bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sphingosine inhibits phorbol 12‐myristate 13‐acetate‐, but not serum‐induced, activation of Na+/H+ exchange in mammalian cells
Authors: Gillies Robert J. , Martinez Raul , Sneider Judith M. , Hoyer Patricia B. 
Published: February 2005

AbstractAddition of serum to quiescent mammalian cells in culture initiates a series of events which culminates in DNA replication and cell division. One of the earliest events in this sequence of events is activation of Na+/H+ exchange, which can result in an increase in intracellular pH (pHin). The regulation of this change in activity is not known. Since treatment of 3T3 cells with activators of protein kinase C (kinase C) can result in an increased pHin, it has been hypothesized that serum stimulation of kinase C is responsible for activation of Na+/H+ exchange. Recently, sphingolipids have been discovered to inhibit kinase C both in vitro and in vivo. Therefore, we undertook the present study to ask whether or not inhibition of kinase C using sphingolipids prevents mitogen‐induced alkalinization in 3T3 cells. Our results indicate that activators of kinase C stimulate Na+/H+ exchange in normal human fibroblasts (BoGi), but not in mouse embryo (3T3) cells. Addition of serum to BoGi cells, on top of saturating doses of phorbol 12‐myristate 13‐acetate (PMA), results in a further cytoplasmic alkalinization. Furthermore, sphingosine prevents the PMA‐induced increase in pHin in BoGi cells, and phosphorylation of an 80 kDa protein in 3T3 cells, but not the serum‐induced alkalinization in either BoGi or 3T3 cells. These data indicate that activation of kinase C does not participate in the physiological activation of Na+/H+ exchange in human fibroblasts or mouse embryo cells by serum.
Insulin receptor and altered glucose transport in a monensin‐resistant mutant of Chinese hamster ovary cell
Authors: Seguchi Tadashi , Yoshimura Akihiko , Ono Mayumi , Shite Satoshi , Kasahara Michihiro , Ebina Yosuke , Rutter William J. , Kuwano Michihiko 
Published: February 2005

AbstractA monensin‐resistant mutant Monr‐31, derived from Chinese hamster ovary (CHO) cell line, has been shown to have a reduced number of insulin receptors and a reduction in glucose uptake in response to insulin. We have further investigated the possibility that altered glucose uptake in Monr‐31 cells is related to an alteration in the activity of the insulin receptor. Uptake of glucosamine, 2‐deoxy‐D‐glucose, and 3‐O‐methyl‐D‐glucose in Monr‐31 cells was one‐half to one‐third that of CHO cells. The cellular content of the glucose transporter in Monr‐31 was reduced to about one‐third that of CHO as assayed by use of an antiglucose transporter antibody. After transfection with the human insulin receptor cDNA, we obtained clones CIR‐0 from CHO, and MIR‐2 and MIR‐15 from Monr‐31; CIR‐0 expressed a tenfold higher level of the insulin‐binding activity than did CHO, and MIR‐2 and MIR‐15 expressed a 20‐fold higher level than did Monr‐31. Glucose uptake in both CHO and CIR‐0 was significantly enhanced by exogenous insulin, but not in Monr‐31, MIR‐2, and MIR‐15. The β‐subunits of insulin receptor in CHO, CIR‐0, Monr‐31, and MIR‐2 were similarly phosphorylated. The decreased glucose transport activity in Monr‐31 cells is discussed in relation to the absence or presence of insulin receptor expression.
Interaction of platelet‐activating factor with endothelial and vascular smooth muscle cells in coculture
Authors: Stoll Lynn L. , Spector Arthur A. 
Published: February 2005

AbstractPlatelet‐activating factor (1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine [PAF]) is a vasoactive ether lipid produced by activated blood cells. To examine the molecular traffic and sites of metabolism of PAF released in the vascular wall, we used a coculture system in which endothelial cells are grown on micropore filters suspended over confluent cultures of vascular smooth muscle cells. The endothelial cells took up PAF 5–7 times more readily from the apical than from the basolateral surface, converting it to 1‐O‐alkyl‐2‐acyl‐sn‐glycero‐3‐phosphocholine (2‐acyl‐PAF) and other minor metabolites. Intact endothelial monolayers effectively shielded the underlying smooth muscle cells from PAF present in the apical fluid; after a 30‐min incubation with [3H]‐PAF, only 1% of the radioactivity was transferred to the interstitial fluid. By contrast, PAF readily entered the interstitial fluid when the endothelial monolayers were injured by exposure to xanthine and xanthine oxidase. PAF did not significantly increase the permeability of endothelial monolayers to albumin. Smooth muscle cells took up and metabolized interstitial PAF more quickly and more completely than did endothelial cells; 65% was converted to 2‐acyl‐PAF in 15 min by the smooth muscle cells. PAF enhanced the proliferative effect of PDGF on smooth muscle cells, as assessed by [3H]‐thymidine incorporation. These findings suggest that endothelial cells form a barrier to PAF released at the luminal surface, but PAF released in the vascular intima interacts primarily with smooth muscle cells, possibly stimulating proliferation in these cells.
Differential responses to growth factors by normal human mesothelial cultures from individual donors
Authors: Lechner John F. , LaVeck Moira A. , Gerwin Brenda I. , Matis Elizabeth A. 
Published: February 2005

AbstractA significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma‐interferon and interleukin‐2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbestos.
Cellular interactions between 3T3 cells and interleukin‐3‐dependent multipotent haemopoietic cells: A model system for stromal‐cell‐mediated haemopoiesis
Authors: Yamazaki K. , Roberts R. A. , Spooncer E. , Dexter T. M. , Allen T. D. 
Published: February 2005

AbstractWith the aid of a multipotent stem cell line (FDCP‐mix cells) co‐cultured with either normal or irradiated Swiss 3T3, cellular interactions between stromal cells and haemopoietic stem cells were studied by electron microscopy and time‐lapse video microscopy. When cultured in the presence of interleukin 3 (IL‐3) but in the absence of stromal cells, the FDCP‐mix cells have a characteristic blast morphology. In the absence of IL‐3, the cells die unless they are co‐cultured with marrow stromal cells or 3T3 cells. In the latter case, they attach, proliferate, and differentiate on both normal and irradiated Swiss 3T3 cell layers without the addition of extrinsic growth factor (IL‐3). At the initial attachment sites of these two cell lines, cellular recognition seemed to be mediated by the formation of microvillus cytoplasmic projections and extracellular matrix. These areas may well be the sites of plasma‐membrane‐bound signalling/adhesional molecules between the interacting cells.
Effects of fluorescent derivatives of TPA on HL60 cells: Dissociation between the differentiation‐induced and protein kinase C activity
Authors: Tran Phuong Lan , Le Peuch Christian , Basset Michele 
Published: February 2005

AbstractThe four fluorescent derivatives of TPA—dansylaza‐TPA, NBDaza‐TPA, and (N)‐and (P)‐dansylamino‐TPA—were synthesized and examined for their ability to induce differentiation in human promyelocytic leukemic HL60 cells. At a concentration of 20 nM, all the derivatives inhibited proliferation and induced 60–80% of the cells to differentiate into macrophage‐like cells. Removal of dansylaza‐TPA from the medium after 5 h did not arrest adherence or the expression of nonspecific esterase activity. However, upon removal of any of the other three compounds after 5 h, HL60 cells became nonadherent and expressed low nonspecific esterase activity after additional culture. To investigate the relationship between protein kinase C (PKC) activation and cell maturation, PKC activity and translocation were measured after 0.5, 5, 24, and 48 h of treatment with each compound. Cells induced to differentiate by dansylaza‐TPA or (N)‐ or (P)‐dansylamino‐TPA exhibited enhanced PKC activity, 50–80% of which was located in the particulate fraction. In cells that differentiated with NBDaza‐TPA, 65–70% of PKC activity remained in the cytosol. After removal of the TPA derivatives, all cells exhibited PKC activity in the cytosol. These results indicate that the fluorescent derivatives are as potent as TPA in inducing HL60 cell differentiation. However, in the case of NBDaza‐TPA and (N)‐ or (P)‐dansylamino‐TPA, their continuous presence in the culture medium was required for the recruitment of cells to differentiate. Consequently, it is suggested that activation and translocation of PKC are among the early biochemical events that trigger HL60 cell differentiation. Nevertheless, these two events alone are not sufficient to induce differentiation.
Serum stimulation of quiescent human fibroblasts induces the synthesis of tissue factor mRNA followed by the appearance of tissue factor antigen and procoagulant activity
Authors: Bloem Laura J. , Chen Lisa , Konigsberg William H. , Bach Ronald 
Published: February 2005

AbstractExpression of the gene for tissue factor, the cell‐surface initiator of blood coagulation, was examined by stimulating growth‐arrested human fibroblasts with serum and measuring changes in the cellular content of tissue factor mRNA, antigen, and activity. Maximum tissue factor mRNA levels were reached within 1 h following serum induction and slowly declined to basal levels from 24 to 48 h after stimulation. The appearance of the tissue factor mRNA was followed by an increase in tissue factor antigen and activity. The parallel rise in antigenically positive protein and procoagulant activity was first observed about 2 h after serum stimulation with a peak at 12 h followed by a slow decline during the next 36 h. The serum‐induced synthesis of the tissue factor mRNA was independent of de novo protein synthesis as demonstrated by the increased tissue factor mRNA levels generated in the presence of cycloheximide. The results of this study suggest that the synthesis of tissue factor in human fibroblasts i s regulated principally at the level of transcription. In one strain of fibroblasts the activity/antigen ratio, during the period of maximum synthesis, was indistinguishable from that of tissue factor which had been immunoaffinity purified from human brain and reconstituted into phospholipid vesicles. However, during serum starvation the activity/antigen ratio in these cells was significantly reduced. Western blot analysis revealed that in serum‐starved cells there was an accumulation of truncated forms of the tissue factor antigen while in the serum‐stimulated cells only the full‐length antigen was observed.
Synchronized human diploid fibroblasts: Progression capabilities of a subpopulation that fails to keep pace with the predominant, rapidly dividing cohort of cells
Authors: Tobey R. A. , Oishi N. , Crissman H. A. 
Published: February 2005

AbstractHighly synchronized cultures of HSF‐55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two‐laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA‐specific stains that differed in the degree of BrdU‐induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S‐phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early‐ and late‐replicating species of human DNA.
Effects of biaxial deformation on pulmonary artery endothelial cells
Authors: Gorfien S. F. , Winston F. K. , Thibault L. E. , Macarak E. J. 
Published: February 2005

AbstractAn apparatus has been designed to subject vascular cells grown on a compliant substrate in vitro to uniform, quantifiable levels of biaxial deformation. The system described can be controlled with respect to strain level, rate, and frequency to mimic the pulsatile force to which vascular cells are exposed in vivo under both physiologic and pathologic conditions. In the experiments presented here, bovine pulmonary artery endothelial cells were grown on a substrate of segmented polyurethane urea (MitrathaneTM). Cell growth and morphology on this substrate were compared with those of cells grown on standard tissue culture polystyrene with no difference noted between the two substrates. Primary cultures of pulmonary artery endothelial cells were seeded onto Mitrathane, which was then subjected to cyclic biaxial deformation‐producing strains of 0.78%, 1.76%, 4.9%, or 12.5% at a frequency of 1 sec−1 and a duty cycle of 0.5 sec−1 for 7 h. Cells subjected to deformations generating strains of either 4.9% or 12.5% secreted significantly less fibronectin than nondeformed cells. Similar results were obtained in experiments using cloned pulmonary artery endothelial cells on Mitrathane subjected to the 4.9% strain; however, total protein synthesis was increased. Cell viability and DNA synthesis were not affected by cyclic biaxial deformation in these experiments.
Interaction of serum and colony‐stimulating factor for survival of a factor‐dependent hemopoietic progenitor cell line
Authors: Böhmer Ralph M. 
Published: February 2005

AbstractThe growth, in vitro of the murine myeloid cell line FDC‐P1 depends on the presenc of serum and a murine hemopoietic growth factor (either granulocyte/ macrophage colony‐stimulating factor (GM‐CSF) or multipotential colony‐stimu‐lating factor (multi‐CSF, IL3)). To determine the differential roles of serum and colony‐stimulating factor (CSF) during the growth of FDC‐P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (> 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re‐addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (>> 105/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum‐free conditioned medium from dense cultures made the survival and growth of FDC‐P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.
Growth, enzyme activity, sugar transport, and hormone supplement responses in cells cloned from a pig kidney cell line LLC‐PK1
Authors: Miller John H. , Heath Lisa N. 
Published: February 2005

AbstractThree clones of the pig kidney cell line LLC‐PK1 were isolated and characterized with regard to morphology, growth, proximal tubule enzyme activity, sugar uptake capacity, and hormone and drug responsiveness in a defined medium. Clone N4 was similar in morphology to the wild type (WT), whereas clone F8 showed loose attachment to the substrate, formed large, sweeping domes, and had an elongated desmosome junction between cells. The third clone, F2, did not form domes and showed a marked reduction in growth rate. Cultures of WT, N4, and F8 had higher specific activities of the enzymes alkaline phosphatase and γ‐glutamyl transpeptidase at confluence relative to growing cells; however, there was no evidence of an increase in activity of either enzyme at confluence in F2. Phlorizin‐sensitive α‐methyl‐D‐glucoside uptake and cytochalasin B‐sensitive 2‐deoxy‐D‐glucose uptake were measured in confluent cultures grown on porous filter supports. None of the clones lacked either of the hexose transport systems, although quantitative differences were evident. N4 cells grown in a defined medium in 96‐well culture plates were tested in situ for their enzyme responses to differentiation inducers, tumor promoters, and hormones. Alkaline phosphatase activity was significantly increased at confluence by serum, parathyroid hormone (PTH), and vasopressin (AVP), and was decreased by tetradecanoylphorbol acetate (TPA) and epinephrine (EPI). Glutamyl transpeptidase activity was decreased at confluence by serum, TPA, and EPI. Similar tests on α‐methyl‐D‐glucoside uptake showed that serum, TPA, PTH, and AVP had no significant effect on phlorizin‐sensitive uptake; however, calcitonin increased uptake by 84% (n = 18). It was concluded that LLC‐PK1 clones maintained in a defined medium are useful models for studying renal cell function.
Regulation of levels of IL‐1 mRNA in human fibroblasts
Authors: Yamato Kenji , El‐Hajjaoui Ziad , Koeffler H. Phillip 
Published: February 2005

AbstractInterleukin‐1 (IL‐1) has a crucial role in host defenses, inflammatory processes, and tissue homeostasis. A wide variety of cells produce this protein in response to a number of extracellular stimuli including microorganisms, antigenic stimuli, and products from other cells. Regulation of IL‐1 production at the molecular level is poorly understood. We studied expression, intracellular signals, and posttranscriptional regulation of IL‐1 mRNA in human mesenchymal cells by using Northern blot analysis. Tumor necrosis factor alpha (TNFα) and activators of protein kinase C including 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and teleocidin induced the accumulation of IL‐1β mRNA in human fibroblasts (WI‐38). Effect of TNFα was not blocked by inhibitors of either protein synthesis (cycloheximide) or protein kinase C activity. Accumulation of IL‐1β mRNA was also increased by a calcium ionophore (A23187) and an inhibitor of the Na+/K+ pump (ouabain); both compounds are known to increase cytoplasmic levels of Ca++. Stability of IL‐1β mRNA in fibroblasts exposed to TPA was more than fourfold greater than after fibroblasts were exposed to either TNFα or cycloheximide. This suggests that posttranscriptional stabilization of IL‐1β mRNA is a major mechanism leading to accumulation of IL‐1β mRNA after activation of PKC in fibroblasts. Fibroblasts did not express IL‐1α mRNA after exposure to stimuli which induced the accumulation of IL‐1β mRNA. In summary, several different pathways regulate levels of IL‐1β mRNA in human mesenchymal cells.
Role of extracellular matrix in the action of basic fibroblast growth factor: Matrix as a source of growth factor for long‐term stimulation of plasminogen activator production and DNA synthesis
Authors: Flaumenhaft Robert , Moscatelli David , Saksela Olli , Rifkin Daniel B. 
Published: February 2005

AbstractWhen bovine capillary endothelial (BCE) cells were treated with 10 ng/ml of basic fibroblast growth factor (bFGF) for 10 or 30 minutes at 37°C, washed extensively with phosphate‐buffered saline (PBS) and incubated in bFGF‐free medium, plasminogen activator (PA) production was stimulated to the same extent as in cells exposed continuously to bFGF. Three methods of removing bFGF from heparin‐like binding sites in the extracellular matrix, but not from bFGF receptors, abolished this long‐term effect of a brief exposure to bFGF. First, BCE cells exposed to bFGF for 30 minutes were washed with 2M NaCI and incubated in bFGF‐free medium. Second, BCE cells were incubated with bFGF for 10 minutes in the presence of heparin, and cells were washed with PBS and incubated in bFGF‐free medium. Third, BCE cell cultures were treated with heparinase and exposed to bFGF. Each of these treatments abolished the long‐term (24‐48 hours) stimulation of PA production normally observed after brief exposure to bFGF. In each of these experiments, incubation of cells in bFGF‐containing medium after the treatments resulted in normal stimulation of PA production, demonstrating that the treatments did not harm the cells. Stimulation of DNA synthesis was observed when cells were exposed to bFGF for 2 hours at 4°C, incubated in bFGF‐free medium for 24 hours at 37°C, and assayed for 3H‐thymidine incorporation. However, no stimulation was observed if the 2 hours incubation at 4°C was carried out in the presence of heparin. Thus, long‐term stimulation of PA activity and DNA synthesis after a brief exposure to bFGF seems to be a consequence of bFGF binding to the extracellular matrix. The extracellular matrixmay act as a physiologic buffer, binding bFGF when concentrations are high and releasing it later for interaction with its receptor. This interaction with matrix may be required for the in vivo action of bFGF.
Maturation‐associated loss and incomplete de novo synthesis of the transferrin receptor in peripheral sheep reticulocytes: Response to heme and iron
Authors: Ahn Jinhi , Johnstone R. M. 
Published: February 2005

AbstractHemin, but not iron, in the culture medium stimulates the maturation‐associated loss of the transferrin receptor from sheep reticulocytes (t1/2for loss ∼ 6 hr) and its appearance in a population of externalized vesicles. A similar pattern is seen with nucleoside binding (a measure of the nucleoside transporter), where hemin increases the loss of binding activity from the cells during culture, concomitant with an increase in nucleoside binding in the externalized vesicles. Sheep reticulocytes retain the ability to synthesize the transferrin receptor, but the 35S‐labeled receptors are not detected in released vesicles. Whereas hemin stimulates the loss of 35S‐labeled transferrin receptors from the cell (t1/2 for loss ∼ 20 hr), nonheme iron is more effective than heme. This difference in response of native and 35S‐labeled receptor to hemin and iron supplements appears to be related to the differences in the two classes of receptors. Although the 35S‐labeled receptor binds transferrin and both native and 35S‐labeled peptides comigrate after chemical deglycosylation, the 35S‐receptor is ∼ 2 kD smaller than the native receptor and fails to acquire its complete size even when chased for up to 24 hr. Moreover, the 35S‐labeled receptor is not expressed at the cell surface, but is retained in a nonrecycling compartment, where it is insensitive to digestion by trypsin at both 0°C and 37°C.