restrain growth of mammary cells in “growth buds” in vivo to preserve their capacity for multiple cycles of secretory differentiation. Differences in the behavior of normal and malignant breast cells in vitro suggest that renewal inhibition, rather than confluence inhibition, may be an important locus of growth control alteration in malignant transformation.