bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of rat proximal tubule epithelial cell growth by fibroblast growth factors, insulin‐like growth factor‐1 and transforming growth factor‐β, and analysis of fibroblast growth factors in rat kidney
Authors: Zhang Guohong , Ichimura Takaharu , Wallin Alf , Kan Mikio , Stevens James L. 
Published: February 2005

AbstractGrowth factors may play an important role in regulating the growth of the proximal tubule epithelium. To determine which growth factors could be involved, we have investigated the mitogenicity of various purified factors in rat kidney proximal tubule epithelial (RPTE) cells cultured in defined medium. Fibroblast growth factors, aFGF (acidic FGF) and bFGF (basic FGF), stimulate DNA synthesis in a dose‐dependent manner, with ED50 values of 4.5 and 3.2 ng/ml, respectively; their effects are not additive. With cholera toxin in the medium, both aFGF and bFGF can replace insulin or epidermal growth factor (EGF) to attain the maximum level of cell growth, but they cannot eplace cholera toxin. Cholera toxin specifically potentiates the effects of FGFs on DNA synthesis. At high cell density, both insulin and insulin‐like growth factor 1 (IGF‐1) induce DNA synthesis more effectively than EGF, FGFs and cholera toxin. The high concentration (0.2‐1.0 μg/ml) of insulin required for cell growth can be replaced by a low concentration of IGF‐1 (10‐20 ng/ml), indicating that insulin probably acts through a low affinity interaction with the IGF‐1 receptor. Transforming growth factor‐β1 (TGF‐β1) inhibits DNA synthesis induced by individual factors and combinations of factors in a concentration‐dependent manner. Northern blot analysis shows that mRNA for TGF‐β1, IGF‐1, and aFGF, but not bFGF are present in rat kidney. Western blot analysis and bioassay data confirmed that the majority of FGF‐like protein in rat kidney is aFGF. The data suggest that in addition to EGF, IGFs, and TGF‐β, FGFs may also be important kidney‐derived regulators of proximal tubule epithelial cell growth in vivo and in vitro.
Caco‐2 cells cultured in serum‐free medium as a model for the study of enterocytic differentiation in vitro
Authors: Jumarie Catherine , Malo Christiane 
Published: February 2005

AbstractCaco‐2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cels are normally cultured in the presence of 15–20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco‐2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush‐border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, γ‐glutamyltransferne, aminopeptidase N, and dipeptidyl‐dipeptidase IV), although the levels of sucrase activity were lower in ITS‐supplemented medium. Coating petri dishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush‐border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 × 10−8M) was added to the ITS‐supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while γ‐glutamyltransfrase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 × 10−6M). On the other hand, hydrocortisone (HC, 106M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco‐2 cells can be maintained in serum‐free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.
Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: Effects of flow rate, thrombin, and cytochalasin D
Authors: Eaton B. M. , Toothill V. J. , Davies H. A. , Pearson J. D. , Mann G. E. 
Published: February 2005

AbstractWe have applied a multiple isotope dilution technique to examine junctional permeability of human umbilical vein endothelial cells (HUVEC) in vitro. Primary cultures were grown to confluence on porous Cytodex‐3 microcarrier beads, packed into 0.3 ml columns (3 × 106 cells) and perfused at varying flow rates (0.3–1.2 ml/min) with HEPES‐buffered Tyrodes solution containing unlabeled cyanocobalamin, insulin, and albumin. Columns were challenged periodically with mixtures of radioactive tracers of different molecular size. Permeability to 22Na+, [57Co]cyanocobalamin (1.3 kD), [125l]insulin (6 kD) or [125l]albumin (66 kD) was assessed relative to [131l]lgG (160 kD, impermeant reference tracer) by comparing column elution profiles. Although the single passage extraction of [125l]albumin by beads alone approximated 40%, the presence of confluent HUVEC rendered these beads effectively impermeable to albumin. High junctional extractions were measured for cyanocobalamin (0.79 ± 0.02, n = 28) and insulin (0.51 ± 0.05, n = 14) in cultures perfused at 0.3–0.4 ml/min, and tracer extraction decreased as perfusion rates increased. Permeability coefficients for cyanocobalamin (9.66 × 10−5 cm/s) and insulin (4.18 × 10−5 cm/s) increased significantly during perfusion with thrombin (10 U/ml) or cytochalasin D (1 μg/ml), whereas permeability to albumin (0.39 × 10−5 cm/s) remained unchanges. Morphological studies, using the glycocalyx stain ruthenium red, revealed that thrombin or cytochalasin D increased the penetration of the stain into junctions between endothelial cells.
Glucose induces lipid peroxidation and inactivation of membrane‐associated ion‐transport enzymes in human erythrocytes in vivo and in vitro
Authors: Rajeswari Pichika , Natarajan Rama , Nadler Jerry L. , Kumar Dinesh , Kalra Vijay K. 
Published: February 2005

AbstractErythrocytes of diabetic subjects (non‐insulin dependent) were found to have eight‐ to ten‐fold higher levels of endogenously formed thiobarbituric acid reactive malonyldialdehyde (MDA), thirteen‐fold higher levels of phospholipid‐MDA adduct, 15–20% reduced Na+‐K+‐ATPase activity with unchanged Ca+2‐ATPase activity, as compared with the erythrocytes from normal healthy individuals. Incubation of normal erythrocytes with elevated concentrations (15–35 mM) of glucose, similar to that present in diabetic plasma, led to the increased lipid peroxidation, phospholipid‐MDA adduct formation, reduction of Na+‐K+‐ATPase (25–50%) and Ca+2‐ATPase (50%) activities. 2‐deoxy‐glucose was 80% as effective as glucose in the lipid peroxidation and lipid adduct formation. However, other sugars, such as fructose, galactose, mannose, fucose, glucosamine and 3‐O‐methylmannoside, and sucrose, tested at a concentration of 35 mM, resulted in reduced (20–30%) lipid peroxidation as the enzyme inactivation occur within 30 min of incubation of erythrocytes with high concentration (15–35 mM) of glucose, while lipid peroxidation product, MDA appears at 4 hr and lipid‐MDA adducts at 8 hr. The lipoxygenase pathway inhibitors, 5, 8, 11‐eicosatriynoic acid and Baicalein (5,6,7‐trihydroxyflavone), reduced the glucose‐induced lipid peroxidation by 30% and MDA‐lipid adduct formation by 26%. Indomethacin, a cyclooxygenase pathway inhibitor, had no discernible effect on the lipid peroxidation in erythrocytes. However, the inhibitors of lipid peroxidation, 3‐phenylpyrazolidone, metyrapone, and the inhibitors of lipoxygenase pathways did not ablate the glucose‐induced reduction of Na+‐K+‐ATPase and Ca+2‐ATPase activities in erythrocytes. Erythrocytes produce 15‐HETE (15‐hydroxy‐eicosatetraenoic acid), which is augmented by glucose. These results suggtest that the formation of lipoxygenase metabolites potentiate the glucose‐induced lipid peroxidation and that the inactivation of Na+‐K+−ATPase and Ca+2‐ATPase occurs as a result of non‐covalent interaction of glucose with these enzymes.
Tumor necrosis factor α and epidermal growth factor regulation of collagenase and stromelysin in adult porcine articular chondrocytes
Authors: Mitchell Peter G. , Cheung Herman S. 
Published: February 2005

AbstractChondrocyte‐derived metalloproteases have been postulated to play a role in the degradation of articular cartilage during the development of chronic arthritic disorders. TNFα (tumor necrosis factor alpha), an inflammatory mediator released by activated macrophages, has been detected in the synovial fluid of patients with rheumatoid diseases. We have found that TNFα is a potent stimulator of collagenase and stromelysin mRNA accumulation, collagenase activity, and immunoprecipitable stromelysin in monolayer cultures of adult porcine articular chondrocytes. In contrast EGF (epidermal growth factor), which stimulates collagenase and/or stromelysin synthesis in fibroblast systems, stimulated minimal amounts of these enzymes at both the message and protein levels. Nuclear run‐on transcription analysis demonstrated that the TNFα‐stimulated increase in stromelysin and collagenase message levels was, at least partially, due to increased transcription. Elevated transcription of these genes, in response to TNFα, was apparent by at least 2 hours post‐stimulation. The degree of c‐fos and c‐jun stimulation by TNFα EGF did not correlate with the levels of collagenase and stromelysin message stimulated by these factors. EGF stimulated significant accumulation of both c‐fos and c‐jun mRNAs while only very low amounts of these messages were stimulated by TNFα. Our data suggests that TNFα may contribute to articular cartilage degradation by stimulating chondrocyte‐derived matrix metalloproteases. In addition the regulation of metalloprotease genes in chondrocytes may be different from their regulation in fibroblasts.
Prostaglandin E2 receptor modulation affects tumor cell adhesion to laminin
Authors: Zhang Shao‐Zeng , Fulton Amy M. 
Published: February 2005

AbstractWe have shown previously that murine mammary adenocarcinoma cells both synthesize prostaglandin E2 (PGE2) and have a high affinity receptor for this ligand. Modulation of either PGE synthesis or PGE receptor function changes the metastatic potential of these cells. Because of the importance of laminin and laminin receptors to the metastatic process, we asked whether or not the PGE receptor participates in tumor cell‐laminin interactions. As has been reported for many other tumor cells, laminin and the laminin‐derived peptide PA22‐2, containing the sequence IKVAV, mediate attachment of line 410.4 mammary tumor cells in vitro. We now demonstrate that the attachment of 410.4 cells to laminin or peptide PA22‐2 was significantly inhibited by three PGE receptor antagonists, LE0101, SC19220, and sodium meclofenamate. LE0101 was most active, inhibiting tumor cell adhesion in a dose‐dependent manner in the absence of nonspecific toxicity. These receptor antagonists had no effect on the PA22‐2‐mediated attachment of a PGE receptor negative tumor cell line, except at the highest concentration of LE0101 tested. No inhibition of adhesion to Type I collagen was seen. These results indicate that the PGE2 receptor modulates tumor cell adhesion to laminin which may subsequently affect the in vivo process of metastasis.
Isoprenoids and astroglial cell cycling: Diminished mevalonate availability and inhibition of dolichol‐linked glycoprotein synthesis arrest cycling through distinct mechanisms
Authors: Langan Thomas J. , Slater Mary C. 
Published: February 2005

AbstractPrimary astroglial cultures were used to compare the relationships to cell cycling of dolichol‐linked glycoprotein synthesis, and of availability of mevalonate, the precursor of dolichol and other isoprenoid lipids. With shift‐up to 10% serum (time 0) after 48 h of serum depletion, the proportion of cells in S phase (bromodeoxyuridine immunofluorescence) remained under 15% for 12 h, then increased by 20 h to 72 ± 10%; DNA synthetic rates (thymidine incorporation) increased 5‐fold. S phase transition was prevented by addition at 10–12 h of tunicamycin, an inhibitor of transfer of saccharide moieties to dolichol. Mevinolin, an inhibitor of mevalonate biosynthesis, also blocked cycle progression when added at this time. However, mevinolin markedly inhibited the isoprenoid pathway, as reflected by over 90% reduction of sterol synthesis, without inhibiting net glycoprotein synthesis. Removal of mevinolin after a 24 h exposure delayed S phase until 48 h, following recovery of sterol synthesis, even though kinetics of glycoprotein synthesis were unaffected. Tunicamycin removal after 24 h spared sterol synthesis, but caused delay of S phase until 72 h, following recovery of glycoprotein synthesis. In mevinolin‐treated cultures, S phase transition was restored by 1 h of exposure to mevalonate at 10 h, although cycling was thereby rendered sensitive to inhibition by cycloheximide and by tunicamycin. Cell cycle progression following hydroxyurea exposure and release was unaffected by mevinolin, tunicamycin, or cycloheximide. Thus, in these developing astroglia, mevalonate and its isoprenoid derivatives have at least two cell cycle‐specific roles: dolichol‐linked glycoprotein synthesis is required at or before the G1/S transition, while a distinct mevalonate requirement is apparent also in late G1.
Insulin‐Like growth factor binding protein (IGFBP) inhibits igf action on human osteosarcoma cells
Authors: Campbell Phil G. , Novak Josef F. 
Published: February 2005

AbstractThe influence of a human insulin‐like growth factor binding protein, hIGFBP‐1, on the action of IGFs on human osteosarcoma cells was examined. hIGFBP‐1 was found to block binding of IGFs to their receptors on MG‐63 cells and subsequent IGF stimulation of DNA synthesis. Concurrent incubation of hIGFBP‐1 with either 125I‐IGF‐I or 125I‐IGF‐II prevented the binding of both 125I‐IGFs to cells in a dose‐dependent manner. hIGFBP‐1 inhibition of IGF binding occurred similarly under both 4° and 37°C conditions. Additionally, hIGFBP‐1 facilitated the dissociation of IGFs bound to cells. The inhibitory effect of hIGFBP‐1 on IGF‐1 mediated 3H‐thymidine incorporation into DNA was dose dependent. hIGFBP‐1 did not inhibit binding to or stimulation of growth in MG‐63 cells by des3‐IGF‐I, an IGF‐I analog with a 100‐fold less affinity for hIGFBP‐I. This confirmed that hIGFBP‐1 competed for IGF receptor binding sites on MG‐63. Since hIGFBP‐1 did not bind to cells, inhibition of IGF action was indirect, presumably through the formation of extracellular soluble bioinactive IGF‐BP complexes.
Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells
Authors: Liteplo Robert G. , Hipwell Sheridan E. , Rosenblatt David S. , Sillaots Sue , Lue‐Shing Helena 
Published: February 2005

AbstractOur aim was to identify the biochemical defect responsible for the inability of highly growth autonomous human tumor cells to proliferate in culture medium devoid of methionine, but containing homocysteine and 5‐methyltetrahydrofolic acid. We have adopted the terms “homocysteine‐responsive” and “homocysteine‐nonresponsive” to describe cells which can or cannot proliferate in methionine‐free homocysteine‐supplemented medium. Using a panel of genetically related homocysteine‐responsive and ‐nonresponsive human melanoma cell lines, the results from a number of experiments indicate that acquisition of the “homocysteine‐nonresponsive phenotype” is associated with the reduced intracellular accumulation of methyl‐cobalamin, a critical cofactor of the methionine synthase enzyme. When in vitro methionine synthase assays were performed in the presence of exogenously added methyl‐cobalamin, specific methionine synthase activity in extracts obtained from homocysteine‐responsive cells was only twofold greater than that observed with extracts prepared from homocysteine‐nonresponsive cells. However, when exogenous methyl‐cobalamin was omitted from the enzyme assays, methionine synthase activity in extracts derived from homocysteine‐nonresponsive cells was dramatically reduced, compared with the small decrease observed with homocysteine‐responsive cell extracts. Compared with their homocysteine‐responsive counterparts, homocysteine‐nonresponsive cells exhibited increased levels of cobalamin efflux and decreased intracellular accumulation of methyl‐cobalamin. There was a clear relationship between the abilities of these related melanoma cell lines to proliferate in methionine‐free homocysteine‐supplemented medium, and the extent of cobalamin loss and capacity of exogenously added methyl‐cobalamin to stimulate in vitro methionine synthase activity. These results indicate a link between alterations in the intracellular trafficking and/or metabolism of cobalamin and the increased growth autonomy of human melanoma cells.
Adipogenic activity produced by hepatocyte‐derived cell lines and by normal hepatocytes in primary culture
Authors: Zaitsu Hirokazu , Serrero Ginette 
Published: February 2005

AbstractCulture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh‐7, PLC/PRF/5, and SKHep‐1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3‐L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100°C, with protease, with 2‐mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cells also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.
Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts
Authors: Liu Alice Y.‐C. , Choi Hueng‐Sik , Lee Yoon‐Kwang , Chen Kuang Yu 
Published: February 2005

AbstractWe examined the induction, by heat shock, of heat shock transcription factor (HSTF) DNA‐binding and hsp 70 gene promoter activities during aging of the IMR‐90 human diploid fibroblasts. Cells with population doubling level (PDL) ranging from 15–48 were heat shocked at temperatures of 39, 42, and 45°C for various time periods; the binding of HSTF to its consensus DNA was determined by gel retardation assay and the promoter activity of the human hsp 70 gene was analyzed by transient expression of reporter gene activity. We observed that the induction of HSE‐binding activity was inversely related to the PDL of the cells used. Importantly, as cells progress through their life span, a higher temperature and a longer period of heat shock were needed to evoke an optimal increase in HSE‐binding activity. A substantial and rapid (within 30 min) increase in HSE‐binding activity was observed when PDL 20 cells were heat shocked at 39, 42, or 45°C. However, PDL 35 cells did not respond to 39°C, and PDL 48 cells responded slowly to heat shock at 45°C, but not 39 or 42°C. Experiments on the heat induced increase in hsp 70 promoter driven reporter gene expression provided similar information on the age‐dependent decrease in transcriptional activation of hsps. These results were further corroborated by quantitation of the abundance of mRNA of hsp 70. Analysis of the cAMP induced expression of the rat somatostatin promoter driven CAT gene provided evidence that the decrease in transcriptional activation of hsps in aging diploid cells was not a reflection of a generalized dysfunction of signal transduction. We conclude that functional changes in the heat shock response occur before cells lose their capacity to replicate, and we suggest that these changes are likely to have a central role in the expression of the aging phenotype.
Heparin inhibits the attachment and growth of Balb/c‐3T3 fibroblasts on collagen substrata
Authors: Antonio James D. San , Lander Arthur D. , Wright Thomas C. , Karnovsky Morris J. 
Published: February 2005

AbstractIn investigating the role of cell‐extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell‐collagen interactions were examined. Exponentially growing Balb/c‐3T3 fibroblasts were radiolabelled with 3H‐thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70–90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10–50% when heparin was present from 0.1–100 μg/ml. Cell‐collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell‐collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (> 40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr ≤ 6KD) with type I collagen was analyzed by affinity co‐electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell–collagen and heparin‐collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative activity in vivo and in vitro.
Effects of pH‐variations on the kinetics of growth and energy metabolism in cultured T‐lymphoma cells: A microcalorimetric study
Authors: Bäckman Per , Kimura Takayoshi , Schön Arne , Wadsö Ingemar 
Published: February 2005

AbstractThe progression of T‐lymphoma cells (CCRF‐CEM) growing in suspension has been monitored during long term (12–28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH, p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 106 cells per ml). The lowering of p(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmol/l, respectively, did not influence Pcell or the growth pattern. No “crowding effect” was observed for the cells in the investigated concentration range (0.6–1.3) × 106 cells per ml.