bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mechanism and regulation of neutrophil priming by platelet‐activating factor
Authors: Gay James C. 
Published: February 2005

AbstractAlthough a weak direct stimulus of superoxide anion (O2−) production, platelet‐activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n‐formyl‐met‐leu‐phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor‐ and PKC‐mediated but is independent of pertussis toxin‐inhibitable G‐proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF‐induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism. © 1993 Wiley‐Liss, Inc.
Transforming growth factor β1 selectively regulates ornithine decarboxylase gene expression in malignant H‐ras transformed fibrosarcoma cell lines
Authors: Hurta Robert A. R. , Greenberg Arnold H. , Wright Jim A. 
Published: February 2005

AbstractNegative growth regulators such as the transforming growth factor beta (TGF‐β) family appear to be important inhibitors in most tissue types. However, inhibition of DNA synthesis and cell proliferation is frequently lost during malignant transformation, and in some cases, tumor cell proliferation is actually stimulated by TGF‐β. The present study demonstrates a novel link between alterations in TGF‐β regulation during malignant conversion, and the expression of ornithine decarboxylase, a key rate‐limiting activity in the biosynthesis of polyamines, and an enzyme that plays an important role in cell growth and differentiation. A panel of radiation and H‐ras transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was investigated for possible TGF‐β1 mediated changes in ornithine decarboxylase gene expression. Selective induction of gene expression was observed since only H‐ras transformed cell lines with malignant potential exhibited marked elevations in ornithine decarboxylase message levels. Ornithine decarboxylase gene expression in nontransformed 10T1/2 cells and cell lines capable of only benign tumor formation was unaffected by TGF‐β1 treatment. H‐ras transformed cells were transfected with a plasmid placing the TGF‐β1 coding region under the control of a zinc sensitive metallothionein promoter. When these cells were cultured in the presence of zinc an elevation of TGF‐β1 mRNA was observed within 30 min. This increase in TGF‐β1 message closely coincided with an elevation in ornithine decarboxylase message, and preceded an induction of jun‐B, an early response gene in cells sensitive to TGF‐β1 stimulation. Evidence for regulation of ornithine decarboxylase gene expression by TGF‐β1 at both transcription and posttranscription was found. Actinomycin D pretreatment of malignant cells prior to TGF‐β1 exposure prevented the increase in ornithine decarboxylase message. Marked differences in the rates of ornithine decarboxylase message decay were observed when cells treated with TGF‐β1 were compared to untreated controls, with the half‐life of ornithine decarboxylase mRNA increasing from 2.5 h in untreated cells to 17.5 h in cells exposed to TGF‐β1. In addition, evidence was obtained for a cycloheximide sensitive regulator of ornithine decarboxylase gene expression, since the presence of this protein synthesis inhibitor increased the levels of ornithine decarboxylase message, and this effect was synergistically augmented by exposure of cells to cycloheximide and induction of TGF‐β1 gene expression together. These results show for the first time that TGF‐β1 can regulate ornithine decarboxylase expression in malignant H‐ras transformed cells, and suggest a mechanism of growth factor stimulation of malignant cells, in which early alterations in the control of ornithine decarboxylase gene expression are important. © 1993 Wiley‐Liss, Inc.
Rapid apoptotic cell death of B‐cell hybridomas in absence of gene expression
Authors: Perreault Josée , Lemieux Réal 
Published: February 2005

AbstractApoptosis is an active form of cell death which plays an important role in different biological processes. The induction of apoptosis usually requires de novo gene expression but has also been observed in certain types of cells in absence of gene expression or following a block of gene expression. We show here that inhibition of macromolecular synthesis induced the rapid apoptotic death of most antibody‐secreting B‐cell hybridomas. The effect was observed in presence of both protein synthesis (cycloheximide [CHX]) and transcription (actinomycin D [Act D]) inhibitors and was characterized by extensive degradation of nucleic acids (DNA and RNA) within 2–3 h of treatment. The CHX treatment not only severely impaired the proliferation of the cells but also resulted in the loss of cell viability (MTT assay) without the need of de novo gene expression. The susceptibility to apoptosis varied among different B‐cell hybridomas and was inherited from the SP2/0 myeloma cell fusion partner. These results indicate the constitutive activation of a death program in B‐cell hybridomas and its inhibition at a late stage by the continuous expression of gene(s) coding for short‐lived protein(s). The occurrence of this phenomenon may well be related to the abundant and deregulated (trans‐location) expression, in this type of cells, of the c‐myc gene which has recently been shown to be a potent inducer of apoptosis in growth‐arrested fibroblasts. © 1993 Wiley‐Liss, Inc.
Platelet‐activating factor stimulates phosphatidic acid formation in cultured rat mesangial cells: Roles of phospholipase D, diglyceride kinase, and de novo phospholipid synthesis
Authors: Kester Mark 
Published: February 2005

AbstractPlatelet‐activating factor (PAF) stimulates phospholipase C (PLC)‐induced hydrolysis of phosphatidylinositol‐4,5‐bisphosphate (Ptdlns‐4,5‐P2). Yet, PAF‐stimulated diglycerides (DG) are still elevated at time points where inositol polyphosphates have returned to basal levels. Thus, other signal transduction pathways that hydrolyze phosphatidylcholine (PtdCho) or phosphatidylethanolamine (PtdEth) and form DG and phosphatidic acids (PA) through either PLC or phospholipase D (PLD) may also mediate PAF‐stimulated cellular responses. Initially the effects of PAF upon 32P‐PA generation in mesangial cells (MC) were assessed. PA formation may be indicative of several metabolic pathways including PLD and DG kinase activities as well as de novo phospholipid synthesis. PAF (10−7 M) increased 32P‐PA formation as early as 5 seconds and this elevation persisted up to 15 minutes. When MC were pretreated with the DG kinase inhibitor‐R59022, PAF‐induced 32P‐PA formation was diminished at early but not late time points, demonstrating that the initial component of PA formation may be due, in part, to PLC activation and subsequent phosphorylation of DG. The reciprocal reaction, PA phosphohydrolase, which dephosphorylates PA to from DG was not stimulated by PAF, suggesting that the sustained elevation of DG induced by PAF is primarily a reflection of PLC. 3H‐glycerol pulse‐labeling experiments suggest that PAF also stimulates de novo phospholipid synthesis which also contributes to PA formation. Conclusive proof for PLD in the generation of PA was obtained by assessing the formation of 3H‐phosphatidylethanol (PEt) from 3H‐alkyl‐lyso‐glycero phosphocholine (GPC) and exogenous ethanol. PAF stimulated alkyl‐PEt generation in the presence but not the absence of 0.5% ethanol. Also, PAF induced a concomitant elevation of alkyl‐PA at 15 minutes and this elevation of alkyl‐PA was reduced when the cells were exposed to exogenous ethanol, reflecting the formation of PEt. Corroborating evidence suggests that PAF stimulates 3H‐choline and 3H‐ethanolamine release, suggesting that PtdCho and PtdEth are substrates for PLD. Thus, these data demonstrate that MC respond to PAF with elevated PLD and DG kinase activities as well as with an increased rate of de novo lipid synthesis which increases PA, a potential intracellular signal. © 1993 Wiley‐Liss, Inc.
Homologous desensitization of bombesin‐induced increases in intracellular Ca2+ in quiescent swiss 3T3 cells involves a protein kinase C‐independent mechanism
Authors: Walsh John H. , Bouzyk Mark , Rozengurt Enrique 
Published: February 2005

AbstractAddition of bombesin to Swiss 3T3 cells causes a rapid and transient increase in the intracellular concentration of Ca2+ ([Ca2+]i), which is followed by desensitization to a subsequent addition of the peptide. The concentrations of bombesin used to study this acute cellular desensitization (0.1–0.5 nM) did not deplete the intracellular pool of Ca2+ released by inositol(1,4,5)trisphosphate, as shown by addition of vasopressin after consecutive additions of bombesin. Two lines of evidence support the conclusion that activation of protein kinase C (PKC) does not mediate the acute homologous desensitization of Ca2+ responses induced by bombesin. First, long‐term treatment (48 h) of Swiss 3T3 cells with phorbol 12, 13‐dibutyrate (PDB) to deplete PKC did not prevent homologous desensitization. The responses to second additions of bombesin at 0.1, 0.25, and 0.5 nM were 42%, 26% and 11% of the initial responses, respectively. Second, the PKC inhibitor GF 109203X did not alter homologous desensitization at concentrations that completely prevented the inhibition of Ca2+ mobilization induced by PDB and blocked PDB‐mediated phosphorylation of the prominent PKC substrate 80K/MARCKS. We conclude that acute homologous desensitization of Ca2+ responses induced by bombesin occurs through a PKC‐independent mechanism. © 1993 Wiley‐Liss, Inc.
Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549
Authors: Koong A. C. , Giaccia A. J. , Hahn G. M. , Saad A. H. 
Published: February 2005

AbstractActive oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves‐tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage‐dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)‐sensitive delayed rectifier channel and the other possessed characteristics of a Tea‐insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N‐Acetyl L‐Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents. © 1993 Wiley‐Liss, Inc.
Estrogen enhances epidermal growth factor‐induced DNA synthesis in mammary epithelial cells
Authors: Vanderboom Russell J. , Sheffield Lewis G. 
Published: February 2005

AbstractEstradiol (E2) priming (1 nM for 48 h) of normal murine mammary gland epithelial cells significantly increased the response of those cells to epidermal growth factor (EGF)‐induced DNA synthesis. The synergism between E2 and EGF was evident in two aspects: After serum‐free synchronization for 24 h, more cells entered the S‐phase of the cell cycle after E2 priming and when treated with 0.17 nM EGF (13%) than did control cells (1.3%) or cells treated with EGF (4%) or E2 (3.5%) alone; further, the dose of EGF required to elicit maximal response was reduced an order of magnitude in estrogen‐primed cells (0.17 nM) compared to controls (1.7 mM). Estrogen alone, however, did not increase DNA synthesis in these cells. Ligand binding studies indicate that these effects of estrogen on proliferating mammary epithelial cells may be explained, at least in part, by a 3.7‐fold increase in the number of high affinity EGF‐receptors observed in estrogen primed cells (7,300 receptors per cell) compared to estrogen deprived cells (1,960 receptors/cell). © 1993 Wiley‐Liss, Inc.
Pool of ligand‐bound platelet‐derived growth factor β‐receptors remain activated and tyrosine phosphorylated after internalization
Authors: Sorkin Alexander , Eriksson Anders , Heldin Carl‐Henrik , Westermark Bengt , Claesson‐Welsh Lena 
Published: February 2005

AbstractWe have examined the state of tyrosine phosphorylation of ligand‐bound, internalized platelet‐derived growth factor (PDGF) β‐receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF‐BB at 370C indicated colocalization of phosphotyrosine, PDGF β‐receptors, and PDGF‐BB in endosome‐like vesicles. Treatment of cells with an acidic buffer, which removed cell surfacebound PDGF‐BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF β‐receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF β‐receptor from metabolically labeled cells showed that a major fraction of the tyrosine‐phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 370C. Under these conditions, about 20‐25% of the total pool of tyrosine‐phosphorylated, receptors were intratcellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine‐phosphorylated, internalized receptors, after 10 min of incubation of the cells at 370C, could also be detected by immunoblotting analysis, using antisera against the PDGF β‐receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF β‐receptors, obtained from PDGF‐BB‐stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF β‐receptors remain active and may participate in signalling a considerable time after internalization. © 1993 Wiley‐Liss, Inc.
Packaging zinc, fibrinogen, and factor XIII in platelet α‐granules
Authors: Marx Gerard , Korner Gil , Mou Xiaode , Gorodetsky Raphael 
Published: February 2005

AbstractZirc(II) accumulated by platelets has profound effects on platelet activity. This study is focused on the distribution of Zn(II) between human platelet subcellular compartments. After incubation with 86Rb+ and platelet lysis, the organelles were separated by sucrose density gradient centrifugation. Fibrinogen served as a marker for α‐granules. 86Rb+ and factor XIII served as markers for the cytoplasmic fractions. Zn(II) was found to be distributed between the cytoplasm and the α‐granules, with variations between different individual units. The total platelet Zn concentration and its relative subcellular distribution were dependent on its extracellular level. Incubation of platelets with 100 μM Zn(II) resulted in a twofold increase of its level in the cytoplasm and by one order of magnitude in the α‐granules. In addition to the anticipated factor XIII activity in the cytoplasmic pool fraction, we found thrombin‐inducible factor XIII activity within the α‐granules. Immunoblotting confirmed the presence of both the a and b subunits of plasma factor XIII (a2b2 form) in the α‐granules. As fibrinogen is not synthesized in the platelet, we propose that by virtue of their mutual binding, fibrinogen, Zn(II) and plasma factor XIII‐a2b2 are simultaneously taken up into the α‐granules by endocytosis, presumably through the vehicle of the GPIIb/IIIa fibrinogen receptor. A rationale for copackaging these components within the α‐granules is that Zn(II) inhibits factor XIII activity and thereby prevents the premature cross‐linking of the concentrated fibrinogen prior to platelet activation and secretion. By contrast, cytoplasmic Zn(II) may increase platelet responsiveness to agonists due to its interaction with cytoplasmic modulators of platelet activity. © 1993 Wiley‐Liss, Inc.
Ligand‐dependent inhibition of myoblast differentiation by overexpression of the type‐1 insulin‐like growth factor receptor
Authors: Quinn Lebris S. , Ehsan Mitra , Steinmetz Brian , Kaleko Michael 
Published: February 2005

AbstractThe insulin‐like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2‐LISN) which expressed 7.5 times higher levels of type‐1 IGF receptors than control (P2‐LNL6) myoblasts, which were infected with a virus lacking the type‐1 IGF receptor sequence. Overexpression of the type‐1 IGF receptor caused the IGF dose‐response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF‐I concentrations, complete inhibition of P2‐LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type‐1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2‐LISN myoblasts constitutively expressed high levels of type‐1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF‐I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF‐BP) release into the culture medium. These results indicate that the type‐1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation‐inducing and differentiation‐inhibitory, which predominate at different ligand concentrations. © 1993 Wiley‐Liss, Inc.
Epidermal growth factor induces terminal differentiation in human epidermoid carcinoma cells
Authors: Konger Raymond L. , Chan Thomas C. K. 
Published: February 2005

AbstractPrevious studies have reported that the proliferation of A431 cells, a human squamous cell carcinoma cell line, was stimulated by picomolar epidermal growth factor (EGF) but inhibited by nanomolar EGF. This biphasic dose‐response phenomenon is not observed in normal human epithelial cells where nanomolar EGF is usually mitogenic. We have examined the effects of inhibitory and stimulatory concentrations of EGF on the growth and differentiation of A431 cells. In the presence of 100 pM EGF, A431 cells showed a mild increase in growth rate (129% of control) compared to cells grown in the absence of EGF. At 10 nM EGF, growth inhibition to 63% of control was observed. EGF at 10 nM stimulates a twofold increase both in cornified envelope formation and in epidermal transglutaminase activity, suggesting that high concentrations of EGF induce terminal differentiation in A431 cells. Mitogenic concentrations of EGF (100 pM) had no significant effect on these differentiation markers. Chronic exposure of A431 cells to 20 or 50 nM EGF resulted in EGF‐resistant A431 variants that are neither growth arrested nor induced to terminally differentiate by 10 nM EGF. Removal of EGF from the growth medium of the EGF‐resistant cells resulted in the reversion of these cells back to the wild‐type A431 biphasic response pattern within 2 weeks. Our results suggest that A431 cells have the capacity to non‐mutatively alter their response pattern to EGF in vitro to maintain themselves in a state of optimum proliferation and away from terminal differentiation. © 1993 Wiley‐Liss, Inc.
Characterization of a γ‐glutamyl transpeptidase positive subpopulation of endothelial cells in a spontaneous tube‐forming clone of rat cerebral resistance‐vessel endothelium
Authors: Wang Bao‐Le , Grammas Paula , De Bault Lawrence 
Published: February 2005

AbstractA spontaneous tube‐forming clone of rat cerebral resistance‐vessel endothelium was characterized in long‐term serial culture. In this study, a clone, RV‐150 ECT, of cerebral resistance vessel endothelial cells in long‐term culture has been shown to have a subpopulation of γ‐GTP positive cells that are present in all cultures regardless of confluency status or tube‐forming stages. In pre‐confluent and confluent cultures, the γ‐GTP positive cells are few in number, stain weakly, and are randomly distributed in the monolayers. In monolayer post‐confluent cultures, γ‐GTP positive cells increase in number, stain strongly, and begin to show signs of non‐random distributions. In early post‐confluent cultures that have become a mixture of monolayer and multilayer cells, there is a further increase in γ‐GTP positive cells which begin to form distinct groupings. In mid post‐confluent fultures, the multilayered areas of the culture have begun clustering to form clear multicellular aggregates. The γ‐GTP positive cells at this stage are reduced in number and are predominately associated with the cell clusters. In late postconfluent cultures, the multicellular clusters develop clear cell cords between/among the clusters. At this stage the γ‐GTP positive cells are associated exclusively with cell clustters. With cord development, the γ‐GTP positive cells are associated with bothe clusters and cords, and are reduced in number apparently because of selective degeneration of these cells. The results of this study demonstrate that a phenotypically distinct subpopulation of endothelial cells exhibits characteristic features of the blood‐brain barrier, namely γ‐GTP. The ability of these cells to express this property in long‐term serial culture suggests that this may represent a useful in vitro model to study the growth and differentiation of bloodbrain barrier vessels. © 1993 Wiley‐Liss, Inc.