bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of cytochrome P450 in rat pleural mesothelial cells in secondary cultures
Authors: Buard Annie , Renier Annie , Jaurand Marie‐Claude , Bignon Jean , Laurent Philippe , Beaune Philippe H.
Published: February 2005

Cultured rat pleural mesothelial cells (RPMC) isolated from male Sprague‐Dawley rats have been shown to metabolize polycylic aromatic hydrocarbons to more oxygenated metabolites. This capacity, which is maintained with passages, suggested the presence of monooxygenase enzymes. In order to clarify the enzymatic pathway, we investigated the expression of cytochromes P450 (CYP) in cultured RPMC by Western and Northern blot analyses. Cells were cultured in Ham's F10 medium supplemented with 10% fetal calf serum. The CYP expression was studied from passage 9 to 16 on different cell strains treated for 48 hours with P450 inducers. CYP1A1 apoprotein expression was very low in untreated cells, but was markedly induced after treatment with 1 μM 3‐methylcholanthrene or 22 μM β‐naphthoflavone. CYP1A1 mRNA was not detected in untreated cells and appeared after 3‐methylcholanthrene treatment. CYP2E1 apoprotein was constitutively expressed in cultured RPMC, and markedly increased by 170 mM ethanol, and 0.1 μM or 1 μM dexamethasone treatments. Unexpectedly, whereas the amount CYP2E1 mRNA was not modified by ethanol treatment, dexamethasone has a marked inductive effect on CYP2E1 mRNA level. The CYP expression pattern was found similar in RPMC issued from different rats, and not dependent on passage number. The CYP expression and the detection of NADPH‐P450 reductase, and of epoxide hydrolase, ascertained that RPMC contain the overall enzymatic pathway required for the biotransformation and activation of procarcinogen compounds, such as polycyclic aromatic hydrocarbons and nitrosamines. Both expression and regulation properties are maintained in long‐term cultures of RPMC. © 1994 Wiley‐Liss, Inc.
EGF modulates phosphoinositide levels in ovarian granulosa cells stimulated by luteinizing hormone
Authors: Hubbard C. J.
Published: February 2005

Hamster granulosa cells were exposed to epidermal growth factor (EGF) and luteinizing hormone (LH) to study cross‐talk between second messenger pathways involving tyrosine kinase, cAMP, and phosphoinositides. Granulosa cells from ovarian preovulatory follicles of PMSG‐primed hamsters were incubated with various additives in serum‐free medium. LH, but not EGF, stimulated inositol phosphate (IP) accumulation; however, when combined with LH, EGF inhibited IP accumulation in a manner that was concentration dependent for both LH and EGF. The inhibitory effects of EGF were significantly reduced by the tyrosine kinase inhibitor genistein and by pertussis toxin suggesting a role for tyrosine kinase and an inhibitory G‐protein (Gi) in this system. EGF stimulated an increase in cAMP, but it does not appear to modulate LH‐stimulated IP levels via cAMP. © 1994 Wiley‐Liss, Inc.
Platelet‐derived growth factor isoforms prevent cell death during starvation of AKR‐2B fibroblasts
Authors: Simm Andreas , Hoppe Viviane , Hoppe Jürgen , Gazit Aviv
Published: February 2005

Confluent AKR‐2B fibroblasts rapidly desintegrate upon removal of serum until a final density of ∼50% of the initial value was reached after 12 h. This density remained unchanged for at least 48 h. Platelet‐derived growth factor (PDGF)‐BB stimulated more than 95% of these cells to divide. PDGF‐AB or ‐BB added immediately after serum removal caused complete survival of the cells, but did not stimulate cell division as demonstrated by two‐dimensional DNA flow cytometry. PDGF‐AA was less effective leading to ∼75% of the initial cell density. This effect could be augmented by the addition of ocadaic acid, a potent phosphatase inhibitor, suggesting that protein phosphorylation plays a role in this process. By using tyrphostin AG807 it was demonstrated that the signaling mechanism for survival requires receptor tyrosine autophosphorylation. © 1994 Wiley‐Liss, Inc.
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

We 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

We 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

We 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

Mast 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

Preincubation 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

One 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

We 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

Agonist 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

The 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

The 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

The 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

The 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

WI‐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.
Control of cell cycle entry and progression in mitogen‐stimulated human B lymphocytes
Authors: Takase Kozo , Terada Naohiro , Lucas Joseph J. , Gelfand Erwin W.
Published: February 2005

Tonsillar B lymphocytes were stimulated to proliferate by the mitogenic combination of phorbol dibutyrate and ionomycin. Progression through the cell cycle was monitored by measurements of cellular DNA and RNA content using flow cytometry. Changes in surface expression of class II MHC antigens and CD20 antigen were also monitored as early parameters of B lymphocyte activation and cell cycle progression. The results showed that about 60% of the population synchronously entered and progressed through the cell cycle. The transition from the resting state, signaled by increased RNA content, occurred about 12 to 24 hr after stimulation; S phase entry occurred at about 36 hr. Small, variable populations of cells appeared to be unresponsive to the stimuli, either because they were “preactivated” before in vitro stimulation or were already dying. The kinetics of appearance and accumulation of several cell cycle regulated/regulatory proteins were followed by immunoblotting. The proliferating cell nuclear antigen (PCNA) cyclin A and p33cdk2 proteins were either absent or present in very low amounts in resting cells and first became detectable in increased amount beginning at about 24 hr after stimulation; increased p34cdc2 protein was not detected until about 36 hr. Increased cellular content and phosphorylation of the p110Rb protein was already obvious by 24 hr after stimulation. The effects of several immunosuppressive agents were examined using purified B cells. Both cyclosporin A and an FK506 analogue were shown to inhibit proliferation of B lymphocytes, at the low doses also inhibitory to T cells. © 1995 Wiley‐Liss, Inc.
Influence of age, sex, and dietary restriction on intracellular free calcium responses of CD4+ lymphocytes in rhesus monkeys (Macaca mulatta)
Authors: Grossmann Angelika , Rabinovitch Peter S. , Lane Mark A. , Jinneman John C. , Ingram Donald K. , Wolf Norman S. , Cutler Richard G. , Roth George S.
Published: February 2005

The influence of aging and dietary restriction on increase in intracellular free calcium ([Ca2+]i) of CD4+ lymphocytes from Macaca mulatta was examined after stimulation with anti‐CD3 mAb. We used a flow cytometric assay with the dye indo‐1 and either direct or reciprocal immunofluorescent staining to dientify CD4+ cells. After stimulation with anti‐CD3 mAb, intracellular free calcium responses were reduced in CD4+ lymphocytes from old male and female ad libitum fed monkeys compared to young and adult male or female monkeys. Old female monkeys had significantly lower [Ca2+]i than did old male monkeys. The reduced responses were in part related to a decreased percentage of responding cells. Dietary restrition of males over a four‐year period did not alter [Ca2+]i response compared to ad libitum fed male monkeys. Female monkeys of all ages (which were restricted only for four months) also had similar [Ca2+]i responses to ad libitum fed controls. Our data suggest that age‐related changes in [Ca2+]i responses are similar between humans and M. mulatta, and that over these intervals, no effects of caloric restrictions can be detected. © 1995 Wiley‐Liss, Inc.
EGF‐mediated phosphorylation of extracellular signal‐regulated kinases in osteoblastic cells
Authors: Zhang Wenyu , Dziak Rosemary M. , Aletta John M.
Published: February 2005

Epidermal growth factor (EGF) induces a rapid increase in the phosphorylation of extracellular signal‐regulated kinases (ERKs) in the human osteosarcoma osteoblastic cell line G292 and in primary cultures of rat osteoblastic cells. This phosphorylation is transient and time‐dependent. Maximal stimulation is attained within 1 min in G292 and within 5 min in rat osteoblastic cells. Enzymatic activity in G292 cells is also induced rapidly after EGF stimulation. Western blot analysis revealed that enhancement of the phosphorylation of ERKs in the EGF‐stimulated cells is not due to an increase in ERK protein, since EGF‐treatment does not lead to an increase in the absolute amount of ERKs present even after 2 days of stimulation. The pattern of expression of the ERKs observed in the two cell types differs in the apparent molecular weights observed. The most slowly migrating immunoreactive protein (∼45 kDa) in normal rat osteoblastic cells is ERK1, identified by an ERK1‐selective antiserum. The same antiserum reacts only weakly with one of the ERK proteins (44 kDa) blotted from the human osteosarcoma cell line G292. Phorbol 12‐myristate 13‐acetate (PMA) is also capable of inducing ERK phosphorylation, albeit to a lesser degree. The combination of PMA and EGF does not produce a greater response than EGF alone. The role of protein kinase C (PKC) in the EGF‐stimulated ERK signaling pathway was further examined by inhibition of PKC with the staurosporine analog, CGP41251, and by down‐regulation of PKC via chronic treatment with PMA. Chronic PMA treatment results in a partial inhibition of the EGF‐mediated phosphorylation. CGP41251 completely abolishes the increased ERK activity produced by PMA, but the effect of EGF in this regard is potentiated. We conclude that PKC and EGF act through parallel pathways to stimulate ERK phosphorylation and activity. The inhibitor studies, in addition, indicate that activation of PKC may moderate the actions of the EGF pathway via a tonic inhibitory feedback. © 1995 Wiley‐Liss, Inc.
No evidence for a tumor necrosis factor α stimulated 2‐methylaminoisobutyric acid uptake in hepatocyte monolayer
Authors: Lim Soo‐Kyung , de Bandt Jean‐Pascal , Aussel Christian , Pernet Pascal , Giboudeau Jacqueline , Cynober Luc
Published: February 2005

This study investigates the short‐tem effects of glucagon and human recombinant tumor necrosis factor α (TNFα) singly and in association on 2‐methylaminoisobutyric acid (MeAIB) transport in hepatocyte monolayers. As expected, glucagon induced a time‐dependent stimulation of MeAIB transport. In our experimental conditions, TNFα did not induce cytolysis. A 2 hour exposure to TNFα (0.05–500 ng/I) with or without glucagon (10−9 to 10−6 M) did not modify the basal or glucagon‐stimulated MeAIB transport. Varying the duration of exposure to TNFα 5 ng/I up to 6 h was equally ineffective. The presence of hydrocortisone potentiated the glucagon‐stimulated transport, but TNFα remained ineffective. Finally, the association of interferon (IFNγ) with TNFα and/or glucagon was unable to modify the transport activity. These data demonstrate that TNFα does not exert a direct effect on MeAIB transport in hepatocytes, at least on a short‐term basis. © 1995 Wiley‐Liss, Inc.
Association of hepatic β2‐adrenergic receptor gene transcript destabilization during postnatal development in the sprague‐dawley rat with a Mr 85,000 protein that binds selectively to the β2‐adrenergic receptor mRNA 3′‐untranslated region
Authors: Baeyens Dennis A. , Cornett Lawrence E.
Published: February 2005

In the liver, transcript destabilization contributes to the decrease in steady‐state levels of β2‐adrenergic receptor mRNA that occurs during early postnatal development in the rat. From genomic DNA, polymerase chain reaction (PCR) was used to amplify a 718‐basepair (bp) fragment of the β2‐adrenergic receptor gene including the entire 3′‐untranslated region. Results from SDS‐gel electrophoresis and autoradiography demonstrated a Mr 85,000 cellular factor present in postnatal day 60, but not fetal day 18 rat liver that was ultraviolet (UV) light‐crosslinked to in vitro transcribed β2‐adrenergic receptor RNA 3′‐untranslated region. Unlabeled β2‐adrenergic receptor RNA 3′‐untranslated region, but not mouse β‐actin RNA, competed with labeled β2‐adrenergic receptor RNA 3′‐untranslated region for binding to the Mr 85,000 protein. Cross‐linking of the β2‐adrenergic receptor RNA 3′‐untranslated region to the Mr 85,000 protein was inhibited by the ribohomopolymer poly(U), with poly(A), poly(C) and poly(G) having little or no effect. Thus, a Mr 85,000 protein has been identified in adult male rat liver that may interact with U‐rich sequences in the 3′‐untranslated region of the β2‐adrenergic receptor mRNA and may account for the decreased stability of hepatic β2‐adrenergic receptor gene transcripts that occurs during development. © 1995 Wiley‐Liss, Inc.
Lipopolysaccharides stimulate Na‐dependent transport in alveolar cells and protect against oxidant injury
Authors: Azarian Réza , Clerici Christine , Couette Sylvianne , Friedlander Gérard , Amiel Claude
Published: February 2005

We have evaluated the effect of lipopolysaccharides (LPS), endotoxins from gram negative bacteria, on sodium‐coupled amino acid and phosphate transport by alveolar epithelial type II cells and on their alteration induced by oxidants. Alveolar type II cells were obtained by enzymatic digestion of rat lung and grown for 24 h prior to incubation with LPS and then exposed or not exposed to H2O2 (2.5 mM; 20 min). LPS (10 μg/ml, 24 h) induced a significant increase in the Na‐dependent component of alanine and phosphate uptake while they decreased Na, K‐ATPase activity measured by ouabain‐sensitive 86Rb influx. We showed that this stimulatory effect i) was independent from macrophage products since it was not mimicked either by supernatant of LPS‐treated alveolar macrophages or by pretreatment with tumor necrosis factor and/or interleukin 1 and ii) was dependent on protein synthesis since it was abolished by protein synthesis inhibitors cycloheximide and actinomycin D. Moreover, LPS blunted H2O2‐induced decrease of Na‐dependent alanine and phosphate uptake. This protective effect of LPS against H2O2 injury i) was independent of macrophage products, ii) was abolished by cycloheximide, and iii) was not associated with either changes in extracellular H2O2 clearance or catalase and glutathione peroxidase activities. We conclude that, in alveolar type II cells, LPS stimulate sodium‐coupled transport by a process involving protein synthesis and partially prevent H2O2‐induced decrease of Na‐coupled transport without discernible change in antioxidant activities. © 1995 Wiley‐Liss, Inc.
Prolactin and epidermal growth factor regulation of the proliferation, morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture
Authors: Darcy Kathleen M. , Shoemaker Suzanne F. , Lee Ping‐Ping H. , Vaughan Mary M. , Black Jennifer D. , Ip Margot M.
Published: February 2005

The epithelial cell‐specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non‐milk‐producing MEC were isolated as spherical end bud‐like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01–10 μg/ml) and EGF (1–100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21‐day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland. © 1995 Wiley‐Liss, Inc.
Binding of platelet‐derived growth factor and low density lipoproteins to glycosaminoglycan species produced by human arterial smooth muscle cells
Authors: Fager Gunnar , Camejo German , Olsson Urban , Östergren‐Lundén Gunnel , Lustig Florentyna , Bondjers Göran
Published: February 2005

The platelet‐derived growth factor (PDGF) binds via a defined amino acid sequence to heparin (Fager et al., 1992, In Vitro Cell. Dev. Biol., 28A:176–180) and the protein moiety of low density lipoproteins (LDL; apo B‐100) via a similar sequence to chondroitin sulfate (Camejo et al., 1988, Arteriosclerosis Thromb., 8:368–377). In this study, synthetic oligopeptides were used to explore the capacity of smooth muscle cell‐derived glycosaminoglycans to bind to the critical sequences of PDGF and apo B‐100. In vitro, proliferating human arterial smooth muscle cells synthesized twice as much proteoglycans as did quiescent cells. The dominating glycosaminoglycan side chains were chondroitin and heparan sulfates in secreted and cell‐associated proteoglycans, respectively. The chondroitin sulfate‐rich proteoglycans had a higher molecular size and were to a larger extent secreted into the culture medium than the heparan and dermatan sulfate‐rich proteoglycans. Heparan, dermatan, and chondroitin sulfates bound to the PDGF‐derived oligopeptide with affinities similar to those of heparin. However, while heparan and dermatan sulfates both inhibited DNA synthesis in human arterial smooth muscle cells, chondroitin sulfate had no such inhibitory effect. Like the PDGF‐derived oligopeptide, the apo B‐100‐derived oligopeptide bound to these glycosaminoglycans. At the same time, both oligopeptides displaced bound LDL from chondroitin sulfate in vitro and released the block on DNA synthesis in smooth muscle cells that heparin induced in culture. Thus, chondroitin, dermatan, and heparan sulfates produced by arterial smooth muscle cells may bind LDL and PDGF competitively in atherogenesis but only heparan and dermatan sulfates inhibit cellular DNA synthesis. LDL and PDGF deposition may occur by binding to similar binding sites on glycosaminoglycans derived from smooth muscle cells within atherosclerotic lesions. © 1995 Wiley‐Liss, Inc.
Inhibition of N‐linked glycosylation induces early apoptosis in human promyelocytic HL‐60 cells
Authors: Pérez‐Sala Dolores , Mollinedo Faustino
Published: February 2005

Inhibition of protein N‐glycosylation by tunicamycin induced morphological changes characteristic of apoptosis in human promyelocytic HL‐60 cells. Internu‐cleosomal DMA fragmentation could be detected after short‐time incubation (between 6 and 9 h) of HL‐60 cells with low doses of tunicamycin (0.05 μg/ml). Under these conditions the synthesis of glycoproteins was reduced to 17% of control values, while no significant changes in the rates of total protein synthesis could be observed. Tunicamycin ability to induce DNA fragmentation was in good correlation with its potency as glycosylation inhibitor in several myeloid cell lines. Tunicamycin‐induced apoptosis was potentiated by activation of protein kinease C (PKC) by phorbol esters and partially prevented by the PKC inhibitor staurosporine. Inhibitors of RNA and protein synthesis displayed a protective effect. Treatment of HL‐60 cells with tunicamycin did not elicit the expression of cell surface differentiation antigens or their ability to generate superoxide anion. In contrast, tunicamycin significantly inhibited these processes during dimethyl sulfoxide (DMSO)‐induced myeloid differentiation. These observations indicate that the main effect of tunicamycin in HL‐60 cells is the induction of apoptosis. © 1995 Wiley‐Liss, Inc.
Differential effects of glucocorticoids on insulin‐like growth factor I action in cultured human fibroblasts
Authors: Conover Cheryl A. , Hintz Raymond L. , Johnson Ben F.
Published: February 2005

Glucocorticoids act synergistically with insulin‐like growth factor I (IGF‐I) to stimulate DNA synthesis and replication of cultured human fibroblasts. In the present study, we further define glucocorticoid and IGF‐I interactive effects on human fibroblast metabolism and growth. IGF‐I stimulated dose‐dependent increases in early metabolic events. Half‐maximal effectiveness was seen at 5–8 ng/ml IGF‐I, with mean maximal responses of 1.5‐, 2‐, and 6‐fold for [3H]2‐deoxyglucose uptake, [14C]glucose incorporation, and [14C]aminoisobutyric acid (AIB) uptake, respectively. A 48‐hour preincubation with 10−7 M dexamethasone markedly enhanced both the sensitivity and maximal effectiveness of IGF‐I stimulation of AIB uptake. In contrast, dexamethasone had no effect on IGF‐I‐stimulated glucose uptake and utilization. Maximum specific binding of [125I]IGF‐I to fibroblast monolayers was identical in ethanol control and glucocorticoid‐treated cells, with 50% displacement at ∼5 ng/ml IGF‐I. In addition to its synergism with IGF‐I, preincubation with dexamethasone augmented insulin and epidermal growth factor (EGF) stimulation of [3H]thymidine incorporation; dexamethasone had no effect on platelet‐derived growth factor or fibroblast growth factor action. Two‐dimensional gel electrophoresis identified two specific glucocorticoid‐induced proteins in human fibroblast cell extracts with molecular weights of 45K and 53K and pls of 6.8 and 6.3, respectively. These data indicate that IGF‐I receptor‐mediated actions in human fibroblasts are differentially modulated by glucocorticoids. Glucocorticoids are synergistic with IGF‐I in stimulating mitogenesis and amino acid uptake, without having any apparent effect on IGF‐I‐stimulated glucose metabolism. Glucocorticoid enhancement of growth factor bioactivity may involve modulation of a regulatory event in the mitogenic signaling pathway subsequent to cell surface receptor activation. © 1995 Wiley‐Liss, Inc.
Activation of junB and c‐myc primary response genes by interleukin 9 in a human factor‐dependent cell line
Authors: Kang Li Ya , Yang Yu‐Chung
Published: February 2005

Interleukin 9 (IL‐9) stimulates the proliferation of various hematopoietic cell types. To elucidate the molecular mechanisms underlying the cell proliferation action, immediate‐early gene expression elicited by IL‐9 in a human factor‐dependent cell line, MO7e, was studied. IL‐9 stimulation resulted in a rapid and transient elevation of primary response genes including junB and c‐myc. The differential effects of protein kinase inhibitors, herbimycin A, genistein, and H‐7 on the steady‐state mRNA level and the transcription rate of junB and c‐myc genes triggered by IL‐9 were also investigated. Herbimycin A, but not genistein, specifically inhibited the expression of junB steady‐state mRNA and the in vitro transcription of the junB gene. IL‐9‐enhanced c‐myc gene expression was completely inhibited by both herbimycin A and genistein at the level of transcriptional initiation. H‐7 failed to inhibit c‐myc, but partially abolished junB mRNA induction. The role of protein kinase C in IL‐9‐mediated junB induction was also examined. The different responses of junB and c‐myc messages to protein kinase inhibitors suggested that more than one pathway may be involved in IL‐9‐mediated signal transduction which leads to the expression of junB and c‐myc genes. © 1995 Wiley‐Liss, Inc.
Induction of bovine bronchial epithelial cell filopodia by tetradecanoyl phorbol acetate, calcium ionophore, and lysophosphatidic acid
Authors: Beckmann Joe D. , Romberger Debra J. , Rennard Stephen I. , Spurzem John R.
Published: February 2005

The morphological responses of primary bovine bronchial epithelial cells (BBECs) cultured in serum‐free medium to protein kinase activators have been examined. When attached to type I collagen‐coated tissue culture dishes, the cells responded to tetradecanoyl phorbol acetate (TPA), calcium ionophore A23187, and lysophosphatidic acid (LPA) by extruding filopodia. In contrast, no morphological changes were elicited by exposures to either epinephrine or dibutyryl‐cAMP. Formation of filopodia was accompanied by actin filament reorganization as demonstrated by staining with labeled phalloidin. Exposures to varied TPA concentrations for 2 h showed maximal stimulation of filopodial extrusions at 10 nM TPA with half‐maximal stimulation at 1 nM. Time‐course measurements with 10 nM TPA showed filopodia formation within 30 min of exposure, with 85% of the BBECs being filopodia positive after 5 h. Filopodia induction in 20‐30% of the cells could be achieved by 1–100 m̈M LPA concentrations. BBECs acquired increasing resistance to TPA‐induced filopodia during the initial 5 days in culture; however, responsiveness to TPA was regenerated by mild treatment with trypsin. Inclusion of fibronectin or vitronectin into the attachment matrix had no effects on the rates or extent of TPA‐induced filopodia formation. © 1995 Wiley‐Liss, Inc.