bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the fibroblast growth factor receptor in human vascular endothelial cells
Authors: Neufeld Gera , Gospodarowicz Denis 
Published: February 2005

AbstractThe fibroblast growth factor (FGF) receptor of human umbilical vein‐derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal growth factor, insulin, or transferrin. The lectin concanavalin‐A does not inhibit the binding of 125l‐bFGF to HUE cell‐surface receptors, whereas it inhibits bFGF binding to BHK‐21 cell‐surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti‐angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor.
Amphiphile‐induced heart muscle‐cell (Myocyte) injury: Effects of intracellular fatty acid overload
Authors: Janero David R. , Burghardt Charles , Feldman Dorothy 
Published: February 2005

AbstractLipid amphiphile toxicity may be an important contributor to myocardial injury, especially during ischemia/reperfusion. In order to investigate directly the potential biochemical and metabolic effects of amphiphile overload on the functioning heart muscle cell (myocyte), a novel model of nonesterified fatty acid (NEFA)‐induced myocyte damage has been defined. The model uses intact, beating neonatal rat myocytes in primary monolayer culture as a study object and 5‐(tetradecyloxy)‐2‐furoic acid (TOFA) as a nonmetabolizable fatty acid. Myocytes incubated with TOFA accumulated it as NEFA, and the consequent NEFA amphiphile overload elicited a variety of cellular defects (including decreased beating rate, depletion of high‐energy stores and glycogen pools, and breakdown of myocyte membrane phospholipid) and culminated in cell death. The amphiphile‐induced cellular pathology could be reversed by removing TOFA from the culture medium, which resulted in intracellular TOFA “wash‐out.” Although the development and severity of amphiphile‐induced myocyte injury could be correlated with both the intracellular TOFA/NEFA content (i.e., the level of TOFA to which the cells were exposed) and the duration of this exposure, removal of amphiphile overload did not inevitably lead to myocyte recovery. TOFA had adverse effects on myocyte mitochondrial function in situ (decoupling of oxidative phosphorylation, impairing respiratory control) and on myocyte oxidative catabolism (transiently increasing fatty acid β oxidation, citric acid cycle flux, and glucose oxidation). The amphiphile‐induced bioenergetic abnormalities appeared to constitute a state of “metabolic anoxia” underlying the progression of myocyte injury to cell death. This anoxic state could be ameliorated to some extent, but not prevented, by carbohydrate catabolism.
Human macrophage colony‐stimulating factor inhibits bone resorption by osteoclasts disaggregated from rat bone
Authors: Hattersley G. , Dorey E. , Horton M. A. , Chambers T. J. 
Published: February 2005

AbstractColony stimulating factors (CSFs) regulate the survival, proliferation and differentiation of haemopoietic progenitor cells, as well as the functional activity of mature cells. Because the osteoclast is derived from haemopoietic tissue, and because osteoblastic cells produce CSFs, we tested the effects of several CSFs on bone resorption by osteoclasts disaggregated from neonatal rat long bone. We found that recombinant macrophage (M)‐CSF was a potent inhibitor of bone resorption, causing significant inhibition at concentrations similar to those required to support the growth of macrophage colonies in agar. Unlike other inhibitors of osteoclastic resorption, M‐CSF did not alter cytoplasmic motility in time‐lapse recordings, suggesting that M‐CSF may inhibit osteoclasts through a different transduction mechanism. None of the remaining cytokines tested (granulocyte‐macrophage CSF, interleukin 3, interleukin 6, or interferon γ) influenced bone resorption. M‐CSF production may be a mechanism by which osteoblastic cells, which produce M‐CSF, may regulate osteoclastic function. Alternatively, inhibition of osteoclastic resorption by a CSF that is responsible for amplification of the macrophage compartment may reflect a close lineage relationship between mononuclear phagocytes, in which M‐CSF induces a diversion of lineage resources away from osteoclastic function.
Transfer of functional EGF receptors to an IL3‐dependent cell line
Authors: Collins Mary K. L. , Downward Julian , Miyajima Atsushi , Maruyama Kazuo , Arai Ken‐Ichi , Mulligan Richard C. 
Published: February 2005

AbstractEpidermal growth factor (EGF) is a small protein that acts as a mitogen for various epidermal, epithelial, and fibroblastic cells that bear specific EGF receptors. The molecule that binds EGF is a 175‐kD transmembrane protein, with an extracellular ligand binding domain and an intracellular domain that possesses tyrosine kinase activity, thought to be involved in the mitogenic signalling process. Here we have constructed a recombinant murine retrovirus that transduces a human cDNA encoding the 175‐kD protein and used this retrovirus to infect BAF3, a murine, bone marrow‐derived cell line, which is dependent on the haematopoietic factor interleukin‐3 (IL3) for its growth in culture. The EGF receptors expressed on the infected cells exhibit two affinity states, as well as EGF‐stimulated autophosphorylation. Furthermore, EGF can replace IL3 in supporting short‐term proliferation of these cells. These data identify functional properties of the EGF receptor upon expression of the 175‐kD EGF binding protein in a haemotopoietic cell that does not express endogenous receptors. They also suggest that gene transfer of growth factor receptors to heterologous cells may allow novel growth stimuli to be exploited.
Quantitation of specific binding of erythropoietin to human erythroid colony‐forming cells
Authors: Sawada K. , Krantz S. B. , Sawyer S. T. , Civin C. I. 
Published: February 2005

AbstractHighly purified human erythroid colony‐forming cells (ECFC), which consist predominately of colony‐forming units‐erythroid (CFU‐F), were prepared from human blood and used to study the binding and processing of erythropoietin (Ep). When radioiodinated human recombinant Ep (125I‐rEp) was incubated with these cells, binding was specific and saturable. Specific binding was directly proportional to cell concentration and did not occur with other human cells. Saturation of specific binding at 3°C occurred at 1 nM (3.9/U/ml), and Scatchard analysis revealed two classes of binding sites on the cell surface. Of a total of 1,050 binding sites per ECFC, one‐fifth had a Kd of 0.10 nM, while the remainder had a Kd of 0.57 nM. Specific binding was twofold greater at 37°C than at 3°C, and removal of surface‐bound Ep with acid indicated that 125I‐rEp was internalized into the cells after incubation at 37°C. Further incubation at this temperature showed a decline of cellular radioactivity, with a release of small molecular weight degradation fragments into the medium. These studies demonstrate two classes of receptors for Ep on normal human ECFC. Internalization and degradation of EP occur, and the biologic effect of the hormone is produced by a small number of Ep molecules, as demonstrated in murine erythroid progenitor cells.
Disturbance of cell proliferation by two model compounds of lipid peroxidation contradicts causative role in proliferative senescence
Authors: Poot Martin , Esterbauer Hermann , Rabinovitch Peter S. , Hoehn Holger 
Published: February 2005

AbstractCumene hydroperoxide (Chp), a lipophilic peroxide, and hydroxy‐nonenal (HNE), a breakdown product of lipid peroxides, were used as model compounds to assess the effects of lipid peroxidation upon cell proliferation. Amniotic fluid fibroblastlike (AFFL) cells and human diploid skin‐derived (HDFL) cells were cultured with the two model compounds and cell proliferation was assayed via bromodeoxyuridine‐Hoechst flow cytometry. At low doses Chp elicited an accumulation of cells in the S and G2 phase, while at higher doses the fraction of nonproliferating cells increased as well. Low doses of HNE caused an accumulation of cells in the G1 and G2 phase, whereas an additional increase of cells in S phase and in the nonproliferating fraction was found at an elevated concentration. A delay of onset of proliferation was obtained with both Chp and HNE. Permanent arrests in the S, G2, and G1 compartment are provoked by Chp only when Chp was applied together with serum. HNE, to the contrary, elicited a permanent arrest in the G2 and the G1 compartment even if added to quiescent cell cultures. Additionally, HNE caused a combination of a prolongation of the G1 phase of the cell cycle and an arrest in this compartment, which is reminiscent of cell differentiation. HDFL cells were much more sensitive toward Chp than were AFFL cells, but both cell types showed similar sensitivities toward HNE. We conclude that lipophilic peroxides exert toxic effects upon cell proliferation distinct from the pattern elicited by aldehydic breakdown products of lipid peroxides. The pattern of cell cycle arrest induced by Chp and HNE makes it unlikely that Chp and HNE, or related products of lipid peroxidation, are responsible for the limitation of the proliferative life span of human fibroblasts in culture.
Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide
Authors: McGowan Joan A. 
Published: February 2005

AbstractHepatocyte DNA synthesis, initiated by epidermal growth factor (EGF), is reversibly inhibited by 2% dimethyl sulfoxide (DMSO). At that concentration, both the survival of the cells in culture and the expression of differentiated functions are prolonged. DMSO does not affect thymidine uptake or EGF receptor binding. Moreover, EGF receptor binding is maintained at 84% of initial 12 hr binding when cells are cultured for several days in the presence of DMSO, whereas specific receptor binding declines to 49% of initial binding under standard culture conditions without DMSO. Studies of hepatocyte functional activity indicate that, during early culture, total cellular export protein synthesis, specific albumin synthesis, and glycogen synthesis are enhanced in the presence of DMSO. Dexamethasone is required for the effect of DMSO on survival, and although dexamethasone alone enhances hepatocyte DNA synthesis in the presence of EGF, it does not reverse the inhibitory effect of 2% DMSO on DNA replication. The correlation of prolonged survival with growth inhibition supports the hypothesis that hepatic growth and differentiated functional activity may be reciprocally regulated.
Insulin‐like growth factor binding protein secretion by muscle cells: Effect of cellular differentiation and proliferation
Authors: McCusker Robert H. , Clemmons David R. 
Published: February 2005

AbstractSeveral cell types have been shown to secrete insulin‐like growth factor binding proteins (IGF‐BP) in vitro. Since IGF‐BP influences cell responsiveness to IGF, three muscle cell types were investigated to determine if they produced IGF‐BP and to identify factors that regulate IGF‐BP secretion. Porcine smooth muscle cells (pSMC), rat L6 skeletal muscle cells, and mouse BC3H‐1 myocytes were used. IGF‐BP activity in serum‐free conditioned media was quantitated with a polyethylene glycol precipitation method. All three cell types secreted IGF‐BP activity into the medium. Insulin was a potent stimulant of IGF‐BP secretion for each cell type. Specifically, 1 ug/ml insulin increased the IGF‐BP concentration in conditioned media from 10.5 ± 1.3 to 15.0 ± 1.5 ng/ml in confluent L6 myotubes, from 42.5 ± 11.1 to 90.5 ± 9.8 ng/ml in confluent BC3H‐1 cells, and from 2.1 ± 0.1 to 3.8 ± 0.1 ng/ml in confluent pSMC. L6 myotubes required more insulin (8 ug/ml) to achieve a half‐maximal stimulation of IGF‐BP secretion than confluent pSMC, differentiation deficient L6.DD cells or BC3H‐1 cells, where half‐maximal stimulation occurred between 125 and 300 ng/ml. L6 myoblasts were 40‐fold more sensitive to insulin stimulation of IGF‐BP secretion than L6 myotubes. IGF‐I, although it interferes with the assay and thereby lowers the amount of detectable IGF‐BP, stimulated the secretion of IGF‐BP from all three cell types. Dexamethasone, (10−7 M) decreased IGF‐BP secretion into the media by approximately 50% for all three cell types. Affinity cross‐linking and ligand blotting of 125I‐IGF‐I to conditioned media from each cell type showed (IGF‐BP)‐(IGF‐I) complexes with molecular weights ranging 32–40 kDa (24–32 kDa for IGF‐BP and 7.5 kDa for IGF‐I). Insulin stimulated cell proliferation for both L6 myoblasts and BC3H‐1 myocytes. This cell proliferative response was associated with an increase in IGF‐BP secretion/cell in response to insulin. In contrast dexamethasone decreased L6 myoblast proliferation and decreased IGF‐BP secretion/cell. We conclude that IGF‐BP is secreted by each muscle cell type and that the state of cellular differentiation or quiescence influences its basal and insulin‐stimulated secretion. Insulin and IGF‐I are stimulators of IGF‐BP secretion, whereas dexamethasone inhibits IGF‐BP secretion. Because these hormones control muscle cell growth and differentiation, the IGF‐BP may play an important regulatory role in these processes.
Properties of binding sites for chloroquine in liver lysosomal membranes
Authors: Colombo Maria Isabel , Bertini Francisco 
Published: February 2005

AbstractChloroquine (CQ) is an antimalarial and antirheumatic drug that accumulates in lysosomes. We purified liver lysosomal membranes of tritosomes from albino mice injected with Triton WR 1339. The membranes were used for the binding assay with CQ in 0.01 M Tris‐HCl buffer (pH 7.4). This binding was saturable, with a KD value of 6.2 μM. To understand the nature of CQ affinity, the binding was done under conditions that alter membrane structure and composition. Changes in pH, high ionic strength, and bivalent cations reversibly decreased the binding, while the effect of non‐ionic detergents was partially reversed. The cationic detergent Hyamine strongly decreased the binding, and its effect was irreversible. Binding increased after treatment with phospholipase C or D, while trypsin and neuraminidase had no effect. The results indicate the existence of binding sites for CQ in liver lysosomal membranes, which were strongly affected by changes of charge in the molecules involved in the binding. The treatment with the enzymes suggests that loss of polar groups of phospholipids increases the affinity of CQ by exposing protein sites located deep in the membrane, or by permiting a closer interaction between the drug and membrane lipids. CQ lysosomotropism and other effects of CQ on the lysosomal apparatus studied by other authors may be due not only to its accumulation inside the acid milieu of the lysosomes, in the same manner as other weak bases, but also to the affinity of CQ for binding sites in the lysosomal membrane.
Induction of cyclic AMP synthesis by forskolin is followed by a reduction in the expression of c‐myc messenger RNA and inhibition of 3H‐thymidine incorporation in human fibroblasts
Authors: Heldin Nils‐Erik , Paulsson Ylva , Forsberg Karin , Heldin Carl‐Henrik , Westermark Bengt 
Published: February 2005

AbstractWe have studied the effect of increased intracellular levels of cyclic AMP on the growth response to platelet‐derived growth factor (PDGF) of human foreskin fibroblasts in culture. It was found that forskolin, a potent stimulator of adenylate cyclase activity, inhibits the stimulatory effect of PDGF on 3H‐thymidine incorporation with a dose dependence similar to that observed with regard to cyclic AMP formation. A time‐course study indicated that forskolin has no effect on ongoing DNA synthesis but affects events in the prereplicative phase. The cell‐cycle block induced by forskolin was found to be reversible; after removal of the drug, DMA synthesis was initiated after a lag period, similar to that of the prereplicative phase of control cells. Forskolin had no effect on PDGF binding, receptor autophosphorylation, or c‐fos mRNA expression. However, a reduction in PDGF‐induced c‐myc mRNA expression was observed in cultures given forskolin. Forskolin was also found to have a marked stimulatory effect on the expression of interferon‐β2 mRNA expression. However, we were unable to demonstrate that the growth‐inhibitory effect of forskolin is mediated by interferon‐β. In conclusion, an increase in cAMP levels leads to a reversible inhibition of PDGF‐induced DNA synthesis in human fibroblasts, which may be related to an inhibition of c‐myc mRNA expression.
Localization of basic fibroblast growth factor to the developing capillaries of the bovine retina
Authors: Hanneken Anne , Lutty Gerard A. , McLeod D. Scott , Robey Frank , Harvey Anita K. , Hjelmeland Leonard M. 
Published: February 2005

AbstractThe basic fibroblast growth factor (FGF) is a potent mitogen that has vascular endothelium as one of its principle target cells. Recent work has provided both the complete amino acid sequence of basic FGF and the nucleotide sequence of the genes for both human and bovine basic FGF. Although capillary endothelial cells have been shown to produce basic FGF in vitro and to deposit basic FGF in their extracellular matrix in vitro as well, no direct evidence yet exists for the distribution of basic FGF in vivo. Antipeptide antibodies were prepared against a 15‐amino‐acid sequence from the amino terminus of basic FGF in order to avoid cross‐reactivity with acidic FGF, a protein with 55% overall homology to basic FGF. After affinity purification, these antisera were used to localize the basic fibroblast growth factor in the fetal and adult bovine retina. Immunoreactive material was found in capillaries of the inner nuclear layer, a capillary network undergoing development during the third trimester in the fetal bovine eye. Although the resolution of the technique does not permit a unique assignment of cellular localization, the presence of stain immediately adjacent to the lumen of capillaries suggests that capillary endothelial cells may produce the basic fibroblast growth factor in vivo during vascular development.
Butyric acid causes morphological changes in cultured chondrocytes through alterations in the extracellular matrix
Authors: Bretton Randolph H. , Pennypacker John P. 
Published: February 2005

AbstractButyric acid induces characteristic changes in the morphology of chick embryo chondrocytes. Chick embryo chondrocytes when cultured in the absence of butyrate exhibit a spherical morphology and synthesize cartilage‐specific chondroitin sulfate proteoglycan (CSPG). When these cultures are initiated and maintained in the presence of butyric acid, chondrocytes exhibit a mesenchymal morphology, a 90% reduction in the synthesis of CSPG, and a 75% reduction in DNA synthesis. The reduced synthesis of CSPG and DNA was shown not to be dependent on the morphological change. Chondrocytes require CSPG in order to express a spherical morphology, since including chondroitinase ABC in the culture media caused the cells to spread. In addition, the treatment of chondrocytes with purified CSPG prior to culture in media containing butyric acid resulted in spherical cells. The butyrate‐induced spreading was shown to require either serum or fibronectin and could be prevented with antiserum against chick cell‐surface fibronectin (cFn). Cell‐surface fibronectin, which was present on both spherical and flattened chondrocytes, organized into fibrils beneath cells which spread. Increased fibronectin synthesis was not responsible for the butyrate‐induced morphological change. From this evidence, it is concluded that the mechanism by which butyrate alters the morphology of these cells in culture involves inhibiting CSPG synthesis, thus preventing CSPG accumulation in the extracellular matrix (ECM). The absence of CSPG in the ECM allows fibronectin to mediate spreading of chondrocytes in culture.
Interaction of heparin with human basic fibroblast growth factor: Protection of the angiogenic protein from proteolytic degradation by a glycosaminoglycan
Authors: Sommer Andreas , Rifkin Daniel B. 
Published: February 2005

AbstractFibroblast growth factors (FGF) are a family of heparin‐binding angiogenic polypeptide mitogens. In the presence of heparin, recombinant human basic fibroblast growth factor (bFGF) is fully protected from tryptic digestion and partially protected from chymotryptic digestion. Complete protection of bFGF by heparin is achieved at ratios of growth factor:heparin of approximately 10 or less (w/w). The protection requires bioactive bFGF because inactivated bFGF is rapidly degraded by trypsin or chymotrypsin in the presence of heparin. The bFGF‐heparin interaction forms hydrophobic complexes which become insoluble in aqueous buffers at bFGF:heparin ratios of 8 to 10 (w/w). The heparin was found to bind up to a tenfold excess of bFGF on a weight basis. bFGF in the presence of heparin is as active as bFGF alone in inducing 3H‐thymidine incorporation into Swiss 3T3 fibroblast DNA.
Bicarbonate abolishes intracellular alkalinization in mitogen‐stimulated 3T3 cells
Authors: Szwergold Benjamin S. , Brown Truman R. , Freed Jerome J. 
Published: February 2005

AbstractAn increase in intracellular pH (pHi) following mitogenic stimulation has been reported in a variety of mammalian cells (W. Moolenaar, Annu. Rev. Physiol., 48:363–376, 1986; E. Rozengurt, Science, 234:161–166, 1986). This increase is currently believed to constitute a “permissive” signal in the process of cell activation (A.E. Lagarde and J.M. Pouyssegur, Cancer Biochem. Biophys. 9: 1–14, 1986). Since the majority of studies of this phenomenon have been conducted in the nonphysiological milieu of bicarbonate‐free solutions, we have undertaken a study of the effects of bicarbonate and CO2 on mitogen‐induced intracellular alkalinization in NIH 3T3 cells.Using nuclear magnetic resonance (NMR) spectroscopy and novel 31P NMR pH indicators (2‐amino‐phosphono‐carboxylic acids) we found that mitogen induces an increase in pHi of 0.16 units only in cells bathed in medium containing low concentrations of bicarbonate (less than 1 mM) and not in cells bathed in medium containing physiological levels of bicarbonate (10–30 mM). In addition to abolishing the mitogen‐induced alkalinization, bicarbonate stabilizes pHi at 7.25 units as the external pH (pHe) is varied from 7.0 to 7.6. In contrast, in a bicarbonate‐free medium pHi increases from 6.9 to 7.3 over the same range of external pHs.At a constant external pH, increasing the bicarbonate/CO2 concentration results in an increase in pHi from 6.9 in bicarbonate‐free solution to 7.25 in a bicarbonate‐buffered medium. This relationship is hyperbolic with half‐maximal effect occurring at a concentration of 0.4 mM bicarbonate at pH 7.05 and 37°C.Our results suggest that the observations of mitogen‐induced alkalinization may be due to the use of nonphysiological bicarbonate‐free media. Since this increase in pHi is not observed in physiological media where bicarbonate concentrations are usually greater than 20 mM, we conclude that an increase in pHi is not an obligatory or usual part of the cellular response to growth factors in vivo.