bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transforming growth factor‐beta and fibroblast growth factor act synergistically to inhibit collagen II synthesis through a mechanism involving regulatory DNA sequences
Authors: Horton Walter E. , Higginbotham Jill D. , Chandrasekhar Srinivasan 
Published: February 2005

AbstractTransforming growth factor‐beta (TGF‐β) and fibroblast growth factor (FGF) are two growth factors that will modulate chondrocyte growth and matrix synthesis. Here we report that these two growth factors act in a synergistic fashion to suppress the synthesis of type II collagen by embryonic chicken sternal chondrocytes. Treatment of chondrocytes with 20 ng/ml TGF‐β or 100 ng/ml FGF (acidic or basic) results in a 60–70% suppression of expression of the pro α1 chain of type II collagen. By comparison, when chondrocytes are exposed to a combination of 1 ng/ml TGF‐β and 10 ng/ml FGF, a complete suppression of type II collagen synthesis was observed. Epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), or insulin‐like growth factor‐1 (IGF‐1) produce no suppression of synthesis either individually or in combination with TGF‐β. The decreased expression of the protein results from a decrease in the steady‐state level of the mRNA transcript coding for type II procollagen, as indicated by a northern analysis. Finally, chondrocytes transfected with a plasmid carrying the CAT gene driven by the collagen II promoter/enhancer sequence displayed high levels of CAT activity when cultured in control media, but treatment of the cells with a combination of the two growth factors resulted in a dramatic reduction of CAT activity, indicating diminished promoter activity. These results suggest that both TGF‐β and FGF can down‐regulate transcription of the collagen II gene through regulatory DNA sequences in the promoter and/or enhancer region. In addition, the finding of synergy suggests that these two growth factors may act through different pathways.
Modulation of growth of human carcinoma SW‐13 cells by heparin and growth factors
Authors: Halper Jaroslava , Carter Bobbie J. 
Published: February 2005

AbstractThis study reports on the effects of heparin, basic and acidic fibroblast growth factors (bFGF and aFGF, respectively), and transforming growth factor type‐e (TGFe) on the growth of a human adrenocortical carcinoma cell line, SW‐13. Heparin has previously been shown to inhibit growth in several cell types, including smooth muscle cells, certain fibroblasts, and epithelial cells, and to modulate the effects of fibroblast growth factors. Whereas bFGF and aFGF bind tightly to heparin and elute from a heparin‐Sepharose column with 2 M NaCl and 1.6 M NaCI, respectively, TGFe binds to heparin with lower affinity and can be eluted from heparin‐Sepharose column with 0.5 M NaCI. TGFe is a polypeptide unrelated to FGF, is present in neoplastic and nonneoplastic tissues, and stimulates the growth of certain epithelial cells and fibroblasts in soft agar and monolayer. Since the growth of SW‐13 cells is stimulated by TGFe and by bFGF, we hypothesized that heparin would inhibit the growth of SW‐13 cells by binding to these growth factors and that the effects of heparin could be overcome with the addition of either growth factor. Our experiments confirmed that heparin inhibits the growth of SW‐13 cells. A dose‐dependent growth inhibition was observed in both monolayer and soft agar. The inhibition in monolayer was partially reversed upon heparin withdrawal. The effects of heparin in both monolayer and soft agar were at least partially overcome by TGFe and by basic or acidic FGF. Overall protein synthesis does not appear to be affected by heparin as measured by [35S]methionine uptake. In contrast, epidermal growth factor (EGF) and insulin‐like growth factor I (IGF‐I) were unable to overcome heparin‐induced inhibition both in monolayer and in soft agar. Heparin also inhibited [3H]thymidine incorporation in AKR‐2B and partially inhibited AKR‐2B cell stimulation by TGFe; however, it further potentiated the already potent stimulation by bFGF. We propose that heparin, TGFe, bFGF, and aFGF modulate the growth of SW‐13 cells and possibly of other epithelial cells in complex ways and that heparin‐like substances present in the extracellular matrix play an important role in the control of epithelial growth.
ATP hydrolysis by ischemic Mitochondria
Authors: Classen J. Barthelow , Mergner Wolfgang J. , Costa M. 
Published: February 2005

AbstractCellular ATP levels are determined by the rates of ATP production and ATP hydrolysis. Both phenomena are affected by ischemia. Mitochondrial enzymes are damaged, inhibiting this organelle's ability to make ATP. Mitochondria are also uncoupled by ischemia and have the ability to hydrolyze ATP. We designed a series of experiments to determine whether decreased production or increased hydrolysis of ATP was the primary effect of mitochondrial damage. Rat hearts were subjected to 45 min of warm ischemia in order to induce irreversible cell damage. ATP or ADP was injected into cuvettes containing mitochondria isolated from normal myocardium or myocardium damaged by ischemia. Luciferin‐luciferase, which fluoresces in the presence of ATP, was also added to the tubes as an indicator of ATP levels. Mixtures of uncoupled and coupled mitochondria were made and compared with the mitochondria damaged by ischemia. The results showed that mitochondria damaged by prolonged ischemia hydrolyze ATP more rapidly than normal mitochondria; however, normal mitochondria can easily compensate for increased ATP hydrolysis when in mixture with equal amounts of uncoupled mitochondria. These data suggests that the low cellular levels of ATP following irreversible ischemia are primarily due to decreased ATP synthesis and not to increased hydrolysis.
Heat shock stimulates the release of arachidonic acid and the synthesis of prostaglandins and leukotriene B4 in mammalian cells
Authors: Calderwood Stuart K. , Bornstein Bruce , Farnum Elizabeth K. , Stevenson Mary Ann 
Published: February 2005

AbstractHeat shock has a profound influence on the metabolism and behavior of eukaryotic cells. We have examined the effects of heat shock on the release from cells of arachidonic acid and its bioactive eicosanoid metabolites, the prostaglandins and leukotrienes. Heat shock (42–45°) increased the rate of arachidonic acid release from human, rat, murine, and hamster cells. Arachidonate accumulation appeared to be due, at least partially, to stimulation of a phospholipase A2 activity by heat shock and was accompanied by the accumulation of lysophosphatidyl‐inositol and lysophosphatidylcholine in membranes. Induction of arachidonate release by heat did not appear to be mediated by an increase in cell Ca+ +. Stimulation of arachidonate release by heat shock in hamster fibroblasts was quantitatively similar to the receptor‐mediated effects of β thrombin and bradykinin. The effects of heat shock and β thrombin on arachidonate release were inhibited by glucocorticoids. Increased arachidonate release in heat‐shocked cells was accompanied by the accelerated accumulation of cyclooxygenase products prostaglandin E2 and prostaglandin F2α and by 5‐lipoxygenase metabolite leukotriene B4. Elevated concentrations of arachidonic acid and metabolites may be involved in the cytotoxic effects of hyperthermia, in homeostatic responses to heat shock, and in vascular and inflammatory reactions to stress.
Heterogeneity of the changes in cytoplasmic pH upon serum stimulation of quiescent fibroblasts
Authors: Bright Gary R. , Whitaker James E. , Haugland Richard P. , Taylor D. Lansing 
Published: February 2005

AbstractAddition of mitogens to quiescent cells results in rapid ionic changes in the cytoplasm, including pH. We studied the changes in cytoplasmic pH in single Swiss 3T3 cells upon serum stimulation using fluorescence ratio imaging microscopy. Quiescence was attained using two approaches, serum deprivation of subconfluent cells and confluence. All measurements were made in the presence of bicarbonate and the absence of other organic buffers. We also used BCECF coupled to dextran to avoid several artifacts associated with using BCECF‐AM, including leakage and phototoxicity. Analysis of the changes in cytoplasmic pH demonstrated a dramatic heterogeneity in the responses of single cells. There were six basic classes of responses, (1) a fast alkalinization, reaching a maximum pH in ∼2–5 min; (2) a slow alkalinization, reaching a maximum pH in 10‐20 min; (3) a very slow alkalinization, not reaching a plateau pH within the measurement time; (4) no apparent change in pH during the measurement time; (5) an early transient acidification, followed by either a fast or slow alkalinization; and (6) an acidification, followed by alkalinization and then by a decrease to some intermediate pH. Subconfluent cells exhibited greater heterogeneity in response than confluent cells, with no single dominant class of response. The dominant (55%) response for confluent cells was a gradual alkalinization of ∼0.01 pH units/min. A larger proportion (52%) of subconfluent cells exhibited an early transient acidification compared to confluent cells (7%). A significant proportion of both types of cells (23% subconfluent, 36% confluent) exhibited no change in cytoplasmic pH upon stimulation. In general, the kinetics of changes in cytoplasmic pH were significantly different from the published results with population averaging methods.
Cell‐type‐specific expression of mouse DNA polymerase β‐gene is regulated by silencer elements
Authors: Yamaguchi Masamitsu , Hayashi Yuko , Ajiro Kozo , Matsukage Akio 
Published: February 2005

AbstractRNA blot hybridization analysis revealed that the steady‐state level of DNA polymerase β‐mRNA in mouse neuroblastoma N18TG2 cells was approximately fivefold higher than that in NIH/3T3 cells. In order to examine the function of DNA polymerase β‐gene silencers in these two cell lines, we employed a chloramphenicol acetyltransferase (CAT)‐transient expression assay using the CAT plasmids containing the silencers linked to various promoter‐enhancers. In NIH/3T3 cells, DNA polymerase β‐gene silencers effectively repressed the function of its own promoter and those of several other heterologous promoter‐enhancers. In contrast, the silencers only marginally affected the CAT expression directed by DNA polymerase β‐gene promoter and heterologous promoter‐enhancers in N18TG2 cells. The extent of the increase of CAT expression by removing silencer elements in NIH/3T3 cells was very similar to the ratio of DNA polymerase β‐mRNA content in N18TG2 cells to that in NIH/3T3 cells. These results indicate that cell‐type‐specific expression of DNA polymerase β‐gene is primarily controled by the function of its silencer elements.
Differential response among cells in the chick embryo myogenic lineage to photosensitization by merocyanine 540
Authors: Nameroff Mark , Rhodes Linda D. 
Published: February 2005

AbstractPrimary cultures of myogenic cells from progressively older embryonic and adult chickens were incubated in medium containing Merocyanine 540 (MC540) and were exposed to white light during the incubation period. After exposure, the cultures were followed to determine cell survival and differentiation. MC540 attached to the surface membranes of all cells. In cultures from 10‐day embryos (E10 cells), concentrations of MC540 ≥ 60 μg/ml resulted in death of nearly all myogenic cells upon exposure to light, but non‐myogenic cells survived and replicated. Below 60 μg/ml, there was a dose‐dependent reduction in muscle differentiation. At concentrations <40 μg/ml, there was no effect on myogenesis. Cultures of cells from 18‐day (E18) embryos (myogenic stem cells) and from adult muscle (satellite cells) were resistant to doses of MC540 that killed E10 cells. E14 myogenic cell populations contained both resistant and sensitive sub‐populations. Terminally differentiated muscle cells were more sensitive to MC540 than precursor cells from any age embryo. Progeny of E18 cells acquired sensitivity to MC540 as differentiation proceeded. In clonal cultures, cells that normally give rise to small muscle clones (committed cells) were selectively destroyed by exposure to the dye. These observations demonstrate that an MC540‐resistant myogenic population is present in low numbers in 10‐day embryonic pectoral muscle. As development proceeds, this population increases such that, by 18 days of gestation, most of the myogenic cells are resistant to MC540. The results also suggest that embryonic chick myogenic stem cells and adult satellite cells have surface membrane properties which differ from those of their committed progeny.
Preadipocyte stimulating factor in rat serum: Evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures
Authors: Li Zhen‐Hua , Lu Zhongding , Kirkland James L. , Gregerman Robert I. 
Published: February 2005

AbstractIn primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (pre‐adipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3‐month‐old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol‐3‐phosphate dehydrogenase (GPDH). Rat serum induces dose‐dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4°C for up to 1 year; inactivated at 100°C (80% loss, 30 min) but stable at 56°C for 1 hr; stable at pH 2–12; non‐dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisin; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl‐300) and 58 kDa (Sephacryl‐300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high‐fat, high‐carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin‐like growth factor‐1 (IGF‐1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony‐stimulating factor (CSF‐1), GM‐CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4–8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3‐L1, 3T3‐F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosy‐lated protein, and may be highly species specific.
GH4 pituitary cell variants selected as nonresponsive to thyrotropin‐releasing hormone‐enhanced substratum adhesion are nonresponsive to epidermal growth factor: Evidence for a common signaling defect
Authors: Ramsdell John S. , Tashjian Armen H. 
Published: February 2005

AbstractThyrotropin‐releasing hormone (TRH) and epidermal growth factor both enhance prolactin synthesis and substrate adhesion (a morphological change called stretching) of GH4 rat pituitary cells. We have examined TRH‐ and EGF‐induced cell stretching using genetic and pharmacologic approaches. We selected and isolated a series of GH4 cell variants nonresponsive to TRH‐induced cell stretching (str−). This selection yielded several variants that were nonresponsive to both TRH‐ and EGF‐induced stretching but were still responsive to stretching induced by several other agents (tetradecanoylphorbol acetate [TPA], butyrate, and Nepla‐nocin A). One of the str− variants (a14) was examined in detail. TRH, EGF, and TPA each enhanced prolactin synthesis in a14 cells, indicating that the a14 variant contained functional receptor binding sites for all 3 ligands as well as the capacity to generate those intracellular signals required for enhanced prolactin synthesis. Because the str− variants were isolated without selective pressure for EGF‐induced stretching and because the possibility of more than one selectable mutation in all the variants is unlikely, we suggest that TRH and EGF share a common mechanism to induce cell stretching. We next examined whether the str− variants had a defect in a signaling pathway or in the biochemical endpoint for TRH‐ and EGF‐induced cell stretching. A pharmacologic approach was utilized to investigate the biochemical basis for induced cell stretching. A synthetic Arg‐Gly‐Asp‐Ser tetrapeptide (RGDS), specific for fibronectin and vitronectin adhesion receptors, inhibited TRH‐, EGF‐, and TPA‐induced GH4 cell stretching and attachment to fibronectin‐ and vitronectin‐coated dishes. These results suggest that the interaction between fibronectin and/or vitronectin and their receptor(s) may be a biochemical endpoint by which several agonists induce stretching of GH4 cells. Because the str− variant has RGDS‐specific binding sites for fibronectin and vitronectin and responds to some agents that induce cell stretching via an RGDS receptor, we conclude that the a14 str− variant has a defect in an intracellular signaling pathway, shared by TRH and EGF, which induces cell stretching.
Characterization of neutral and cationic amino acid transport in Xenopus oocytes
Authors: Campa Michael J. , Kilberg Michael S. 
Published: February 2005

AbstractAmino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+‐dependent and saturable Na+‐independent processes. Na+‐dependent transport of 2‐aminoisobutyric acid (AIB) was insensitive to cis‐ or trans‐inhibition by the System A‐defining substrate 2‐(methylamino)‐isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+‐dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+‐dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo, + was obtained through inhibition analysis of Na+‐dependent arginine transport as well arginine inhibition of Na+‐dependent threonine uptake. The Na+‐independent transport of leucine was subject to trans‐stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2‐amino‐(2,2,1)‐bicycloheptane‐2‐carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+‐independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.
Agarose‐selected variants of two human tumor cell lines exhibit altered methionine auxotrophy
Authors: Liteplo Robert G. , Hipwell Sheridan E. 
Published: February 2005

AbstractOur aim was to determine if the selection of human tumor cells with enhanced anchorage‐independent growth capacity was associated with alterations in methionine auxotrophy. Cells with an increased ability to form colonies on soft agarose were selected from human melanoma (MeWo) and neuroepithelioma (SK‐N‐MC) cell lines. In contrast to their respective parental lines, a high proportion of the agarose‐selected variants were completely unable to proliferate in methionine‐free medium containing its immediate precursor homocysteine. The variants exhibited no significant change in their total DNA 5‐methylcytosine content and showed no stimulation of either RNA or DNA synthesis upon the addition of homocysteine when the cells were cultured in methionine‐free medium. These variants were unable to synthesize [3H]S‐adenosylmethionine from [3H]adenine and homocysteine. The failure to detect the accumulation of [3H]S‐adenosylmethionine in these variant lines was not likely due to the enhanced turnover of S‐adenosylmethionine but rather to a reduced ability to synthesize methionine from homocysteine and 5‐methyltetrahydrofolic acid. These results support our hypothesis that alterations in the metabolism of methionine and/or intracellular transmethylating activities may contribute to, or be associated with, the autonomous growth of malignant human tumor cells.
Growth‐inhibiting effect of tumor necrosis factor on human umbilical vein endothelial cells is enhanced with advancing age in vitro
Authors: Shimada Yoshiya , Kaji Kazuhiko , Ito Hideki , Noda Kouichi , Matsuo Mitsuyoshi 
Published: February 2005

AbstractWe have examined the effects of in vitro aging on the growth capacity of human umbilical vein endothelial cells (HUVECs) under the influence of tumor necrosis factor (TNF) with or without interferon‐γ (IFN‐γ). The growth and colony‐forming abilities of control cells were impaired with advancing age in vitro, especially at later stages (more than 70–80% of life span completed). It was found that treatment with TNF inhibited growth and colony‐forming efficiency at any in vitro age. The effects of TNF were shown to increase with increasing in vitro age, as reflected by a more pronounced increase in doubling times, a decrease in saturation density, and a reduction in colony‐forming efficiency. However, the characteristics of TNF receptors, including the dissociation constant, and the number of TNF‐binding sites per cell‐surface area remained rather constant. The effect of TNF was augmented by IFN‐γ at a dose that alone affected growth and colony formation only slightly. The augmentation by IFN‐γ was also found to depend on in vitro age; the synergy with TNF in the deterioration of colony‐forming ability was observed only in “aged” cells. These results suggest that the intrinsic responsiveness of HUVECs to growth‐inhibiting factors, as well as to growth‐stimulating factors, changes during aging in vitro.