bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modulation of myogenic determination factor 1 expression by cell‐cell contact
Authors: Song An , Daub Raymond , Harrington Maureen A. 
Published: February 2005

AbstractMyogenic determination factor 1 (MyoD1) expression is modulated by a variety of agents including growth factors and activated cellular proto‐oncogenes. However, little is known about the effect of cell‐cell contact, which increases as myoblasts terminally differentiate, on the control of MyoD1 expression. Steadystate levels of MyoD1 transcripts decline over a 6–12 hour time period when myoblasts plated at a single cell density are incubated in media supplemented with 0.2% serum; by 48 hours MyoD1 mRNA levels have returned to the initial basal level. The decline in MyoD1 transcripts is diminished, but not prevented in myoblasts which maintain cell‐cell contacts (at least 50% of cells with two or more sites of contact). MyoD1 transcript levels do not change if single cell cultures are maintained in 10% serum or are cocultured with fibroblasts. Analysis of conditioned media revealed that myoblasts plated at the single cell density or at a density which allowed multiple sites of cell‐cell contact are not producing an activity(s) responsible for modulating MyoD1 mRNA levels. The changes in MyoD1 expression are mediated at the transcriptional level. Thus changes in the degree of cell‐cell contact in cultures of myogenically determined cell lines effect changes in MyoD1 gene expression. Consequently when the influence of cytokines or other pharmacological agents on commitment to terminal myogenic differentiation is examined, the degree of cell‐cell contact within the culture system may affect the response elicited. © 1993 Wiley‐Liss, Inc.
Phenotypic effects of aldosterone and dexamethasone in a SV40‐transformed mammalian cortical ascending limb cell line exhibiting mineralocorticoid receptors
Authors: Vandewalle A. , Rafestin‐Oblin M.‐E. , Hagege J. , Géniteau‐Legendre M. , Cassingéna R. , Ronco P. M. 
Published: February 2005

AbstractWe have analyzed the functional and morphological effects of corticosteroid hormones in a SV40‐transformed rabbit cortical‐ascending‐limb (CAL) cell line (RC.SV2, Vandewalle et al., 1989) having mineralocorticoid (MR) and glucocorticoid (GR) receptors (Rafestin‐Oblin et al., 1993). Both aldosterone and dexamethasone (5 × 10−8M) induced a marked increase in (3H)ouabain binding (used to quantify membrane Na+ ‐K+ ATPase) detectable as early as 6 hours and maximal at 24 hours (+56‐57%) (due to a 1.6‐1.8‐fold increase in cell membrane binding sites without Kd alteration), and significantly augmented the ouabainsensitive component of Rb+ influx. Triiodothyronine (T3, 10−9 M) also stimulated ouabain binding by 21% but was not permissive for steroid action, whereas 5 μg/ml insulin had no effect. Both steroid hormones, T3 and insulin induced the formation of domes that was tightly correlated with ouabain binding (r = 0.949) except for insulin. The effects of aldosterone and dexamethasone on cell monolayers and cell ultrastructure were, however, strikingly different as aldosterone induced a marked amplification of basolateral areas with appearance of large intercellular spaces, reminiscent of the changes observed in deoxycorticosteronetreated rats, whereas dexamethasone predominantly influenced cell height. This discrepancy might be due to specific occupancy of MR and GR by aldosterone and dexamethasone, respectively, and/or to nongenomic effects of dexamethasone. We have thus characterized a cell culture model making it possible to analyze the actions of mineralocorticoid and glucocorticoid hormones in the mammalian kidney. © 1993 Wiley‐Liss, Inc.
High‐level expression of cytokine‐induced neutrophil chemoattractant (CINC) by a metastatic rat cell line: Purification and production of blocking antibodies
Authors: Wittwer Arthur J. , Carr Linda S. , Zagorski John , Dolecki Gregory J. , Crippes Barbara A. , De Larco Joseph E. 
Published: February 2005

AbstractSignificant levels of cytokine‐induced neutrophil chemoattractant (CINC) were found in serum‐free medium conditioned by a highly metastatic rat cell line, RC20. To study CINC's role in inflammation and metastasis, CINC was purified from this source for use in in vitro assays and for antibody production in goats and rabbits. CINC was a potent chemoattractant for rat neutrophils (EC‐50 0.5 nM). A fusion protein of glutathione‐S‐transferase and CINC (GST‐CINC) was produced in E. coli. Anti‐CINC polyclonal IgG was purified from immune goat and rabbit sera by protein A and GST‐CINC affinity chromatography. Both goat and rabbit anti‐CINC antibody preparations at 4 μg/mL (an 11‐fold molar excess) were found to completely block the activity of 2.5 nM CINC in a rat neutrophil chemotaxis assay. These antibodies have been used to develop a sensitive immunoassay for CINC. The availability of large amounts of affinity‐purified blocking anti‐CINC antibody will allow investigations into the role played by CINC in rodent inflammation models and in the metastasis of RC20 cells. © 1993 Wiley‐Liss, Inc.
Dibutyryl cyclic adenosine monophosphate differentially regulates cell proliferation in low and high alkaline phosphatase SaOS‐2 human osteosarcoma cells: Evidence for mediation by the insulin‐like growth factor‐II system
Authors: Mohan S. , Strong D. D. , Hilliker S. , Malpe R. , Lee K. , Farley J. , Baylink D. J. 
Published: February 2005

AbstractIn the present study, we have sought to determine whether a given signal transduction pathway can have diverse effects on subpopulations of cells of a lineage depending upon the stage of differentiation. To test this hypothesis, we selected the cyclic adenosine monophosphate (cAMP) signal transduction pathway because of its recognized importance in mediating the actions of many hormones, e.g., parathyroid hormone which acts on the bone‐forming cells, the osteoblasts. Subpopulations of human osteosarcoma SaOS‐2 cells with low (LSaOS) and high (HSaOS) alkaline phosphatase (ALP) content were chosen as model systems for preosteoblasts (pre‐OB) and osteoblasts (OB), respectively. Dibutyryl cyclic AMP (DBcAMP) treatment of serum free cultures produced a differential effect on the proliferation of LSaOS cells (40 ± 5% of control at 1 mM DBcAMP, P < 0.001) compared with HSaOS cells (no statistically significant effect). The finding supports the hypothesis. Next, we sought evidence for mediation, at least in part, by the insulin‐like growth factor (IGF)‐II regulatory system. We report that the basal expression of IGF‐II, IGF binding protein (IGFBP)‐3, and IGFBP‐4 was higher in LSaOS cells than in HSaOS cells with the opposite true for type I IGF receptor. DBcAMP treatment of LSaOS cells decreased IGF‐II and IGFBP‐3 but increased IGFBP‐4 and type I IGF receptor; no effect was observed for the type II IGF receptors. DBcAMP treatment of HSaOS cells had no detectable effect on IGF‐II; IGFBP‐3, or type I and type II IGF receptor expression; only IGFBP‐4 expression increased with DBcAMP. These observations suggest that the differential regulation of cell proliferation by the cAMP signal transduction pathway may be mediated, at least in part, by the IGF‐II regulatory system. © 1993 Wiley‐Liss, Inc.
Cell surface expression of DBA binding sites in LLC‐PK1 cells increases at confluence and is enhanced by PKA activation
Authors: Amsler Kurt 
Published: February 2005

AbstractPost‐confluent populations of LLC‐PK1 cells express many glycoproteins at the surface, including glycoproteins which bind the lectin Dolichos biflorus agglutinin (DBA). DBA‐binding glycoproteins are localized preferentially to the apical cell surface and exhibit molecular weights ranging from less than 30 kDa to greater than 200 kDa. Subconfluent cell populations exhibit little surface DBA binding. Upon attaining confluence DBA binding capacity increases progressively over several days. This correlates roughly with increasing activities of several differentiated apical membrane functions. Expression of DBA binding sites at the surface occurs on a cell‐by‐cell basis. An increasing level of surface DBA binding in the cell population corresponds to an increasing proportion of the cell population expressing binding sites. Activation of cAMP‐dependent protein kinase in post‐confluent cell populations, and to a lesser extent in subconfluent populations, increases surface DBA binding sites by a protein synthesis‐dependent mechanism after a lag period of at least 24 h. This results from increases in both the proportion of cells expressing surface binding sites and the level of binding sites on individual cells. © 1993 Wiley‐Liss, Inc.
Importance of cell aggregation for expression of liver functions and regeneration demonstrated with primary cultured hepatocytes
Authors: Yuasa Chie , Tomita Yumiko , Shono Masayuki , Ishimura Kazunori , Ichihara Akira 
Published: February 2005

AbstractAdult rat hepatocytes aggregated to form floating multicellular spheroids when cultured in Primaria dishes, which have a positively charged surface, in serum‐free Williams' medium E (WE) supplemented with insulin and epidermal growth factor (EGF). These hormones were essential for maintenance of the spheroids, whereas the size of the spheroids depended on the inoculum cell density. The spheroids retained in vivo levels of expressions of albumin and glucokinase and synthesized scarcely any DNA even in the presence of insulin and EGF. On transfer to type I collagen‐coated dishes, the spheroids gradually disaggregated and the cells formed monolayers, in which the expressions of albumin and glucokinase were suppressed and DNA synthesis and hexokinase activity were increased. DNA synthesis of hepatocytes in monolayer culture was maximal 24 hr after transfer of the spheroids, ∼80% of the hepatocyte nuclei were labelled with bromodeoxyuridine during culture for 48 hr, and the mitotic index was ∼70% after 60 hr. These results suggest that, in spheroids, hepatocytes remained in the G0 phase, but that when they formed monolayers, they progressed to the G1 phase and proceeded through the cell cycle in the presence of insulin and EGF. This work shows that the cell cycle of hepatocytes in culture can be manipulated by providing conditions for quiescence as spheroids or growth as monolayers and that the shape of hepatocytes is important for regulating their growth and liver‐specific functions. © 1993 Wiley‐Liss, Inc.
Thrombospondin mediates migration and potentiates platelet‐derived growth factor‐dependent migration of calf pulmonary artery smooth muscle cells
Authors: Yabkowitz Rachel , Mansfield Pamela J. , Ryan Una S. , Suchard Suzanne J. 
Published: February 2005

AbstractA precipitating factor in the development of atherosclerotic lesions is the inappropriate migration and proliferation of vascular smooth muscle cells (SMC) within the intima of the vessel wall. Focusing on the role of extracellular matrix proteins in SMC migration, we have demonstrated that thrombospondin (TSP) itself is a potent modulator of SMC motility and acts to potentiate platelet‐derived growth factor (PDGF)‐mediated SMC migration as well. Migration of SMC to TSP was dose dependent. Interestingly, maximal SMC migration to TSP exceeded that to either PDGF or basic fibroblast growth factor (bFGF). The distal COOH terminus of TSP was shown to mediate SMC migration as demonstrated by complete inhibition of the response by monoclonal antibody (mAb) C6.7. Nevertheless, proteolytic fragments of TSP were not as potent as intact TSP in mediating SMC migration. Only by combining the heparin‐binding domain (HBD) with the 140 kD COOH terminal fragment was SMC migration restored to levels seen with intact TSP. Based on antibody inhibition studies, an αv‐containing integrin receptor, but not αvβ1 or αvβ3, appeared to be involved in SMC migration to TSP. The coincidental expression of PDGF and TSP at sites of vascular injury and inflammation led us to evaluate the effect of suboptimal levels of TSP on SMC responsiveness to PDGF. SMC migration in response to PDGF was enhanced nearly 60% in the presence of suboptimal concentrations of TSP. This effect was specific for PDGF and dependent on the concentration of TSP with maximal potentiation obtained between 50–100 nM TSP, concentrations tenfold lower than those necessary for SMC migration to TSP itself. mAb C6.7 completely inhibited enhancement but, as with SMC migration to TSP alone, TSP proteolytic fragments did not possess the effectiveness of the intact molecule. Additional experiments assessing SMC migration to PDGF demonstrated that PDGF stimulated SMC motility indirectly by inducing TSP synthesis. These studies suggested that TSP functions as an autocrine motility factor to modulate SMC migration, which in conjunction with PDGF could serve to aggravate and accelerate development of atherosclerotic lesions at sites of vascular injury or inflammation. © 1993 Wiley‐Liss, Inc.
Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control
Authors: Vicart Patrick , Testut Patrice , Schwartz Bertrand , Llorens‐Cortes Catherine , Perdomo Juana J. , Paulin Denise 
Published: February 2005

AbstractMarkers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII‐related antigen. Treatment of IVEC cells with Interleukin‐1β (IL‐1 β) at 10 U.ml−1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM‐1), intercellular adhesion molecule‐1 (ICAM‐1), and vascular cell adhesion molecule‐1 (VCAM‐1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 μM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT‐PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium. © 1993 Wiley‐Liss, Inc.
Adenine nucleotides and adenosine metabolism in pig kidney proximal tubule membranes
Authors: Blanco Julià , Canela Enric I. , Sayós Joan , Mallol Josefa , Lluis Carme , Franco Rafael 
Published: February 2005

AbstractExogenous adenosine triphosphate (ATP) added to brush‐border membrane vesicles was rapidly degraded mainly to inosine according to the high ecto‐nucleotidase activities in these vesicles. In the absence of phosphate, inosine was slowly transformed into hypoxanthine, and xanthine oxidase and dehydrogenase activities were not detected. The presence of ecto‐adenosine deaminase and ecto‐adenosine monophosphate (AMP) nucleotidase was shown. The ecto‐adenosine deaminase was inhibited by deoxycoformycin and was also detected in rat renal brush‐border membrane vesicles. Using orthovanadate, levamisole, and α, β‐methylene adenosine diphosphate as possible inhibitors, alkaline phosphatase was shown to be the main agent responsible for ecto‐AMP nucleotidase activity. In pig renal basolateral membrane vesicles and in whole cell extracts from pig renal cortex, ecto‐AMP nucleotidase was the limiting factor in ATP degradation. Comparing the ATP catabolism in the whole cell cortical extract with the catabolism in the same sample precleared of membranes, it was shown that ectonucleotidase activity is mainly bound to the membranous components. It is also shown that the whole cell extract of pig renal cortex has hypoxanthine phosphoribosyl transferase activity, and it seems probable that the rapid and specific formation of luminal inosine and its transport into the cell in competition with adenosine may start the purine salvage pathway through the synthesis of IMP from hypoxanthine. © Wiley‐Liss, Inc.
Use of p112‐deficient myoblasts to determine the temporal order of the in vitro expression of myogenic components
Authors: Chen Xiao Yong , Lo Theodore C. Y. 
Published: February 2005

AbstractThe present investigation examines the function and site(s) of involvement of an ecto‐protein kinase and its substrate protein (a cell surface 112 kDa protein) in the in vitro myogenic pathway. The phosphorylated 112 kDa protein (p112) has recently been shown to be involved in myogenesis. Not much information is currently available on the role of the ecto‐protein kinase and the 112 kDa protein in modulating the expression of the myogenic factors and various muscle‐specific proteins. Five different p112‐deficient rat myoblasts were used to examine the temporal order of the in vitro expression of the myogenic components namely, L6 myoblasts treated with BrdUrd or phloretin, a conditional p112‐defective mutant (clone D1), an ecto‐protein kinase‐deficient mutant (clone F72), and a mutant defective in the 112 kDa protein (clone D1/S4). All these p112‐deficient myoblasts were also impaired in myogenesis. The absence of p112, ecto‐protein kinase, and/or the 112 kDa protein was found to have no effect on the Myf‐5 mRNA level. However, the expected increase in NCAM and Myf‐4 mRNAs was not observed in any of the p112‐deficient myoblasts examined. This suggests that the p112 site of action is probably located upstream of the Myf‐4 and NCAM sites in the myogenic pathway. While 7–28 fold increases in the MLC, MHC, and TnT transcripts were observed during myogenesis, such increases were not observed in the p112‐deficient myoblasts. However, when mutant D1/S4 was transfected with the myf‐4 cDNA, expression of Myf‐4 in the transfectant resulted in increased level of the MLC, MHC, and TnT mRNAs, and in myotube formation, even though the Myf‐5 and NCAM mRNA levels and p112 were not altered. This suggests that p112 may function by activating transcription of Myf‐4, which will subsequently promote the expression of muscle‐specific proteins and myotube formation. In the absence of p112, Myf‐5 cannot activate the expression of Myf‐4, NCAM, MLC, MHC, TnT, and myotube formation. If all these components are involved in the same myogenic pathway, then p112 may be acting downstream from Myf‐5, and upstream from NCAM and Myf‐4. © 1993 Wiley‐Liss, Inc.
Anti‐integrin antibodies induce type IV collagenase expression in keratinocytes
Authors: Larjava Hannu , Lyons J. Guy , Salo Tuula , Mäkelä Marja , Koivisto Leeni , Birkedal‐Hansen Henning , Akiyama Steven K. , Yamada Kenneth M. , Heino Jyrki 
Published: February 2005

AbstractDuring wound healing, pericellular proteolysis is thought to be essential for the detachment of keratinocytes from basement membrane and in their migration into the wound bed. We have characterized integrin‐type cell adhesion/migration receptors in human mucosal keratinocytes and examined their function in the regulation of type IV collagenase gene expression. Two major integrins of the β1 class, α2β1 and αβ1, were found to function as collagen and fibronectin receptors, respectively. Antibodies against β1 and α3 integrin subunits were found to stimulate the expression of the 92 kDa type IV collagenase severalfold in a dosedependent manner. Keratinocytes expressed also the 72 kDa type IV collagenase, the synthesis of which remained, however, unchanged in keratinocytes treated with anti‐integrin antibodies. Stimulation of 92 kDa enzyme was found to be caused directly by antibody binding to integrins, since Fab‐fragments of anti‐β1 antibodies alone were able to induce collagenase expression in the absence of secondary, clustering antibodies. Antibodies against α2β1 integrin caused no stimulation. Keratinocytes seeded on different substrata (plastic, collagen, fibronectin, laminin, or vitronectin) showed equal induction of type IV collagenase expression. Expression of 92 kDa type IV collagenase could not be induced by peptides (GRGDS, GRGES), proteins (fibronectin, laminin, fibrinogen., albumin), or antibodies to fibronectin. We suggest that proteolytic processes around keratinocytes can be regulated by extracellular factors signalling through integrin‐type receptors. © 1993 Wiley‐Liss, Inc.
Differential heat stress stability of epidermal growth factor receptor and erbB‐2 receptor tyrosine kinase activities
Authors: Liu Samuel M. , Carpenter Graham 
Published: February 2005

AbstractThe epidermal growth factor (EGF) and erbB‐2 receptors are structurally related membrane‐bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH‐3T3 or NR6 cells transfected with wild‐type EGF‐R and incubated at 37°C or heat shocked at 46°C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46°C, however, the basal tyrosine kinase activity of the wild‐type erbB‐2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF‐R extracellular domain and intracellular domain of erbB‐2 were heat stressed, 125I‐EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF‐stimulated chimeric erbB‐2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46°C. The loss of chimeric receptor erbB‐2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB‐2 receptors. Sensitivity of erbB‐2 to heat stress is also not a result of denaturation of this receptor's carboxy‐terminal domain. Insertion of the erbB‐2 tyrosine kinase domain into the EGF‐R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF‐R and erbB‐2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners. © 1993 Wiley‐Liss, Inc.