bluuebunny/crossref_metadata_2025_split huggingface dataset:
Growth factor responsiveness of human articular chondrocytes: Distinct profiles in primary chondrocytes, subcultured chondrocytes, and fibroblasts
Authors: Guerne Pierre‐André , Sublet Arthur , Lotz Martin 
Published: February 2005

AbstractThe objectives of this study were to establish a growth factor response profile for adult human articular chondrocytes, to determine whether this is unique for chondrocytes or influenced by the differentiation status of the cells, and to characterize growth factor interactions. It is shown that transforming growth factor‐β (TGF‐β) is the most potent mitogen among a variety of factors tested. All three isoforms of TGF‐β caused similar dose‐dependent increases in chondrocyte proliferation. Other members of the TGF‐β family, including bone morphogenetic protein 2B (BMP2B), activin, and inhibin, did not detectably increase chondrocyte proliferation. Platelet‐derived growth factor‐AA (PDGF‐AA), basic fibroblast growth factor (bFGF), and insulin‐like growth factor 1 (IGF‐1) also stimulated proliferation but were less effective than TGF‐β. In contrast to findings with other cell types, the effects of TGF‐β on chondrocyte proliferation were not dependent on the endogenous production of PDGF. The cytokines Interleukin 1 (IL‐1) and tumor necrosis factor‐α (TNF‐α) gave no stimulation, but IL‐1 inhibited chondrocyte proliferation induced by TGF‐β or serum. This response profile was characteristic for primary chondrocytes from human adults and distinct from subcultured (dedifferentiated) chondrocytes or skin fibroblasts. The latter preferentially responded to PDGF, and IL‐1 caused greater increases in proliferation than TGF‐β. In summary, these results describe growth factor responses that are characteristic for chondrocytes and provide a basis for the analysis of changes in chondrocyte growth proliferation that occur in aging and tissue injury. © 1994 Wiley‐Liss, Inc.
Termination of endothelin signaling: Role of nitric oxide
Authors: Goligorsky Michael S. , Tsukahara Hirokazu , Magazine Harold , Andersen Thomas T. , Malik Asrar B. , Bahou Wadie F. 
Published: February 2005

AbstractCellular mechanisms responsible for the termination of ET‐1 signal are poorly understood. In order to examine the hypothesis that nitric oxide serves as a physiological brake of ET‐ 1 signaling, Chinese hamster ovary (CHO) cells stably transfected with the ETA receptor cDNA (CHO‐ET) were studied. CHO‐ET responded to ET‐1 with robust [Ca2+], transients and developed a long‐lasting homologous desensitization. Donors of nitric oxide (NO), 3‐morpholino‐sydnonimine HCl(SIN‐1), or sodium nitroprusside (SNP) reduced the amplitude of these responses, accelerated the rate of [Ca2+], recovery, and counteracted the development of homologous desensitization by a cyclic GMP‐independent mechanism, suggesting an alternative mode for NO modulation of ET‐1 responses. Stimulation of CHO‐ET cells with mastoparan, a wasp venom acting directly on G proteins (bypassing receptor activation), was inhibited by NO, revealing a postreceptoral target for NO‐induced modulation of [Ca2+] mobilization. Using a lys9‐biotinylated ET‐1 (ET‐1 [BtK9]), binding sites were “mapped” in CHO‐ET cells. Receptor‐ligand complexes did not exhibit spontaneous dissociation during 60min observations. Quantitative fluorescence microscopy revealed that SNP or SIN‐1 caused a rapid, concentration‐dependent, and reversible dissociation of biotinylated ET‐ 1 from ETA receptor (EC50 = 75 μM and 6 μM, respectively), an effect that was not mimicked by 8‐bromo‐cyclic GMP. “Sandwich” co‐culture of endothelial cells with CHO‐ET showed that activation of NO production by endothelial cells similarly resulted in dissociation of ET‐1 [BtK9] from ETA receptors. We hypothesize that NO plays a role in physiological termination of ET‐1 signalling by dual mechanisms: (1) displacement of bound ET‐1 from its receptor, thus preventing homologous desensitization, and (2) interference with the postreceptoral pathway for [Ca2+] mobilization, hence inhibiting end‐responses to ET‐1. © 1994 Wiley‐Liss, Inc.
Binding and activation of plasminogen on the surface of osteosarcoma cells
Authors: Campbell Phil G. , Wines Karen , Yanosick Thomas B. , Novak Josef F. 
Published: February 2005

AbstractPlasmin (Pm) is a broad action serine protease implicated in numerous physiological functions. In bone, Pm may play a role in growth, resorption, metastasis, and the activation of growth factors. The various components of the Pm system are known to bind and function on the cell surface of various cell types, but no pertinent data are available describing membrane‐bound Pm or its zymogen, plasminogen (Pg), in either normal or neoplastic bone cells. We report here that Pg binds to the surface of the human osteosarcoma cell line MG‐63 and is activated to Pm by endogenous urokinase plasminogen activator (uPA). These conclusions are based on experiments utilizing radiolabeled compounds and a cell surface proteolytic assay measuring amidolytic activity of Pm. 125I‐Pg binding to cells was time dependent, saturable, reversible, and specific. Binding was characterized by a relatively low affinity (Kd ∼0.9 μM) and a high capacity (∼7.5 x 106 sites/cell). The binding of 125I‐Pg was associated with lysine binding sites of the plasminogen molecule. Activation of 125I‐Pg to 125I‐Pm occurred on the cell surface and was dependent upon cell bound uPA, as determined by inhibitory antibodies. Binding of Pg to MG‐63 monolayers represented ∼80% bound specifically to the cell surface and the remainder to the surrounding extracellular matrix. Either co‐incubation with uPA or pre‐incubation with Pm resulted in increased 125I‐Pg binding to osteosarcoma cells. Cell surface Pm proteolytic activity was confirmed by an amidolytic chromogenic assay. Both Pm and Pg bound to cells with Pg being activated by endogenous uPA. Plasmin activated on the cell surface was partially protected from inhibition by α2‐antiPm (requiring Pm lysine binding site interaction) but inhibited by aprotinin, (interacting directly with the Pm catalytic site). Resistance of cell bound Pm to α2‐antiPm inhibition suggests that cell surface proteolysis can occur in the presence of a soluble Pm inhibitor known to exist in the extracellular space. Based on these results, we speculate that the various bone physiological processes implicating Pm may occur at or near the bone cell surface. © 1994 wiley‐Liss, Inc.
Heterogeneity in collagen biosynthesis by sprouting retinal endothelial cells
Authors: Canfield A. E. , Schor A. M. 
Published: February 2005

AbstractBovine retinal microvascular endothelial cells can display two distinct and reversible morphologies in culture: ‘cobblestone’ and ‘sprouting’. The cobblestone morphology resembles the resting cells lining the lumen of mature vessels while the sprouting morphology resembles the angiogenic cells involved in the formation of new vessels. Retinal cells displayed some heterogeneity in the shape of the cells making up the cobblestone monolayer. In contrast, all cell lines displayed an identical sprouting morphology. We have investigated the synthesis of matrix macromolecules by retinal endothelial cells displaying either the cobblestone or the sprouting morphology. Type IV was the only collagen synthesised by eight different lines of early‐passage (between one and six) cobblestone endothelial cells. Collagen types I and III were not detected in these cultures. In contrast, heterogeneity was observed in the types of collagen synthesised by four lines of early‐passage cells displaying the sprouting morphology. That is, two lines synthesised collagen types I, III and IV, whereas two other lines continued to synthesise only type IV collagen. Both cobblestone and sprouting cells synthesised fibronectin and thrombospondin, although the relative amounts of these macromolecules varied with culture conditions. The pattern of collagen synthesis by cobblestone cells was also affected by in vitro „ageing”︁: 4/5 lines examined above passage eight synthesised collagen types I, III and IV. Our results indicate that there is heterogeneity in the sprouting phenotype displayed by retinal endothelial cells, and that this phenotype is not necessarily associated with the synthesis of type I collagen. We suggest that differences in the spectrum of matrix macromolecules synthesised by sprouting endothelial cells may play a role in the control of angiogenesis. © 1994 wiley‐Liss, Inc.
Identification of an IP3 receptor in endothelial cells
Authors: Bourguignon Lilly Y. W. , Iida N. , Sobrin L. , Bourguignon C. J. 
Published: February 2005

AbstractIn this study we have used saponin to permeabilize bovine endothelial cell membranes in order to directly test the involvement of IP3 in regulating internal Ca2+ release. Our results indicate that the release of internal Ca2+ occurs as early as 1–3 seconds after IP3 addition. This IP3‐induced internal Ca2+ release can be inhibited by heparin (an IP3 receptor antagonist). Further binding of [3H]IP3 to saponin‐permeabilized bovine endothelial cells reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of ≈0.50 (±0.03) nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the bovine endothelial cell IP3 receptor (≈260 kDa) displays immunological cross‐reactivity with the rat brain IP3 receptor. Immunofluorescence data indicates that the IP3 receptor is preferentially located at the perinuclear region of the cells. In addition, PCR analysis of first‐strand cDNAs from both bovine endothelial cells and rat brain tissues reveals that the IP3 receptor transcript in bovine endothelial cells belongs to the short non‐neuronal form and not the long neuronal form detected in rat brain tissue. These findings suggest that the IP3 receptor in endothelial cells is both structurally and functionally analogous to that reported in non‐neuronal cell systems and probably plays an important role in agonist‐induced endothelial cell activation. © 1994 wiley‐Liss, Inc.
Plasma membrane K+/H+‐ATPase From leishmania donovani
Authors: Jiang Suping , Anderson Steven A. , Winget G. Douglas , Mukkada Antony J. 
Published: February 2005

AbstractLeishmania donovani has an active K+/H+ exchange system on the surface membrane. Modulation of external K+ concentration resulted in a corresponding change in internal pH (pHi) suggesting a link between proton and potassium transport. Although a Na+/H+ antiporter is present on the plasma membrane, its sensitivity to amiloride suggests that it operates independent of K+/H+ exchange. Reduction of cellular ATP with NaN3 and KCN inhibits K+/H+ exchange showing thereby that the process is energy dependent. The K+/H+ exchange is sensitive to inhibitors of the gastric K+/H+‐ATPase. It is concluded that the H+‐ATPase previously reported on the plasma membrane of L. donovani is in fact a K+/H+‐ATPase. © 1994 wiley‐Liss, Inc.
Regulation of gamma‐glutamyl transpeptidase and alkaline phosphatase activities in immortalized rat brain microvessel endothelial cells
Authors: Roux F. , Durieu‐Trautmann O. , Chaverot N. , Claire M. , Mailly P. , Bourre J.‐M. , Strosberg A. D. , Couraud P.‐O. 
Published: February 2005

AbstractRat brain microvessel endothelial cells were immortalized by transfection with a plasmid containing the E1A adenovirus gene. One clone, called RBE4, was further characterized. These cells display a nontransformed phenotype and express typical endothelial markers, Factor VIII‐related antigen and Bandeiraea simplicifolia binding sites. When RBE4 cells were grown in the presence of bFGF and on collagen‐coated dishes, confluent cultures developed sprouts that extend above the monolayer and organized into three‐dimensional structures. The activity of the blood‐brain barrier‐associated enzyme, gamma‐glutamyl transpeptidase (γGTP), was expressed in these structures, not in the surrounding monolayer. Similar results were obtained with the microvessel‐related enzyme alkaline phosphatase (ALP). Addition of agents that elevate intracellular cAMP reduced the formation of three‐dimensional structures, but every cell inside the aggregates still expressed γCTP and ALP activities. Such structures, associated with high levels of γCTP and ALP activities, were also induced by astroglial factors, including (1) plasma membranes from newborn rat primary astrocytes or rat glioma C6 cells, (2) C6 conditioned media, or (3) diffusible factors produced by primary astrocytes grown in the presence of, but not in contact with RBE4 cells. RBE4 cells thus remain sensitive to angiogenic and astroglial factors for the expression of the blood‐brain barrier‐related γCTP activity, as well as for ALP activity, and could constitute the basis of a valuable in vitro model of the blood‐brain barrier. © 1994 wiley‐Liss, Inc.
Depleted level of heparan sulfate proteoglycan in the extracellular matrix of endothelial cell cultures exposed to endotoxin
Authors: Colburn P. , Kobayashi E. , Buonassisi V. 
Published: February 2005

AbstractExposure of cultured endothelial cells to endotoxin causes an increase in the amount of cellular heparan sulfate proteoglycan and a depletion of this molecule in the extracellular matrix. Concomitant with the decrease in the extracellular matrix is the appearance of a fraction of proteoglycan bearing altered carbohydrate moieties in the culture medium, β‐mereaptoethanol, mannitol, and dimethyl sulfoxide bring back to normal the structural properties of the proteoglycan in the medium and restore the matrix content in proteoglycan to a level comparable to that of control cells but do not affect the increase in the amount of proteoglycan on the cell. This “uncoupling” suggests that two independent chains of events underlie the synthetic and structural changes of the proteoglycan triggered by endotoxin in the endothelial cell. © 1994 wiley‐Liss, Inc.
Induced expression of p52(PAI‐1) in normal rat kidney cells by the microfilament‐disrupting agent cytochalasin D
Authors: Higgins Paul J. , Ryan Michael P. , Providence Kirwin M. 
Published: February 2005

AbstractIn established normal rat kidney (NRK) cells, synthesis of the 52 kDa type‐1 inhibitor of plasminogen activator [p52(PAI‐1)] is stimulated by the cell shape—modulating fungal metabolite cytochalasin D (CD). Induction paralleled the time course of morphologic change and reflected relatively specific increases in sapo‐nin‐resistant p52(PAI‐1) protein accumulation (approximating ten‐ to thirty‐fold over control) and mRNA abundance (seven‐ to nine‐fold). Augmented p52(PAI‐1) mRNA levels closely correlated with increases in 43 kDa p52(PAI‐1) core protein biosynthesis. Sensitivity to tunicamycin indicated that N‐linked post‐translational modifications to this 43 kDa core species generated the full complement of 50 kDa (intermediate) and 52 kDa (mature) p52(PAI‐1) glycosylated isoforms. CD‐induced p52(PAI‐1) expression occurred efficiently in quiescent NRK cells maintained under serum‐free conditions as well as in fully serum‐supplemented actively growing cultures. While 8‐bromo‐cAMP reduced both constitutive and transforming growth factor‐beta—induced p52(PAI‐1) synthesis by >50%, no such inhibition was evident in short‐term (4 h) CD‐stimulated cultures. Long‐term (24 h) exposure of NRK/CD cells to 8‐bromo‐cAMP did result in an approximately 34% reduction in stimulated p52(PAI‐1) expression, however, levels expressed by NRK/CD + cAMP populations remained markedly elevated relative to control values. These data suggest the existence of a cell shape—dependent aspect of p52(PAI‐1) expression control distinct from both the constitutive and growth factor—mediated pathways of gene regulation. © 1994 wiley‐Liss, Inc.
Type‐1 insulin‐like growth factor receptor overexpression produces dual effects on myoblast proliferation and differentiation
Authors: Quinn Lebris S. , Steinmetz Brian , Maas Adolf , Ong Luan , Kaleko Michael 
Published: February 2005

AbstractUsing a retroviral vector, we developed a line of C2 mouse skeletal myoblasts, C2‐LISN, which expressed high levels of the human type‐1 insulin‐like growth factor (IGF) receptor. When switched to low serum medium, C2‐LISN myoblasts underwent terminal differentiation extremely rapidly compared to control C2 myoblasts. In high serum conditions which were not permissive for differentiation, C2‐LISN myoblasts expressed ten‐fold higher levels of the myogenic transcription factor myogenin than did control C2 myoblasts. When cultured in low serum medium with both transforming growth factor‐β (TGF‐β) and high concentrations of IGF‐I, C2‐LISN myoblasts failed to differentiate and grew to very high saturation densities, forming multilayers. Upon removal of TGF‐β, multilayered C2‐LISN myoblasts differentiated within 2 days. These results demonstrate that overexpression of the type‐1 IGF receptor can amplify signals which stimulate myogenic differentiation. Overexpressed type‐1 IGF receptors can also mediate strong mitogenic signals if differentiation is inhibited by TGF‐β. The C2‐LISN myoblast cell line may be a useful model to investigate the intracellular pathways which stimulate myogenic differentiation. Additionally, overexpression of the type‐1 IGF receptor could provide a strategy to expand populations of differentiation‐competent myoblasts for experimental or clinical applications. © 1994 wiley‐Liss, Inc.
Human osteosarcoma (U‐2 OS) cells express both insulin‐like growth factor‐I (IGF‐I) receptors and insulin‐like growth factor‐II/mannose‐6‐phosphate (IGF‐II/M6P) receptors and synthesize IGF‐II: Autocrine growth stimulation by IGF‐II via the IGF‐I receptor
Authors: Raile K. , Höflich A. , Kessler U. , Yang Y. , Pfuender M. , Blum W. F. , Kolb H. , Schwarz H. P. , Kiess W. 
Published: February 2005

AbstractRecently, insulin‐like growth factor‐I and ‐II (IGF‐I and ‐II) have been implicated in the growth promotion of tumors in vivo and tumor cells in vitro. We have studied the human osteosarcoma cell line U‐2 OS in order (1) to gain more insight into the growth promoting actions of the IGFs and (2) to establish an in vitro tissue culture model of IGF action in human tumor cells. Specific binding of 125I‐IGF‐I and 125I‐IGF‐II to IGF‐I receptors and IGF‐II/mannose‐6‐phosphate (M6P) receptors on U‐2 OS cells was demonstrated in competitive binding experiments and in affinity crosslinking experiments. Western blotting of cell extracts confirmed the expression of the IGF‐II/M6P receptor. In addition, in Northern blotting experiments using total RNA from U‐2 OS cells IGF‐I receptor RNA of 11 kb and IGF‐II/M6P receptor RNA of approximately 9 kb were detected. Solution hybridization experiments confirmed the presence of IGF‐I receptor and IGF‐II/M6P receptor RNA. In a subset of experiments DNA synthesis was measured as 3H‐thymidine uptake into cellular DNA of U‐2 OS cells. Normal rat serum stimulated DNA synthesis maximally. IGF‐I‐deficient serum from hypophysectomized rats as well as IGF‐I or IGF‐II without serum were approximately twofold and tenfold, respectively, less potent than serum in stimulating 3H‐thymidine uptake. The concentrations of IGF‐I and IGF‐II needed for half maximal stimulation of DNA synthesis corresponded well with the respective concentrations required for half maximal inhibition of 125I‐IGF‐I binding to U‐2 OS cells. The anti‐IGF‐I receptor antibody alphaIR3 blocked the IGF‐I and IGF‐II stimulated increase of 3H‐thymidine uptake. In addition, basal DNA synthesis was partially inhibited by the anti‐IGF‐I receptor antibody. These data suggest that U‐2 OS cells synthesize and secrete IGF‐like peptides. Northern blotting experiments confirmed that U‐2 OS cells express an IGF‐II RNA species of 5.3 kb but no IGF‐I transcripts. In a series of RNase protection assays, protected RNA fragments were detected with an IGF‐II riboprobe. © 1994 wiley‐Liss, Inc.
Both viral (adenovirus E1B) and cellular (hsp 70, p53) components interact with centrosomes
Authors: Brown C. Randell , Welch William J. , Doxsey Steven J. , White Eileen 
Published: February 2005

AbstractHuman 293 cells, transformed by and expressing the early region of the adenovirus genome (i.e., E1A and E1B), contain a phase‐dense cytoplasmic structure situated in close proximity to the nucleus. Via indirect immunofluorescence studies such structures have been previously shown to contain both the adenovirus E1B (55 kDa) protein as well as the tumor suppressor gene product p53. Here we show that such structures also stain positive for the cytoplasmic hsp 70 proteins. Such phase‐dense structures containing hsp 70, p53, and adenovirus E1B are not unique to 293 cells but also are observed in rodent cell lines stabily transfected with the early region of the adenovirus genome. Using an antibody against a centrosomal protein, pericentrin, we show that these cytoplasmic phase‐dense structures are in close proximity to the centrosome. Cell fractionation studies revealed such structures to be highly detergent insoluble. However, like the centrosome, the cytoplasmic phase‐dense structures could be rendered detergent soluble following treatment of the cells with agents that disrupt the integrity of the cytoskeleton. While the phase‐dense structures appear in close proximity to the centrosome in interphase cells, during mitosis the centrosome and the phasedense bodies separate from one another. Owing to these observations we examined whether hsp 70 and p53 might also co‐localize with the centrosome in other cell types not expressing the adenovirus E1A/E1B proteins. We show that a portion of both hsp 70 and p53 indeed are present within the centrosome in Hela, COS, and 3T3 cells. These observations raise the possibility that components like hsp 70 and p53 may participate in the mechanism(s) controlling cell division in mammalian cells. © 1994 Wiley‐Liss, Inc.
Chronic hypoxia impairs the differentiation of 3T3‐L1 fibroblast in culture: Role of sustained protein kinase C activation
Authors: Sahai Atul , Patel Mona S. , Zavosh Aryana S. , Tannen Richard L. 
Published: February 2005

AbstractThe effect of hypoxia on 3T3‐L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM). Control cultures remained in GM throughout the incubation period. Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity. Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM. By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 ± 4% of cells accumulating fat deposits. Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype. Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively. In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme. In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H7 exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls. These studies indicate that chronic hypoxia impairs the differentiation of 3T3‐L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process. © 1994 Wiley‐Liss, Inc.