bluuebunny/crossref_metadata_2025_split huggingface dataset:
Environment‐dependent growth inhibition of human epidermal keratinocytes by recombinant human transforming growth factor‐beta
Authors: Rollins Barrett J. , O'Connell Therese M. , Bennett Gregory , Burton L. Eugene , Stiles Charles D. , Rheinwald James G. 
Published: February 2005

AbstractTransforming growth factor‐beta (TGF‐β) purified from platelets is a potent growth inhibitor of several normal epithelial cell types in culture. In contrast, some carcinoma cell lines derived from tumors of these same tissues are resistant to this factor. Using recombinant human TGF‐β, the authors have confirmed these results with six normal human epidermal keratinocyte strains and four human epidermal squamous carcinoma cell lines. However, the sensitivity of normal cells to TGF‐β was found to depend on the culture conditions. When grown in a specialized nutrient medium supplemented with pituitary extract, keratinocytes were completely inhibited by the addition of 0.3 ng/ml TGF‐β. In contrast, when their growth was supported by cocultivation with 3T3 fibroblast feeder cells, 30‐ to 100‐fold higher concentrations of TGF‐β were required to achieve comparable growth inbibition. This differential sensitivity occurred despite the fact that in both culture systems TGF‐β in the culture medium had a half‐life of about 50 minutes, becoming tightly bound to the surface of the culture dish. Bound TGF‐β proved to be biologically active and stable for about a week in the absence of 3T3 feeder cells. Incubating 3T3 cells on TGF‐β‐coated dishes, however, resulted in nearly quantitative removal and degradation of the TGF‐β within 2 days, permitting normal rates of keratinocyte growth. The binding of TGF‐β to surfaces and the ability of fibroblasts to attenuate its inhibitory activity for epithelial cells must be considered when evaluating in vitro models and in planning strategies for the use of this factor in vivo.
Effects of interleukin‐1 on fibroblast extracellular matrix, using a 3‐dimensional culture system
Authors: Qwarnströ E. E. , MacFarlane S. A. , Page R. C. 
Published: February 2005

AbstractThis study describes the alterations induced by Interleukin‐1α and ‐β (IL‐1α and IL‐1β) on fibroblast‐synthesized extracellular matrix. Fibroblasts were grown between pieces of dentin or in collagen‐coated Terasaki wells for 3 or 6–9 weeks to create 3‐dimensional cell‐containing matrices constituted primarily of proteoglycans and collagens, respectively. Following incubation with IL‐1α or IL‐1β (10−9 M) at 37°C for 24 or 72 hr, samples were prepared for light and electron microscopy. Both IL‐1α and IL‐1β induced collapse of the extracellular matrix by 72 hr, as manifested by a decrease of the cross‐sectional area and an increased density of the matrices. Three‐week matrices were reduced 26% and 45% by using IL‐1α and IL‐1β, respectively. Comparable values obtained by using 6‐week matrices were 14% and 30%. Cells within the matrix, normally stellate in shape with numerous extended processes, attained a more rounded or spindle shape with few and reduced processes and showed apparent alterations at cell matrix attachment sites and rearrangement of the cytoskeleton. Elongated cells at the top of the matrix appeared more compressed. The alterations were more pronounced in cultures incubated with IL‐β than with IL‐1α. Immunocytochemistry of extracellular matrix components revealed a decrease in staining intensity of chondroitin and dermatan sulfate in the 3‐week matrix following IL‐1β incubation. There was also a decrease in collagen type I staining of 9‐week matrices treated with IL‐1α or IL‐1β. These studies show that IL‐1 has an effect on fibroblast‐synthesized extracellular matrix and indicate that the effects of IL‐1α and IL‐1β may differ. The resulting collapse of the matrix appears at least in part to be due to changes in proteoglycans and collagens.
Modulation of endothelial fibronectin synthesisby polymorphonuclear granulocytes
Authors: Macarak Edward J. , Gorfien Stephen , MacGregor Rob Roy 
Published: February 2005

AbstractThe attachment of polymorphonuclear granulocytes (PMNs) to vascular endothe‐lial cells occurs continually in normal tissues; however, knowledge of the factors that control leukocyte margination is incomplete. In the present study, we used cell cultures of pulmonary artery endothelium to study their interaction with PMNs. Endothelial cells were seeded in Costar 24‐well plates following which PMNs were inoculated onto the endothelial monolayers and incubated for 2 to 20 hours. During this period, fibronectin synthesis by endothelial cells was estimated by ELISA. In wells to which PMNs had been added, supernatant fibronectin concentration was increased at all time points during the 20 hour incubation. At 20 hours, supernatants from wells to which PMNs had been added contained approximately 2 1/2 times the control level of fibronectin. Since the amount of fibronectin, as determined by ELISA, adsorbed onto the added PMNs was negligible, these data suggest that PMNs can modulate the synthesis of fibronectin by pulmonary artery cells. Pulse labeling experiments and measurements of endothelial intracellular fibronectin also suggest this possibility. The endothelial response does not appear to be owing to nonspecific physical interaction since similarly sized polystyrene beads did not cause any change in supernatant fibronectin levels while glutaraldehyde‐fixed PMNs caused only a 20–25% increase in fibronectin levels.
Lipopolysaccharide‐induced expression of the competence gene KC in vascular endothelial cells is mediated through protein kinase C
Authors: Shen Xin‐Yi , Hamilton Thomas A. , Dicorleto Paul E. 
Published: February 2005

AbstractThe KC gene is a cell cycle‐dependent competence gene originally identified in platelet‐derived growth factor‐stimulated BALB/c‐3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady‐state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1‐oleoyl‐2‐acetyl‐glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA‐stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1‐(5‐isoquinoline‐sulfonyl)‐2‐methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS‐treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation‐independent process. Second, unlike LPS‐induced KC expression in macrophages and platelet‐derived growth factor‐induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to require activation of protein kinase C.
Effect of mitogen and lymphokine stimulation on proteoglycan synthesis by lymphocytes
Authors: Bartold P. Mark , Haynes David R. , Vernon‐Roberts Barrie 
Published: February 2005

AbstractThe ability of mouse thymocytes and peripheral blood lymphocytes from rats to synthesize and secrete proteoglycans in the presence of a variety of mitogens and lymphokines was studied in vitro, and it was confirmed that such lymphocytes synthesize and secrete significant quantities of proteoglycans. Mitogenic stimulation of the cells with phytohaemagglutanin (PHA) induced a fourfold increase in proteoglycan synthesis; stimulation with interleukin‐1 stimulated proteoglycan synthesis up to fivefold. Proteoglycan synthesis could also be stimulated by culturing the cells in the presence of interleukin‐2. To determine if this response was related to cell proliferation, the cells were cultured in the presence of PHA and either cyclosporine or prostaglandin E2 two agents that inhibit lymphocyte proliferation. Under these conditions, proteoglycan synthesis remained elevated, indicating that this effect may be independent of cell proliferation. Chemical analysis of the proteoglycans indicated them to be composed of chondroitin sulfate and heparan sulfate. Their molecular size was small compared with cartilage proteoglycans but similar to the small dermatan sulfate proteoglycans synthesized by fibroblasts. On the basis of molecular size, three proteoglycan populations were identified, and their relative proportions were altered by mitogenic stimulation of the cells. Taken together, these findings imply that proteoglycan synthesis is intimately associated with lymphocyte activation and may be related to cellular function in immune responses.
Protein kinase C modulates effects of prostanoids on cyclic adenosine monophosphate in guinea pig chief cells
Authors: Raufman Jean‐Pierre , Cosowsky Laurey 
Published: February 2005

AbstractIn the course of examining the role of protein kinase C in signal transduction in dispersed chief cells from guinea pig stomach, we observed that phorbol esters inhibit prostaglandin (PG)‐stimulated increases in cyclic adenosine monophos‐phate (cAMP). Phorbol 12‐myristate 13‐acetate (PMA), an activator of protein kinase C, decreased maximal levels of PGE2‐stimulated cAMP by 40%. This dose‐dependent effect was observed within 30 sec and was maximal by 1 min of incubation at 37°C. Phorbols that do not activate protein kinase C did not have this effect. Adding H7, a protein kinase C inhibitor, abolished the inhibitory effects of PMA. Adding phosphodiesterase inhibitors did not alter the inhibitory effects of PMA, indicating that these effects are not caused by activation of cyclic nucleotide phosphodiesterases. PMA did not alter the increase in cellular cAMP caused by cholera toxin, forskolin, secretin, or vasoactive intestinal peptide. Hence the site of these prostanoid‐specific actions of protein kinase C does not appear to be stimulatory or inhibitory guanine nucleotide binding proteins or the catalytic component of the adenylyl cyclase system. In dispersed chief cells, activation of protein kinase C may inhibit prostanoid‐induced stimulation of the adenylyl cyclase system by a direct effect on prostaglandin receptors.
Down‐regulation of proteolytic activity in 12‐O‐tetradecanoyl‐phorbol‐13‐acetate‐induced k562 leukemia cell cultures: Depletion of active urokinase by excess type 1 plasminogen activator inhibitor
Authors: Alitalo Riitta , Andersson Leif C. , Tapiovaara Hannele , Sistonen Lea , Vaheri Antti , Stephens Ross 
Published: February 2005

AbstractThe human chronic myeloid leukemia cell line K562 acquires several megakaryoblastoid features when cultured in the presence of the tumor promoter 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA). We observed strongly increased secretion of several proteins into the culture media of K562 cells within a few hours of TPA treatment. Two of the major secreted polypeptides were identified by immunoprecipitation from media of metabolically labeled cultures as the tissue inhibitor of metalloproteinases (TIMP) and the type 1 plasminogen activator inhibitor (PAI‐1). Maximal amounts of PAI‐1 mRNA and secretion of PAI‐1 polypeptides were observed after 24 hr of TPA treatment and PAI‐1 persisted at elevated levels for several days. The induction of PAI‐1 mRNA was dependent on de novo protein synthesis. Uninduced and induced cells secreted urokinase plasminogen activator in its single‐chain proenzyme form (pro‐u‐PA), which was cleaved extracellularly to the active two‐chain form as shown by pulse‐chase labeling experiments. Upon TPA induction, the secretion of u‐PA polypeptides increased severalfold, and there was a transient accumulation of pro‐u‐PA in the culture medium. However, this did not lead to increased u‐PA activity in the cultures, since active u‐PA was removed by complex formation with the large excess of coinduced PAI‐1. Induction of u‐PA mRNA was biphasic: The first peak of about tenfold increase in steady‐state u‐PA mRNA at 3 hr was followed by a steep decline to the baseline level at 12 hr, and a second, slower accumulation of u‐PA mRNA occurred over the next few days. The biphasic accumulation of u‐PA mRNA was also reflected in u‐PA protein synthesis. We conclude that concerted changes in favor of a nonproteolytic extracellular environment occur in TPA‐induced K562 cultures undergoing megakaryoblastoid differentiation. These changes include excessive secretion of TIMP and inhibition of the induced u‐PA by the simultaneous accumulation of PAI‐1.
TGF‐β stimulates primary human skin fibroblast DNA synthesis via an autocrine production of PDGF‐related peptides
Authors: Soma Yoshinao , Grotendorst Gary R. 
Published: February 2005

AbstractTransforming growth factor‐β (TGF‐β) stimulates DNA synthesis in human foreskin fibroblasts after a prolonged lag period as compared with other growth factors. The mechanism of induction of DNA synthesis appears to be dependent on the synthesis and secretion of PDGF‐related proteins as antibodies which are specific for PDGF can block the TGF‐β‐induced DNA synthesis. Other growth factors such as PDGF, EGF, or FGF do not induce the synthesis of these PDGF‐related proteins. Additionally, TGF‐β treatment of human foreskin fibroblasts induces the expression of the PDGF A‐chain gene but not the B‐chain gene. This phenomenon appears to function in vivo, as subcutaneous injection of TGF‐β in rat skin induces the expression of the PDGF A‐chain gene. These data suggest that TGF‐β may stimulate the growth of fibroblastic cells via an autocrine production of PDGF‐related proteins.
Electric stimulation of human fibroblasts causes an increase in Ca2+ influx and the exposure of additional insulin receptors
Authors: Bourguignon Gerard J. , Jy Wenche , Bourguignon Lilly Y. W. 
Published: February 2005

AbstractPreviously we reported that treating human fibroblasts in cell culture with high‐voltage, pulsed galvanic stimulation (HVPGS) can significantly increase cellular protein and DNA synthesis (Bourguignon and Bourguignon: FASEB J., 1:398–402, 1987). In this study we have identified two of the early cellular events which occur following exposure to HVPGS: (1) an increase in Ca2+ uptake from the external medium and (2) an increase in the number of insulin receptors on the fibroblast cell surface. The increase in Ca2+ uptake begins within the first minute of electric stimulation while increased insulin binding is not detected until the second minute of stimulation. The HVPGS‐induced increase in insulin binding can be inhibited by bepridil, a specific Ca2+ channel blocker, suggesting that the Ca2+ influx is required for the exposure of additional insulin receptors on the cell surface.Furthermore, we have determined that the addition of insulin to electrically stimulated cultures results in (1) an immediate, second increase in Ca2+ uptake and (2) significant increases in both protein and DNA synthesis compared to cells which were not stimulated. All three of these insulin‐dependent effects are also inhibited by bepridil.Based on these results, we propose that HVPGS initially triggers the opening of voltage‐sensitive calcium channels in the fibroblast plasma membrane. The increased level of intracellular Ca2+ then induces the exposure of additional insulin receptors on the cell surface. If insulin is available to bind the additional receptors, the fibroblasts will significantly increase both protein and DNA synthesis.
Protamine selectively inhibits collagen synthesis by human intestinal smooth muscle cells and other mesenchymal cells
Authors: Perr Hilary A. , Drucker David E. M. , Cochran David L. , Diegelmann Robert F. , Lindblad William J. , Graham Martin F. 
Published: February 2005

AbstractCollagen synthesis is a major function of human intestinal smooth muscle (HISM) cells and contributes to intestinal fibrosis in chronic inflammatory bowel disease. As an extension of previous in vitro studies of the role of heparin in regulating HISM cell proliferation and collagen synthesis, the effect of protamine sulfate was studied. Protamine decreased collagen production by 50% in confluent and proliferating cultures. This effect was concentration‐dependent and was selective for collagen in that neither noncollagen production nor DNA accumulation in the culture plates was affected. Other human mesenchymal cells which produce collagen, such as dermal fibroblasts and aortic smooth muscle cells, responded to protamine in a similar fashion. Protamine has a strong cationic charge and is rich in lysine and arginine. To determine which of these properties was important in decreasing collagen production, the effect of protamine was compared to that of other polyionic compounds. Poly‐L‐lysine decreased collagen production to a lesser degree than protamine. Poly‐L‐arginine was toxic to the cells. Poly‐L‐glu‐tamic acid, which has an opposite charge to protamine, had no effect. These findings suggest that both the number and the arrangement of lysyl residues, in addition to positive charge, are important. Binding assays demonstrated that protamine did not inhibit collagen production by binding to ascorbate in the culture medium. Electrophoretic separation and chromatography of collagen types expressed following protamine treatment showed that the ratio of type I to type III collagen remained 2:1. This observation suggests that suppression of collagen production is not specific to a particular collagen type. The selective inhibition of collagen production by protamine provides an important tool to study the regulation of collagen production in human cells and may also provide potential therapy of fibrotic disorders.
Evidence that alterations in small molecule permeability are involved in the Clostridium perfringens type a enterotoxin‐lnduced inhibition of macromolecular synthesis in vero cells
Authors: Hulkower Keren I. , Wnek Andrew P. , McClane Bruce A. 
Published: February 2005

AbstractThe mechanism by which Clostridium perfringens enterotoxin (CPE) simultaneously inhibits RNA, DNA, and protein synthesis is unknown. In the current study the possible involvement of small molecule permeability alterations in CPE‐induced inhibition of macromolecular synthesis was examined. Vero cells CPE‐treated in minimal essential medium (MEM) completely ceased net precursor incorporation into RNA and protein within 15 minutes of CPE treatment. However, RNA and protein synthesis continued for at least 30 minutes in Vero cells CPE‐treated in buffer (ICIB) approximating intracellular concentrations of most ions. Addition of intracellular concentrations of amino acids to ICIB (ICIB‐AA) caused a further small but detectable increase in protein synthesis in CPE‐treated cells. ICIB did not affect CPE‐specific binding levels or rates. Similar small molecule permeability changes (i.e., 86Rb‐release) were observed in cells CPE‐treated in either ICIB or in Hanks' balanced salt solution. Collectively these findings suggest that CPE‐treatment of cells in ICIB‐AA ameliorates CPE‐induced changes in intracellular concentrations of ions and amino acids and permits the continuation of RNA and protein synthesis. These results are consistent with and support the hypothesis that permeability alterations for small molecules are involved in the CPE‐induced inhibition of precursor incorporation into macromolecules in Vero cells.
ATP‐resistant variants of transformed mouse fibroblasts
Authors: Belzer Ilana , Friedberg Ilan 
Published: February 2005

AbstractAddition of ATP to cultures of transformed mouse fibroblasts, 3T6 cells, resulted in cell growth inhibition, whereas the growth of the non‐transformed counterparts, 3T3 cells, was only slightly affected. The inhibition was found to be specific for adenine nucleotides, and concentration dependent. At relatively low concentrations (e.g., 0.1 mM) the effect of ATP was cytostatic, whereas at higher concentrations (e.g., 1.0 mM) a cytotoxic effect was exerted. ATP‐resistant variants of 3T6 cells were selected by exposure of cultures to gradually elevated concentrations of ATP. The variants were found to resemble the non‐transformed counterparts, 3T3 cells, more than the 3T6 parent cells, by the following criteria: ATP‐induced alte rations in the membrane potential, changes in membrane permeability, cell growth inhibition, and colony formation on soft agar. The data indicate that long exposure of the transformed cells to external ATP results in redifferentiation and reduction in their tumorigenicity.
Retinoic acid modulates dome formation by MDCK cells in defined medium
Authors: Taub Mary 
Published: February 2005

AbstractRetinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K‐1). After 4–5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7days with 3 × 10−7 M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 × 10−3 μ M2 in area, whereas in control medium the majority of the domes were between 0 and 9 × 10−3 μm2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K‐1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1‐independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K‐1 lacking PGE1 This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1‐independent MDCK cells. Dibutyryl cAMP‐resistant MDCK cells, which normally do not form domes in medium K‐1, were also studied. Remarkably, the dibutyryl cAMP‐resistant MDCK cells were observed to form domes at a significant frequency when medium K‐1 was supplemented with retinoic acid. However in medium K‐1 lacking PGE1,an effect of retinoic acid on dome formation by dibutyryl cAMP‐resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP‐resistant MDCK cells to form domes in medium K‐1 has previously been attributed to their decreased cAMP‐dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP‐dependent protein kinase or activation of a separate pathway.