bluuebunny/crossref_metadata_2025_split huggingface dataset:
Changes in promoter utilization in human and mouse c‐myc genes upon transformation induction in temperature‐sensitive cell lines
Authors: Luo Yi , Krause Margarida O. 
Published: February 2005

AbstractWe have previously reported accelerated transcription and rapid accumulation of c‐myc mRNAs upon induction of transformation in a temperature‐sensitive mouse cell line (Gallant et al., 1989, Oncogene Res., 4:39–46). Here we have used both mouse and human cell lines transformed with a temperature‐sensitive mutant of the Simian virus 40 (SV40) virus to investigate whether a shift in promoter utilization within the c‐myc gene locus is part of a general mechanism that deregulates c‐myc expression during transformation induction. We devised a simple and sensitive method using reverse transcription followed by radioactive polymerase chain reaction (RT‐PCR) to measure the relative change in c‐myc mRNAs arising from each of the four known promoters. We show that a three to fivefold increase in c‐myc transcripts from the P1 and P3 promoters occurs in both human and mouse cell lines within 30 min of the shift to the permissive temperature. The major P2‐initiated transcripts are not significantly effected. However, exon 3‐containing RNAs increase more gradually up to 24 h postinduction and P1 and P3 transcripts, while remaining elevated, still contribute relatively little to the total c‐myc RNA population. These and other results, demonstrating a transient activation of P1 and P3 promoters, suggest an indirect role of the minor transcripts in the deregulated expression of the c‐myc gene in transformed cells. © 1994 Wiley‐Liss, Inc.
Microtubule inhibitors differentially affect translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 cells
Authors: Thatte Hemant S. , Bridges Kenneth R. , Golan David E. 
Published: February 2005

AbstractWe used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein‐conjugated transferrin (FITC‐Tf). Coordinated decreases in surface fluorescence counts, the photobleachig parameter K, and transferrin receptor fractional mobility were observed as FITC‐Tf was cleared from the cell surface by receptor‐mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC‐Tf uptake at 37°C and 21°C were calculated to be 0.10‐0.15 min−1 and 0.02–0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by β‐lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtuble inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule‐stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug‐resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor‐mediated transferrin uptake in K562 cells. © 1994 Wiley‐Liss, Inc.
Heterogeneity and contact‐dependent regulation of amylase release by individual acinar cells
Authors: Bosco Domenico , Soriano Jesús V. , Chanson Marc , Meda Paolo 
Published: February 2005

AbstractWe have used a reverse hemolytic plaque assay to investigate the amylase release of single and aggregated pancreatic acinar cells. We have found that a minority of single acinar cells released detectable amounts of amylase under basal conditions and were modestly stimulated, in a dose‐dependent manner, during a 30‐min exposure to concentrations of carbamylcholine (CCh) ranging from 10−8 to 10−5 M. This stimulation was largely accounted for by the recruitment of additional secreting cells, rather than by a significant increase in their individual secretory output. We have also observed that aggregates comprising two to five acinar cells secreted more frequently and released more amylase than single acinar cells in the presence of each of the CCh concentrations tested. Under both basal conditions and following CCh stimulation, the proportion of secreting aggregates and their amylase output increased linearly with the aggregate size. Under basal conditions as well as in the presence of secretagogue concentrations in the 10−8−10−7 M range, individual cells contributed similarly to amylase secretion whether they were single or part of aggregates. By contrast, following stimulation by 10−6−10−5 M CCh, aggregated cells showed a much higher average secretion than single cells. Investigating the mechanism of this contact‐dependent effect, we found that 10−3 M heptanol did not significantly modify the secretion of single cells and markedly promoted the basal amylase release of acinar cell pairs. This effect was associated with a marked reduction in gap junctional communication between acinar cells, as evaluated by microinjection of Lucifer yellow, and was not observed during exposure to high concentrations of CCh, which also reduced junctional communication. These data show that pancreatic acinar cells are intrinsically heterogeneous in their ability to release amylase and that their basal as well as stimulated secretion are promoted by the establishment of direct intercellular contacts. Our experiments also suggest that junctional coupling contributes to the contact‐dependent mechanism which enhances the recruitment of secreting cells and their individual output. These observations strengthen the view that direct interactions between acinar cells are essential in the control of pancreatic secretion. © 1994 Wiley‐Liss, Inc.
Paracrine Ca2+ signaling in vitro: Serotonin—mediated cell—cell communication in mast cell/smooth muscle cocultures
Authors: Weintraub William H. , Cleveland‐Wolfe Patricia , Fewtrell Clare 
Published: February 2005

AbstractMast cells are tissue‐resident immune cells that are capable of signaling many different cell types in vascularized tissue including epithelia and smooth muscle. We have developed an in vitro coculture system in which secretion of serotonin by a mucosal mast cell line (RBL‐2H3) can be studied at a single cell level by measuring Ca2+ transients in fura‐2 loaded mast cells and serotonin‐sensitive A7r5 smooth muscle cells using fluorescence video microscopy and digital image processing. A7r5 cells elevate intracellular Ca2+ via 5HT2 receptors in response to bath‐applied serotonin with an ED50 for serotonin of 550nM. Crosslinking lgE receptors with antigen caused Ca2+ transients in the mucosal mast cells. Ca2+ responses in the smooth muscle were detected ≈︁ 30–240 sec after the initiation of the mast cell Ca2+ responses. Smooth muscle Ca2+ responses were dependent on preloading mast cells with serotonin and were blocked by the 5HT2 antagonist ketanserin. The timing and magnitude of the smooth muscle responses indicated that secretion from mast cells can lead to local concentrations of serotonin in the range of 300 nM within 1 min of antigen stimulation. This coculture technique has allowed the first direct demonstration of serotonin‐mediated signaling between immune cells and vascular elements. © 1994 Wiley‐Liss, Inc.
Separation of agonist‐stimulated arachidonate mobilization from subsequent leukotriene B4 synthesis in human neutrophils: Different effects of oleoylacetylglycerol and phorbol myristate acetate as priming agents
Authors: Rosentha Miriam D. , Franson Richard C. 
Published: February 2005

AbstractPreincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f‐Met‐Leu‐Phe (fMLP)‐stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3‐dioctanoylglycerol (1,3‐diC8) is equipotent with 1,2‐sn‐dioctanoylglycerol (1,2‐diC8) as a priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79–86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12‐myristate 13‐acetate (PMA), on agonist‐stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f‐Met‐Leu‐Phe (fMLP)‐stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 μm) and PMA (10 nM) are equipotent in increasing fMLP‐stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5‐lipoxygenase (5‐LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP‐stimulated synthesis of platelet‐activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 μM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist‐stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (<0.1 μM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5‐LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC‐independent manner. © 1994 Wiley‐Liss, Inc.
Regulation of phenol sulfotransferase expression in cultured bovine bronchial epithelial cells by hydrocortisone
Authors: Beckmann Joe D. , Illig Mary , Bartzatt Ronald 
Published: February 2005

AbstractOne conjugative pathway for the inactivation of endogenous and exogenous hydroxylated aromatic compounds is catalyzed by phenol (aryl) sulfotransferases (PSTs), which esterify phenolic acceptors with sulfate. The tracheobronchial epithelium is commonly exposed to phenolic drugs and pollutants, and metabolic sulfation and PST activity in this tissue have been previously demonstrated. To determine what factors may control PST expression, extracts of serum‐free, growth factor‐supplemented cultures of bovine bronchial epithelial cells were assayed for PST activity and PST antigen. The most significant finding was dose‐dependent, apparent stimulated expression by hydrocortisone (EC50 = 4 nM, maximal stimulation at 20 nM). Time‐course experiments, however, revealed progressive loss of PST in the absence of corticosteroid. After decay of extant PST in steroid‐free medium, hydrocortisone reinduced the expression of PST three to fivefold. Western blots using mouse anti‐bovine PST revealed corresponding increases in 32 kDa PST protein levels in response to hydrocortisone. Steady state kinetic analyses indicated apparent Km values of 1—3 μM for 2‐naphthol regardless of culture conditions. These results suggest that detoxification of phenolic compounds by sulfation may be regulated by corticosteroids.
Proliferative responses of epithelial cells to 8‐bromo‐cyclic AMP and to a phorbol ester change during breast Pathogenesis
Authors: Starzec Anna B. , Spanakis Emmanuel , Nehme Alissar , Salle Valerie , Veber Nathalie , Mainguene Claire , Planchon Philippe , Valette Annie , Prevost Gregoire , Israel Lucien 
Published: February 2005

AbstractWe have explored the relationship of changes in proliferative responses of human mammary epithelial cells to a phorbol ester (TPA) and to 8‐Br‐cAMP, which modulate the activities of protein kinases A and C (PKA and PKC), with breast tumour progression. Treatment with TPA had no effect on nontumorigenic cell lines established from human fibrocystic biopsies and apparently normal tissue around a tumour. In contrast, TPA strongly inhibited the proliferation of numerous human tumorigenic breast cell lines. Treatment with 8‐Br‐cAMP decreased the proliferation of all studied nontumorigenic and tumorigenic cell lines. We have also studied the effect of TPA and 8‐Br‐cAMP on growth of epithelial cells in short‐term culture obtained from surgical human mammary biopsies with different states of breast disease. Both drugs enhanced growth of normal breast cells but had no significant effects on cells from biopsies with benign breast disease. In contrast, all examined cuitures from breast cancer biopsies were strongly inhhited by 8‐Br‐cAMP. Otherwise, TPA had an inhibitory effect only in the case of invasive ductal carcinoma of grade III. Malignant Ha‐ras‐transformation of nontumorigenic TPA‐insensitive breast HBL‐100 cells induced an inhibitory effect of TPA. In addition, a TPA‐insensitive MCF7 clone was much less tumorigenic in athymic mice than the parental strain shown to be inhibited by TPA. These data suggest that the two intracellular transduction pathways change at different stages of breast pathogenesis. Alterations in the PKA pathway are early events and are probably important to cell immortalization but do not necessarily lead to malignant development. In contrast, changes in PKC pathway are rather later events associated with advanced malignant transformation. © 1994 Wiley‐Liss, Inc.
Enhancement of β‐Adrenergic‐induced cAMP accumulation in activated T‐Cells
Authors: Carlson Sonia L. , Trauth Konrad , Brooks William H. , Roszman Thomas L. 
Published: February 2005

AbstractAgonist stimulation of the β‐adrenergic receptor on T‐cells results in the production of cAMP, which has been correlated with modulation of T‐cell function. In previous studies, we have demonstrated that the mitogen PHA can synergistically enhance the accumulation of cAMP in T‐cells in response to the agonist isoproterenol. In this report we have investigated the mechanisms by which dual stimulation of T‐cells acts to synergistically enhance cAMP accumulation. The results demonstrate that increasing the levels of intracellular calcium with ionomycin or thapsigargin enhanced isoproterenol‐induced cAMP accumulation in T‐cells. In contrast, PHA enhanced isoproterenol‐induced cAMP by a calcium‐independent mechanism as evidenced by stimulation with isoproterenol plus PHA in calcium‐free medium. Further studies revealed that PHA prevented both sequestration of the β‐adrenergic receptor and its dissociation from Gs protein in response to isoproterenol stimulation. In contrast, PHA did not prevent the functional uncoupling of the β‐adrenergic receptor from adenylyl cyclase, suggesting that additional mechanisms are likely involved. In summary, these studies demonstrate that dual receptor signalling of T‐cells increases cAMP accumulation and offers a potential mechanism for catecholamine modulation of T‐cell function. © 1994 Wiley‐Liss, Inc.
Extracellular calcium modulates prereplicative cyclic AMP surges in EGF‐stimulated primary neonatal rat Hepatocytes
Authors: Armato U. , Ribecco Maria , Guerriero Chiara , Testolin Lucia , Whitfield J. F. 
Published: February 2005

AbstractThe cells in nearly pure (96‐98%) primary cultures of hepatocytes from neonatal rat liver in high (1.0 mM)‐Ca2+, serum‐free, synthetic HiWo5Ba2000 medium initiated DNA synthesis and entered mitosis between 11 and 30 h after the addition of 10 ng/ml EGF. During the 10‐h prereplicative period, the cultured hepatocytes, like regenerating rat liver cells, generated two large cyclic AMP transients, one peaking between 30 min and 2 h and the other around 6 h. Hepatocytes stimulated by the same concentration of EGF in low (0.02 mM)‐Ca2+ medium increased cyclic AMP synthesis as much as the EGF‐treated hepatocytes in high‐Ca2+ medium, but they released the additional cyclic AMP into the medium and could not generate prereplicative internal cyclic AMP surges, initiate DNA replication, or enter mitosis. These results suggest that one of the ways external Ca2+ controls prereplicative development of hepatocytes is to restrain the release of cyclic AMP and thus enable the cell to accumulate enough internal cyclic AMP to stimulate events required to initiate DNA replication. © 1994 Wiley‐Liss, Inc.
Profile of energy metabolism in a murine hybridoma: Glucose and glutamine utilization
Authors: Petch D. , Butler M. 
Published: February 2005

AbstractThe antibody‐secreting murine hybridoma, CC9C10, was grown in batch culture in a medium containing 20 mM glucose and 2 mM glutamine. After 2 days of exponential growth, the glutamine content of the medium was completely depleted, whereas the glucose content was reduced to 60% of the original concentration. The glucose and glutamine metabolism was analyzed at midexponential phase by use of radioactively labelled substrates. Glycolysis accounted for the metabolism of most of the glucose utilized (> 96%) with flux through the pentose phosphate pathway (3.6%) and the TCA cycle (0.6%) accounting for the remainder. Glutamine was partially oxidised via glutaminolysis to alanine (55%), aspartate (3%), glutamate (4%), lactate (9%), and CO2 (22%). Calculation of the theoretical ATP production from these pathways indicated that glucose could provide 59% and glutamine 41% of the energy requirement of the cells. © 1994 Wiley‐Liss, Inc.
Regulation of the mouse mammary tumor virus receptor by phosphorylation and internalization in mammary epithelial cells
Authors: Bolander Franklyn F. 
Published: February 2005

AbstractThe mouse mammary tumor virus enters mammary epithelial cells via a plasma membrane protein that binds to a viral envelope glycoprotein, gp52. In intact cells, this gp52 receptor can be phosphorylated by activators of protein kinase A and protein kinase C (PKC), but this modification does not occur in response to epidermal growth factor, whose receptor is a tyrosine kinase, or to gp52. Phosphorylation of the gp52 receptor rapidly leads to internalization and gradual loss of binding activity. Both the phosphorylation and the intrnalization induced by PKC are abolished by prior downregulation of this kinase. Although the physiological function of the gp52 receptor is unknown, its binding to gp52 can stimulate several biological activities, including amino acid accumulation. Receptor processingimpairs this gp52‐induced amino acid uptake, as well as viral infection, by depleting the binding protein at the cell surface. In contrast, PKC augments insulin‐induced amino acid transport, and PKC downregulation abolishes the action of insulin, suggesting that insulin and gp52 utlize partially separate pathways leading to amino acid transport. These data further suggest that PKC may be involved in this insulin‐stimulated activity. © 1994 Wiley‐Liss, Inc.
Effects of glucose supply on myeloma growth and metabolism in chemostat culture
Authors: Meijer J. J. , van Dijken J. P. 
Published: February 2005

AbstractThe effects of the glucose supply on growth and metabolism of an SP2/0 derived recombinant myeloma cell line were studied in chemostat culture during growth on IMDM medium at a fixed dilution rate of 0.032 h−1. Lowering of the feed medium glucose concentration from 25.0 to 1.4 mmol/L resulted in a decrease of steady‐state viable cell concentration from 1.9 × 109 L−1, whereas viability remained above 90%. Mass balances indicated that only a minor amount of glucose was utilized via the TCA cycle irrespective of the glucose concentration in the feed medium. The apparent biosynthetic yield of cells from ATP was independent of the ratio between the specific glucose and glutamine consumption rate. It is concluded that the primary role of glucose is the provision of intermediates for anabolic reactions. In addition, glucose may play an indirect catabolic role in the process of glutaminolysis by providing the pyruvate for the transamination of glutamate to alanine and α‐ketoglutarate. At low glucose concentrations in the feed medium, glutamine is probably the sole energy source for this myeloma in chemostat culture. © 1995 Wiley‐Liss, Inc.
Regulation of human ornithine decarboxylase expression following prolonged quiescence: Role for the c‐Myc/Max protein complex
Authors: Peña Angel , Wu Shujan , Hickok Noreen J. , Soprano Dianne Robert , Soprano Kenneth J. 
Published: February 2005

AbstractWI‐38 cells can remain quiescent for long periods of time and still be induced to reenter the cell cycle by the addition of fresh serum. However, the longer these cells remain growth arrested, the more time they require to enter S phase. This prolongation of the prereplicative phase has been localized to a point early in G1, after the induction of “immediate early” G1 genes such as c‐fos and c‐jun but before maximal expression of “early” G1 genes such as ornithine decarboxylase (ODC). Understanding the molecular basis for ODC mRNA induction can therefore provide information about the molecular events which regulate the progression of cells out of long‐term quiescence into G1 and subsequently into DNA synthesis. Studies utilizing electrophoretic mobility shift assays (EMSA) of nuclear extracts from short‐ and long‐term quiescent WI‐38 cells identified a region of the human ODC promoter at −491 bp to −474 bp which exhibited a protein binding pattern that correlated with the temporal pattern of ODC mRNA expression. The presence of a CACGTG element within this fragment, studies with antibodies against c‐Myc and Max, the use of purified recombinant c‐Myc protein in the mobility shift assay, and antisense studies suggest that these proteins can specifically bind this portion of the human ODC promoter in a manner consistent with growth‐associated modulation of the expression of ODC and other early G1 genes following prolonged quiescence. These studies suggest a role for the c‐Myc/Max protein complex in regulating events involved in the progression of cells out of long‐term quiescence into G1 and subsequently into S. © 1995 Wiley‐Liss, Inc.