bluuebunny/crossref_metadata_2025_split huggingface dataset:
Okadaic acid‐induced actin assembly in neutrophils: Role of protein phosphatases
Authors: Downey Gregory P. , Takai Akira , Zamel Ricardo , Grinstein Sergio , Chan Chi Kin 
Published: February 2005

AbstractActivation of neutrophils results in morphological and functional alterations including changes in cell shape and initiation of motile behavior that depend on assembly and reorganization of the actin cytoskeleton. Phosphoproteins are thought to be key intermediates in the regulation of cytoskeletal alterations and whereas much attention has been directed at the role of protein kinases, relatively little information is available on the importance of phosphatases. To elucidate the role of protein phosphatases, we studied the effects of the phosphatase inhibitors okadaic acid and calyculin A on the actin cytoskeleton of human neutrophils. Exposure of cells to okadaic acid resulted in assembly and spatial redistribution of actin, which peaked at 25 min and returned to baseline levels by 45 min, as assessed by flow cytometric analysis of NBD‐phallacidin stained cells and confocal fluorescence microscopy, respectively. These effects correlated with an increase in protein phosphorylation, determined by incorporation of 32P into cellular proteins using SDS‐PAGE and autoradiography. Similar but more rapid responses were observed in electropermeabilized cells treated with okadaic acid or calyculin A. The dose dependence of these effects was compatible with a role for phosphatase type 1 as the target enzyme. These findings also suggested the presence of constitutively active protein kinases capable of effecting actin polymerization. Phosphorylation of myosin light chain (MLC) has been postulated to promote actin assembly, but myosin light chain kinase (MLCK) appeared not to be involved because: (1) the effect of okadaic acid was not inhibited by the MLCK inhibitor KT5926 and (2) in permeabilized cells suspended in medium with free calcium [Ca2+] < 10 nM (conditions under which MLCK is inactive), the effect of okadaic acid persisted. The role of phosphatases in stimulus‐induced actin assembly was assessed in cells preincubated with okadaic acid for 45 min, after F‐actin levels had returned to baseline. Under these conditions, okadaic acid completely abrogated actin assembly induced by phorbol myristate acetate, platelet activating factor, and leukotriene B4, whereas the effects of the chemotactic peptide fMLP and opsonized zymosan (OpZ) were unaffected. We conclude that serine and threonine phosphatases exert a tonic negative influence on actin assembly and organization. Furthermore, divergent pathways seem to mediate the response to lipidic stimuli, on one hand, and fMLP and OpZ, on the other, as evidenced by the differential susceptibility to inhibition by okadaic acid. © 1993 Wiley‐Liss, Inc.
Mitogen‐activated Ca++ channels in human B lymphocytes
Authors: Brent Lawrence H. , Gong Qihua , Ross James M. , Wieland Steven J. 
Published: February 2005

AbstractTwo complementary experimental methods have been used to examine mitogen‐induced transmembrane conductances in human B cells using the Daudi cell line as a model for human B cell activation. Spectrofluorometry was used to investigate mitogen‐induced changes in [Ca++]i and transmembrane potential. Activation of human B cells with anti‐μ antibodies resulted in a biphasic rise in [Ca++]i, the second phase being mediated by the influx of extracellular Ca++. Ca++ influx was inhibited by high [K+]e, suggesting that this influx was transmembrane potential sensitive. Membrane currents of Daudi cells were investigated using voltage clamp techniques. Before mitogenic stimulation, the cells were electrically quiet. Within several minutes of the addition of anti‐μ antibodies to the bath solution, inward currents were observed at negative voltages. Whole‐cell currents changed instantly with voltage steps and were transmembrane potential sensitive in that at potentials more positive than −40 mV no currents were detectable. A similar conductance was also activated by the introduction of IP3 into the intracellular solution, suggesting that IP3 generation after surface IgM crosslinking is involved in the activation of this conductance. Both anti‐μ and IP3 induced currents were blocked by 1 mM La+++, which is known to block Ca++ channels. These results strongly support the presence of membrane Ca++ channels in human B cells that function in the early stages of activation. Changes in transmembrane potential appear to be important in regulating Ca++ influx. These mechanisms work in concert to regulate the level of [Ca++]i during the early phases of human B cell activation. © 1993 Wiley‐Liss, Inc.
Regulation of insulin‐like growth factor‐binding‐protein‐1, 2, 3, 4, 5, and 6: Synthesis, secretion, and gene expression in estrogen receptor‐negative human breast carcinoma cells
Authors: Sheikh M. Saeed , Shao Zhi‐Ming , Hussain Arif , Clemmons David R. , Chen Jian‐Chyi , Roberts Charles T. , Leroith Derek , Fontana Joseph A. 
Published: February 2005

AbstractInsulin‐like growth‐factors I and II (IGF‐I, II) are potent mitogens for breast carcinoma proliferation. In extracellular fluids, most of the IGF‐I and II is associated with specific IGF‐binding proteins (IGFBPs). The role of these IGFBPs in IGF action is still not clear, but it has been demonstrated that these proteins may either enhance or inhibit IGF‐mediated cellular effects. Synthesis and secretion of IGFBPs have been demonstrated in breast carcinoma cells. In this study, we examined retinoic acid (RA) and IGF‐I modulation of IGFBP mRNA and IGFBP levels in two ER‐negative human breast carcinoma cell lines. Treatment of MDAMB‐231 and MDA‐MB‐468 cells with RA increased the levels in conditioned media of a Mr 42‐46‐kDa IGFBP, which was immunoprecipitated by an IGFBP‐3 antibody. IGF‐I also increased the accumulated levels of IGFBP‐3 in the conditioned media of both cell lines. Both cell lines expressed high basal levels of IGFBP‐3 mRNA; the addition of RA increased IGFBP‐3 mRNA levels by 1.5‐fold, whereas the addition of IGFI had no effect on IGFBP‐3 mRNA levels in either cell line. The difference in the magnitude of the RA enhancement of IGFBP‐3 mRNA levels (1.5‐fold) and RA stimulation of IGFBP‐3 levels in conditioned media (3.5–4‐fold) suggests that some of the effect of RA is at a posttranscriptional level. IGF‐I increased the levels of IGFBP‐2 and IGFBP‐5 in conditioned media by greater than tenfold but had no effect on IGFBP‐2 and IGFBP‐5 mRNA levels, again suggesting the involvement of posttranscriptional controls. Pretreatment of MDA‐MB‐468 and MDA‐MB‐231 cells with IGF‐I receptor antibody (αIR3) blocked the IGF‐I effect on IGFBP‐3 levels in the media in both cell lines and IGFBP‐2 and IGFBP‐5 secreted levels in MDA‐MB‐468 cell conditioned media. The addition of RA also blocked IGF‐I stimulation of IGFBP2 and IGFBP‐5 levels. Cycloheximide treatment completely blocked the RA and/or IGF‐I‐mediated modulation of these binding proteins, suggesting that these agents enhance IGFBP‐3, IGFBP‐2, and IGFBP‐5 synthesis and consequent secretion. MDA‐MB‐468 cells expressed IGFBP‐5 mRNA, whereas both MDA‐MB‐231 and MDA‐MB‐468 expressed IGFBP‐6 mRNA. RA enhanced IGFBP‐6 gene expression by threefold in MDA‐MB‐231 cells, whereas IGF‐1 had no effect on IGFBP‐6 gene expression in either cell line. These results demonstrate that RA and IGF‐I modulate IGFBP levels in a number of breast carcinoma cell lines. Such modulation of IGFBPs may in turn affect IGF mediated cellular responses and thus the biologic behavior of these malignant cells.
Isolation of a temperature‐sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment
Authors: McGraw Timothy E. , Dunn Kenneth W. , Maxfield Frederick R. 
Published: February 2005

AbstractWe have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)‐ricin and Tf‐diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with mor‐phologically aberrant endocytic phenotypes. One of the cell lines identified, B104‐5, has a striking temperature‐induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40°C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild‐type endocytic compartment is closely aligned with the microtubule‐organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104‐5 receptor recycling compartment morphology can be phenocopied in wild‐type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104‐5 cells appears normal in immunofluorescent staining. B104‐5 cells, unlike the parental cells, do not proliferate at 40°C. The mutation in B104‐5 cells is recessive, as fusion with wild‐type cells results in a reversion of the B104‐5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104‐5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines. © 1993 Wiley‐Liss, Inc.
Transforming growth factor‐β1 stimulates macrophage urokinase expression and release of matrix‐bound basic fibroblast growth factor
Authors: Falcone Domenick J. , McCaffrey Timothy A. , Haimovitz‐Friedman Adriana , Garcia Michelle 
Published: February 2005

AbstractMacrophage expression of urokinase‐type plasminogen activator (uPA) appears to play a role in their release of matrix‐bound basic fibroblast growth factor (bFGF) and transforming growth factor‐β (TGF‐β). In experiments reported here, we have examined the potential regulatory effects of bFGF and TGF‐β1 on macrophage uPA expression. TGF‐β1 stimulated in a dose‐ and time‐dependent manner the expression of secreted membrane and intracellular uPA activities by a macrophage cell line (RAW264.7). When examined at similar concentrations, bFGF had little effect, and interleukin‐1α, tumor necrosis factor‐α, and monocyte colony stimulating factor had no effect on macrophage uPA expression. Exposure of macrophages to TGF‐β1 led to a rapid and sustained increase in the steady‐state levels of uPA mRNA that was independent of de novo protein synthesis and was completely inhibited by actinomycin D. However, the TGF‐β1‐induced increase in uPA mRNA was largely unaffected by subsequent incubation of cells with actinomycin D. The protein kinase C inhibitior H7 markedly reduced the ability of TGF‐β1 to stimulate expression of uPA activity. Likewise, okadaic acid and microcystin, inhibitors of serine/threonine phosphatases, potentiated the ability of TGF‐β1 to upregulate macrophage uPA expression. TGF‐β1 primed cells converted nearly all added plasminogen to plasmin and expressed sixfold more membrane‐bound plasmin than control cells. Preincubation of TGF‐β1 with either serum or methylamine‐modified α2‐macroglobulin did not affect its ability to induce macrophage uPA expression. When control and TGF‐β1‐primed macrophages were cultured on matrices containing bound125I‐bFGF, their release of 125I‐bFGF was increased five and tenfold, respectively, in the presence of plasminogen. The ability of TGF‐β to induce macrophage uPA expression and the plasmin‐dependent release of matrix‐bound bFGF may provide an indirect mechanism by which TGF‐β stimulates angiogenesis. © 1993 Wiley‐Liss, Inc.
Cellular resistance to oxidative stress is accompanied by resistance to cisplatin: The significance of increased catalase activity and total glutathione in hydrogen peroxide‐resistant fibroblasts
Authors: Spitz Douglas R. , Phillips John W. , Adams Donna T. , Sherman C. Michael , Deen Dennis F. , Li Gloria C. 
Published: February 2005

AbstractStudies designed to better understand the involvement of cellular resistance to oxidative stress in mechanisms of cellular resistance to cisplatin were undertaken using H2O2‐resistant variants of the HA 1 Chinese hamster fibroblast cell line. H2O2‐resistant cell lines were resistant to clonogenic inactivation mediated by cisplatin with dose modifying factors at 10% survival of 1.5–3.0, relative to HA 1 cells. The most cisplatin resistant of these cell lines (OC5) also demonstrated fewer DNA–DNA crosslinks induced by cisplatin, relative to HA 1. Since H2O2‐resistant cells contained increased catalase activity as well as total glutathione (GSH) content, the involvement of these cellular antioxidants in the resistance to cisplatin toxicity was evaluated. Treatment of HA 1 and H2O2‐resistant cell lines (OC5, OC14) with 9 mM aminotriazole reduced catalase activity by 60–65% but had no effect on the cytotoxicity of cisplatin. In contrast, treatment with 5 mM buthionine sulfoximine reduced total GSH by 90% and sensitized the cells to cisplatin cytotoxicity. Furthermore, extracellular reaction of GSH with cisplatin prior to treating HA 1 cells reduced the toxicity of the compound, indicating that this reaction is capable of participating in the detoxification of cisplatin. These results indicate that cellular adaptation to oxidative stress renders cells resistant to DNA damage as well as to cytotoxicity associated with cisplatin treatment. Furthermore, increases in total GSH content (but not catalase activity) appear to partially account for cisplatin resistance demonstrated by H2O2‐resistant cells. © 1993 Wiley‐Liss, Inc.
Decrease in cellular replicative potential in “giant” mice transfected with the bovine growth hormone gene correlates to shortened life span
Authors: Pendergrass William R. , Li Yi , Jiang DeZhao , Wolf Norman S. 
Published: February 2005

AbstractAdult mice, (C57BL/6 × Sjl)F1 hybrids, transfected with the bovine growth hormone gene (bGH) grow to twice normal size, but have a mean life span less than 50% that of control siblings without the transgene. The replicative potentials of cells from six different tissue sites (tail skin and ear skin dermal fibroblasts, tail subdermal connective tissue fibroblasts, kidney medulla epithelial cells, bone marrow myofibroblasts, and spleen myofibroblasts) were assayed in vitro using clone size distribution analysis. Cells from all of the above bGH+ tissues produced a smaller fraction of large clones, relative to age‐matched controls, in all of these cell types. The loss of replicative potential did not appear to be the result of negative conditioning of the cloning media by the bGH+ cells, and was tightly correlated to the period of accelerated growth in these animals (3–12 weeks), a time when additional GH receptors are expressed. © 1993 Wiley‐Liss, Inc.
Novel action of transforming growth factor β1 in functioning human pancreatic carcinoid cells
Authors: Ishizuka Jin , Beauchamp R. Daniel , Sato Kazuo , Townsend Courtney M. , Thompson James C. 
Published: February 2005

AbstractWe have shown recently that 5‐HT is an autocrine growth stimulatory factor for a cell line (BON) that is derived from a human pancreatic carcinoid tumor. This action is mediated by a 5‐HT receptor‐linked decrease of cyclic adenosine monophosphate (AMP) production, but not mediated by a 5‐HT receptor‐linked stimulation of phosphatidylinositol hydrolysis. The BON cells also express transforming growth factor betas (TGFβs) (1, 2, and 3) and release TGFβ into their medium. In this study, we examined the effects of TGFβ1 on the secretion of 5‐HT, on signal transduction pathways involved in 5‐HT secretion, and on growth of BON cells. TGFβ1 inhibited basal and acetylcholine‐stimulated release of 5‐HT, but did not inhibit isobutylmethylxanthine‐stimulated release of 5‐HT. TGFβ1 inhibited both basal and acetylcholine‐stimulated hydrolysis of phosphatidylinositol in a dose‐dependent manner, but did not affect cyclic AMP production. TGFβ1 inhibited growth of BON cells in culture; this effect was reversed by exogenously administered 5‐HT. Three different specific and saturable TGFβ1 binding sites were identified; binding assays performed after mild acid wash (0.1% acetic acid, pH 2.5) conditions uncovered TGFβ receptors that were apparently occupied by endogenously produced TGFβ species. Affinity cross‐linking assay showed that BON cells had three different TGFβ binding proteins. These results suggest that TGFβ1 can inhibit growth of BON cells by altering secretory responses of 5‐HT by means of receptor‐mediated inhibition of phosphatidylinositol hydrolysis. We conclude that growth of BON cells is regulated, at least in part, by the opposing receptor‐mediated autocrine actions of 5‐HT and TGFβ. © 1993 Wiley‐Liss, Inc.
Receptors for Escherichia coli heat stable enterotoxin in human intestine and in a human intestinal cell line (Caco‐2)
Authors: Cohen Mitchell B. , Jensen Nancy J. , Hawkins Jennifer A. , Mann Elizabeth A. , Thompson Michael R. , Lentze Michael J. , Giannella Ralph A. 
Published: February 2005

AbstractEscherichia coli heat stable enterotoxin (STa) and the newly identified endogenous ligand guanylin bind to an intestinal receptor and activate membrane bound guanylate cyclase. We compared STa binding and affinity crosslinking of STa receptors in human small intestine to those in the Caco‐2 human colon carcinoma cell line. STa had similar kinetics of binding in human intestinal and Caco‐2 brush border membranes. In both human intestine and Caco‐2 brush border membranes, multiple specifically radiolabeled bands, including a 140–165 kDa band, were identified by affinity crosslinking. However, in human intestine the most prominent autoradiographic species was a 60 kDa band. A 60 kDa protein was also specifically immunoprecipitated from solubilized human brush border membranes using antisera raised against a cloned STa receptor fusion protein. Our observations of multiple crosslinked proteins in human intestine and Caco‐2 cells could be explained by the existence of several members of a family of STa receptors and/or the existence of smaller STa binding proteins generated by the protease cleavage of a larger complete STa receptor. © 1993 Wiley‐Liss, Inc.
Activation of phospholipase C by heat shock requires GTP analogs and is resistant to pertussis toxin
Authors: Calderwood Stuart K. , Stevenson Mary Ann , Price Brendan D. 
Published: February 2005

AbstractThe heat shock response in mammals consists of a complex array of intracellular reactions initiated by stress, although its regulation is poorly understood. We have investigated the role of transmembrane signal transduction in the response, examining mechanisms involved in the activation of phospholipase C (PLC) by heat shock. In rodent fibroblasts permeabilized with digitonin, heat shock and receptor‐mediated PLC activity exhibited a strict GTP analog dependency. This indicates that heat shock‐mediated phopholipase activation, in common with receptor mediated stimulation, does not involve direct effects on the phospholipases and suggests the participation of GTP binding (G) proteins in the activation process. When cells were treated with the inhibitor pertussis toxin (PTX), the phospholipases retained their inducibility by heat shock, but became refractory to thrombin treatment, indicating that heat shock may influence PLC activity through a distinct population of G proteins compared to thrombin. The data seem to exclude a role for PTX sensitive G proteins in the production of IP3 after heating and suggest a pathway involving the direct thermal activation of the Gq class of G proteins, which are coupled to the PLCβ1 isoform. © 1993 Wiley‐Liss, Inc.
Heat shock in human neutrophils: Superoxide generation is inhibited by a mechanism distinct from heat‐denaturation of NADPH oxidase and is protected by heat shock proteins in thermotolerant cells
Authors: Maridonneau‐Parini Isabelle , Malawista Stephen E. , Stubbe Hiltrud , Russo‐Marie Françloise , Polla Barbara S. 
Published: February 2005

AbstractIndependently of the stimulating agent used, generation of O2− by human neutrophils is transiently inhibited when the cells have been exposed to elevated temperatures. This phenomenon is concomitant with the synthesis of heat shock proteins (HSPs). We have investigated a possible relationship between HSPs and modulation of NADPH oxidase activity in human neutrophils exposed to heat. HSPs were not involved in the inhibition of O2− generation since 1) in enucleated cytoplasts, which are unable to synthesize proteins, the generation of O2− was inhibited after exposure to 43°C, 2) using actinomycin D (Act D) in intact cells, it was possible selectively to inhibit the synthesis of HSPs without modifying the inhibition of NADPH oxidase activity that followed HS. Furthermore, the recovery of NADPH oxidase activity was not under the control of HSPs because the enzyme recovered as well in Act D‐treated neutrophils. The NADPH oxidase activity was reconstituted in a cell‐free assay by combining the cytosol with the plasma membrane‐enriched fraction in the presence of arachidonic acid (AA) and NADPH. Subcellular fractions obtained from control or heated neutrophils exhibited similar oxidase activities suggesting that heat exposure did not induce denaturation of the oxidase components but rather altered the mechanisms of translocation and/or assembly of these components with the plasma membrane. This hypothesis was supported by the inhibition of the granule release in heated cells, a process which also requires translocation and association fusion with the plasma membrane. On the other hand, preexposure of neutrophils to HS prevented the inhibition of O2− generation during a second challenging HS. This acquired thermotolerance was abolished when the synthesis of HSPs was inhibited during the first HS with Act D, indicating a direct relationship between protection of O2− generation and synthesis of HSP. Here we demonstrate that synthesis of HSPs and inhibition or recovery of NADPH oxidase activity are concomitant but unrelated phenomena. In contrast, accumulation of HSPs in thermotolerant neutrophils appeared to play an important role in the prevention of NADPH oxidase inhibition. These results provide further insights into the behavior of human neutrophils and NADPH oxidase upon heat injury. © 1993 Wiley‐Liss, Inc.