bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of both type 1 and type 2 plasminogen activator inhibitors in human cells
Authors: Schleef Raymond R. , Wagner Nancy V. , Loskutoff David J. 
Published: February 2005

AbstractThis report describes the development and use of functional immunoradiometric assays that distinguish the activity of β‐migrating endothelial‐type plasminogen activator inhibitor (PAI‐1) from that of placental‐type plasminogen activator inhibitor (PAI‐2). These assays are based upon the binding of PAI‐1 and PAI‐2 to immobilized single‐chain tissue‐type plasminogen activator (tPA) and to immobilized urokinase (UK), respectively. The extent of binding of each PAI is quantified by incubating the PAI‐PA complex first with rabbit antiserum specific for the individual PAI and then with 125I‐labeled goat antirabbit IgG. In control experiments, the assays were shown to be sensitive, dose‐dependent over a wide range, and specific for each PAI. These assays were employed to establish the PAI profile of a variety of human cells. Neither PAI‐1 nor PAI‐2 could be detected in Bowes melanoma cells or in a renal adenocarcinoma cell line (ACHN), while the histiocytic lymphoma cell (U‐937) produced only PAI‐2. Five cell lines, including two that were previously shown to contain one or the other PAI (e.g., umbilical vein endothelial cells and a fibrosarcoma cell line, HT‐1080) in fact contained both PAIs. The cells containing both PAIs were studied in more detail. In each case, SDS treatment of CM was shown to enhance PAI‐1 activity (by converting the latent form of this inhibitor into its active form) and to destroy PAI‐2 activity. Various compounds including interleukin 1, dexamethasone, and phorbol myristate acetate were found to selectively influence the cellular production of one PAI without concomitantly affecting the production of the other, suggesting that the synthesis of these inhibitors is not coordinately regulated.
A phorbol ester‐nonproliferative variant of Swiss 3T3 cells is deficient in Na+ K+ Cl− cotransport activity
Authors: O'Brien Thomas G. , Prettyman Ralph , George Kenneth S. , Herschman Harvey R. 
Published: February 2005

AbstractThe identity of the genetic defect(s) in Swiss 3T3 TNR‐2 and TNR‐9 that confers nonresponsiveness to the proliferative effect of 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) is not known. In BALB/c 3T3 cells, loss (via mutation) of a specific membrane ion transport system, the furosemide‐sensitive Na+ K+ Cl− cotransporter, is associated with decreased responsiveness to TPA. In this study, the transport properties of parental Swiss 3T3 cells and the TPA‐nonresponsive lines TNR‐2 and TNR‐9 were determined in the presence and absence of TPA. When the rate of 86Rb+ efflux (as a tracer for K+) was measured from each of the three cell lines, a furosemide‐ and TPA‐inhibitable component of efflux was clearly evident in parental and TNR‐9 cells but was virtually absent in TNR‐2 cells. 86Rb+ influx measurements indicated the presence in parental 3T3 cells and the TNR‐9 line of a substantial furosemide‐sensitive flux that could be inhibited by TPA. In contrast, much less furosemide‐sensitive influx was present in 3T3‐TNR‐2 cells, and it was relatively unaffected by TPA. In both parental 3T3 and 3T3‐TNR‐2 cells, most of the furosemide‐sensitive 86Rb+ influx is dependent on extracellular Na+ and Cl−. The apparent affinities of the transporter for these two ions, as well as for K+, were similar in both cell lines. In parental cells, the inhibition of furosemide‐sensitive 86Rb+ influx was quite sensitive to TPA (K1/2 ≅ 1 nM) and occurred very rapidly after phorbol ester exposure. As expected because of its volume‐regulatory role, inhibition of Na+ K+ Cl− cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of cotransport activity in TNR‐2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3‐TNR‐2 cells (but not TNR‐9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+ K+ Cl− cotransport by TPA in parental cells caused a substantial reduction in cell volume (25%). In contrast, because of the reduced level of contransport activity in TNR‐2 cells, TPA had only a slight effect on cell volume. These results suggest that the genetic defect in 3T3‐TNR‐2 cells (but not TNR‐9 cells) responsible for nonresponsiveness to phorbol esters may be the reduction of Na+ K+ Cl− contransport activity. Thus this membrane transport system may be an important component of the signal transduction pathway used by phorbol esters in 3T3 cells.
Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix
Authors: Schuetz Erin G. , Li Donna , Omiecinski Curtis J. , Muller‐Eberhard Ursula , Kleinman Hynda K. , Elswick Barbara , Guzelian Philip S. 
Published: February 2005

AbstractFreshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver‐specific functions over time in culture. We incubated hepatocytes in a serum‐free medium on a reconstituted basement membrane gel, “matrigel” (prepared from an extract of extracellular matrix of the murine Engelbreth‐Holm‐Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver‐specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, contained higher amounts of glutathione peroxidase, and, as judged by Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver‐specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P‐450b, and cytochrome P‐450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P‐450c by 3‐methylcholanthrene and for P‐450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P‐450d by 3‐methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P‐450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte “dedifferentiation.” None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H‐thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of mRNAs for the cytoskeleton proteins actin and tubulin in hepatocytes on both matrices during the first 2 days in culture. However, the continuously flattening hepatocytes on Vitrogen maintained substantially higher levels of cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver‐specific functions.
Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases
Authors: Montesano R. , Pepper M. S. , Belin D. , Vassalli J.‐D. , Orci L. 
Published: February 2005

AbstractWe have previously shown that capillary endothelial cells grown on the surface of three‐dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary‐like tubular structures in response to tumor‐promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine‐specific phosphatases, to determine whether this agent could induce the expression of an anglogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasrninogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase‐type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.
Expression of the CSF‐1 gene in the blast cells of acute myeloblastic leukemia: Association with reduced growth capacity
Authors: Wang Chen , Kelleher C. A. , Cheng G. Y. M. , Miyauchi Jun , Wong Gordon G. , Clark Steven C. , Minden Mark D. , McCulloch Ernest A. 
Published: February 2005

AbstractMyelopoietic growth factors are known to influence the growth in culture of malignant blast cells from human Acute Myeloblastic Leukemia (AML). We have used cDNA clones for the factor CSF‐1 and its receptor fms to study DNA and RNA from the blasts of 25 AML patients. The CSF‐1 gene was always in the germline configuration. CSF‐1 mRNA was found in about half the blast populations. The cells were also studied for their growth properties in culture. A highly significant association was found between CSF‐1 expression and poor growth in suspension culture. Most blast populations expressed fms; the number of fms expression negative samples was too small to permit the detection of any association between fms expression and growth or any interaction between the effects of the expression of the growth factor and its receptor. We propose that CSF‐1 may be an important part of the mechanism determining the balance between self‐renewal and determination in AML blast clones.
End products of glucose and glutamine metabolism by cultured cell lines
Authors: Lanks Karl W. , Li Ping‐Wu 
Published: February 2005

AbstractRates of CO2 production from glucose and glutamine, intracellular metabolite levels, and release of metabolic end products into the culture medium were determined for 13 cultured cell lines, including a glycolysis‐defective mutant. All the non‐mutant lines synthesized pyruvate, lactate, alanine, proline, aspartate, and citrate, so that the metabolism of glucose and glutamine resulted mainly in the production of these compounds and only to a lesser extent in complete oxidation to CO2. These data and the pattern of metabolites produced by the mutant line were consistent with a model characterized by incomplete glutamine oxidation leading to end product accumulation. Multiple linear regression analysis identified the metabolite levels most highly correlated with the intracellular citrate level and with the amount of citrate released into the medium. The analysis also showed that the rates of CO2 production from glucose and glutamine were themselves positively correlated, suggesting that the oxidation of the two substrates is coordinately controlled under normal culture conditions.
Transforming growth factor beta (TGF‐β) inhibits hepatocyte DNA synthesis independently of EGF binding and egf receptor autophosphorylation
Authors: Russell William E. 
Published: February 2005

AbstractSubpicomolar concentrations of human platelet‐derived transforming growth factor beta (TGF‐β) inhibited growth factor‐stimulated DNA synthesis in primary cultures of adult rat hepatocytes. This inhibition was not the result of changes in the size of intracellular pools of 3H‐thymidine and was not dependent on the state of confluence of the cells. A 24‐hr exposure to TGF‐β either before or after insulin/EGF stimulation was as inhibitory on DNA synthesis between 48 and 72 hr of culture as was TGF‐β present throughout 72 hr of culture. From 12 hr in culture to 24 hr, hepatocyte EGF binding sites dropped from about 230,000 to 85,000 per cell with no significant change in Kd, but with a loss in capacity for EGF‐induced receptor down‐regulation. Maximally inhibitory concentrations of TGF‐β did not compete with EGF for the EGF receptor, and a 4‐to 24‐hr exposure to TGF‐β did not alter subsequent EGF binding. Coincubation of hepatocytes with TGF‐β and EGF did not influence the 60% reduction in EGF binding sites produced by EGF alone. In addition, TGF‐β did not prevent EGF‐induced autophosphorylation of the 170,000 dalton EGF receptor in membranes form whole liver. Our studies suggest that TGF‐β regulates hepatocyte growth independently of changes in EGF receptor number, ligand affinity, or postbinding autophosphorylation.
Inositol is a required nutrient for keratinocyte growth
Authors: Gordon Philip R. , Mawhinney Thomas P. , Gilchrest Barbara A. 
Published: February 2005

AbstractBovine hypothalamus is known to contain a growth‐promoting activity for human epidermal keratinocytes. By sequential purification, the substance was isolated and found to be myo‐inositol. The identity of the substance as myo‐inositol was confirmed by ion modified partition, gas liquid, thin layer chromatography, by mass spectrometry, and quantitative bioassay. The inositol content of the crude hypothalamic extract and of an active acetone precipitate (the first step in the purification) was determined to be sufficient to account for their observed bioactivity. At an optimal concentration of 55 μM (10 μg/ml), myo‐inositol approximately tripled keratinocyte yield compared to paired cultures in basal medium containing 0.3 μM, although this yield was only half that produced by a crude saline extract of hypothalamus, suggesting that there are additional growth‐promoting activities in the tissue extract. No other skin‐derived cell type tested was stimulated by supplemental inositol. These results establish that the inositol requirement for cultured human keratinocytes is markedly higher than for any other normal or malignant cell type investigated to date, and expand the list of brain‐derived phospholipid precursors known to stimulate epithelial proliferation in vitro. These data suggest that inositol may subserve quantitatively or qualitatively different functions in the keratinocyte than in other cell types.
Hypertonic sucrose inhibition of endocytic transport suggests multiple early endocytic compartments
Authors: Park Ro Dong , Sullivan Peter C. , Storrie Brian 
Published: February 2005

AbstractIncubation of animal cells with hypertonic sucrose and polyethylene glycol (PEG) 1,000 renders endosomes senstive in situ to hypotonic shock (Okada and Rechsteiner, 1982). We found that: (1) in vitro endosomes were osmotically insensitive; and (2) hypertonic sucrose inhibited transport from very early endosomes to lysosomes. Endocytic vesicles were labeled by incubating Chinese hamster ovary (CHO) cells for 1–10 min at 37°C with horseradish peroxidase (HRP) and/or fluorescein isothiocyanate‐conjugated dextran (FITC‐dextran). Cell fractions prepared in 0.25 M sucrose were hypotonically shocked by dilution with 5 mM Na phosphate buffer, pH 6.7, to a final sucrose concentration of 0.05 M. After hypotonic shock, endocytized HRP and FITC‐dextran pelleted with membrane while lysosomal hydrolases did not. The HRP activity in the pellet was latent, suggesting that endosomes were resistant to osmotic shock. Uptake in the presence of hypertonic sucrose had little effect on the subsequent osmotic sensitivity of the endosomes. Uptake in the presence of hypertonic sucrose and PEG 1,000 rendered endosomes fragile to cell homogenization. Unexpectedly, the inclusion of hypertonic sucrose in the uptake and chase media inhibited the appearance of HRP in lysosomes. HRP internalized during a 10‐min uptake appeared as if it were present in two physically distinct compartments, one accessible to transport inhibition by exogenous sucrose (“very early” endosomes) and the other not (“early” endosomes). After a brief uptake (1–3 min), postincubation of CHO cells in 0.25 M sucrose‐containing media completely blocked transport of internalized HRP to lysosomes. This blockage could be partially relieved by cointernalization of invertase with HRP. These results suggest that transport between multiple early endosome populations is sensitive to intraorganellar osmotic conditions.
Regulation of the proliferation of primary rat hepatocytes by eicosanoids
Authors: Skouteris George G. , Ord Margery G. , Stocken Lloyd A. 
Published: February 2005

AbstractDNA synthesis in primary adult rat hepatocyte cultures was promoted by epidermal growth factor (EGF), arachidonic acid, and prostaglandins E2 and F2α (PGE2 and PGF2α). Growth promotion by EGF was blocked by 0.1 mM indomethacin and 1 mM aspirin, without affecting cell viability. If verapamil was present in the medium when EGF was added, the growth response was inhibited. Hepatocytes stimulated by EGF or arachidonic acid released PGE2 and PGF2α into the culture medium. This was diminished if 0.1 mM indomethacin was also in the medium. The importance of autocrine regulation of hepatocyte growth by prostaglandins is discussed.
Heparin‐like molecules regulate the number of epidermal growth factor receptors on vascular smooth muscle cells
Authors: Reilly Christopher F. , Fritze Linda M. S. , Rosenberg Robert D. 
Published: February 2005

AbstractThe effect of heparin on the binding of epidermal growth factor (EGF) to vascular smooth muscle cells (SMC) was examined. Heparin pretreatment of SMC obtained from bovine aortic explant tissue resulted in significant reductions in the amount of EGF bound. Decreases in mitogen binding were observed with both growth arrested as well as exponentially growing cultures. The heparin concentrations (10–100 μg/ml) and pretreatment times (48–72 h) necessary for suppression of EGF binding correlated with the concentrations and temporal requirements necessary for growth inhibition. Chondroitin sulfate, which has negligible antiproliferative activity, had no effect on EGF binding. However, a highly inhibitory heparan sulfate species obtained from postconfluent SMC suppressed EGF binding by 45%. Platelet‐derived growth factor and insulin‐like growth factor‐1 binding were unaffected by heparin. Scatchard analysis revealed that heparin induced 50 to 60% reductions in the numbers of high and low affinity EGF receptors without detectable changes in the binding affinity or ratio of high to low receptors. Experiments were also performed with enzymatically dispersed SMC. These cultures were inhibited by heparin in a time dependent manner which was partially reversible in the presence of EGF. Subsequent studies revealed that heparin suppressed EGF binding in these cultures by 20 to 40%. In summary, heparin reduces the number of EGF receptors on both explant and enzyme dispersed SMC by a mechanism which closely parallels the antiproliferative effects of this glycosaminoglycan.
Stimulation by tumor necrosis factor of HL‐60 thymidine salvage pathway metabolism dissociated from proliferation
Authors: Elias Laurence 
Published: February 2005

AbstractThe effect of human tumor necrosis factor (TNF) on early‐passage HL‐60 cells was studied. A transient phase of increased [3H]thymidine (TdR) incorporation was noted at 20‐24 hr of exposure to TNF. This increase was disproportionate to the much slighter stimulation of the percentage of S‐phase cells, which was measured by flow cytometry. Evidence for increased metabolic trapping of [3H] TdR following TNF treatment was apparent from whole cell uptake experiments. The salvage pathway enzyme TdR kinase was therefore measured and was found to be elevated comparably to [3H]TdR uptake. The mechanism of TNF regulation of TdR kinase was further investigated by a series of combination treatment experiments using other biologic factors and pharmacologic inhibitors of various intracellular steps. The response to TNF was not potentiated or reproduced by IL‐1, IL‐2, IL‐3, IL‐4, G‐CSF, M‐CSF, GM‐CSF or α‐ or γ‐interferon. Blockers of early signal transduction steps, including H7, W7, sphingosine, and pertussis toxin, failed to inhibit TNF stimulation of [3H]TdR incorporation. mRNA synthesis inhibition with α‐amanitin blocked this TNF effect, as did cAMP but not cGMP analogues. A sensitizing effect was noted with amiloride or cytochalasin B, characterized by greater relative increases of [3H]TdR incorporation and TdR kinase activity in response to TNF. In the presence of cytochalasin B, TNF treatment resulted in no change or slight decreases in the percentage of S‐phase cells. Regulation of TdR kinase could thereby be dissociated from the usual cell cycle control. This study thus documents a unique example of stimulation of thymidine salvage pathway metabolism by a biologic factor, dissociable from overall cell cycle regulation.
The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts
Authors: Dell'Orco R. T. , Worthington M. I. 
Published: February 2005

AbstractHistone variant synthesis patterns from human diploid fibroblast‐like cells of different in vitro ages were determined during exponential growth, at confluence, and during low serum arrest. The results are reported as the ratios of H2A variant synthesis (H2A.1 and H2A.2/H2A.x and H2A.z) and H3 variant synthesis (H3.1 and H3.2/H3.3) that have been used to characterize individual cell cycle states. Hydroxyurea was employed in some experiments to reduce S phase cells. The results indicate that high population doubling level (PDL) cells move through the G1 phase of the division cycle during exponential growth and exist in the G0 cell cycle state at confluence and during low serum arrest. Low PDL cells, however, exist in the G1 cell cycle state at confluence and revert to a G0 state only after maintenance as quiescent populations. This would suggest that when stimulated high PDL cells cannot enter into S phase, they revert to a GO cell cycle state.
Regulation of neuroblast proliferation by hormones and growth factors in chemically defined medium
Authors: Wu Doris K. , Maciag Thomas , de Vellis J. 
Published: February 2005

AbstractPure neuronal cultures prepared from 6‐day‐old embryonic chick brains incorporated [3H]‐thymidine in serum‐free medium up to the 4th day in culture. The addition of insulin any time within this culture period caused an increase in thymidine incorporation. This increase in [3H]‐thymidine was correlated with an increase in cell number and percentage of labeling index. Triiodothyronine and endothelial cell growth factor were also active in stimulating [3H]‐thymidine incorporation into chick neuroblasts. The effect of these trophic agents is unique since a variety of known mitogens tested were negative.