bluuebunny/crossref_metadata_2025 huggingface dataset:
Overexpression of manganous superoxide dismutase (MnSOD) in pulmonary endothelial cells confers resistance to hyperoxia
Authors: Lindau‐Shepard Barbara , Shaffer Jacquelin B. , Del Vecchio Peter J.
Published: February 2005

Treatment of cells or organisms with agents that increase the expression of MnSOD confers resistance to certain types of oxidative damage. However, since these treatments also affect other cellular systems with antioxidant capabilities, the role of MnSOD remains uncertain. To better determine whether increased MnSOD expression confers increased resistance to oxidant stress, a eukaryotic expression vector harboring a mouse MnSOD cDNA was constructed. Bovine lung microvessel endothelial cells were co‐transfected with the MnSOD expression vector and pSV2‐neo, which contains the neo' gene and provides a dominant selectable marker. Control clones were generated by transfecting the cells with pSV2‐neo alone. Stably transfected cell lines were selected and cell lines overexpressing MnSOD were confirmed by Northern blotting, immunoblot analysis, and activity gels. The activities of copper/zinc superoxide dismutase, catalase, and glutathione peroxidase were examined, and no increase in activity of any of these enzymes was detected. Cells were exposed to hyperoxic challenge by treatment with 95% O2 and 5% CO2 for 24 h. Viability was assessed by a clonogenic assay. The cell lines that overexpressed MnSOD showed a twofold increase in survival compared to control cells. These results demonstrate a significant resistance to hyperoxia induced oxidative stress in endothelial cells overexpressing MnSOD. © 1994 Wiley‐Liss, Inc.
Reorganization of endothelial cord‐like structures on basement membrane complex (Matrigel): Involvement of transforming growth factor β1
Authors: Kuzuya Masafumi , Kinsella James L.
Published: February 2005

The formation of capillary‐like network structures by cultured vascular endothelial cells on reconstituted basement membrane matrix, Matrigel, models endothelial cell differentiation, the final step of angiogenesis (Kubota et al., 1988; Grant et al., 1989). When endothelial cells derived from bovine aorta and brain capillaries were plated on Matrigel, DNA synthesis was suppressed and a network of capillary‐like structures rapidly formed in 8–12 h. With time, the network broke down, resulting in dense cellular cords radiating from multiple cellular clusters in 16–24 h. Finally, multicellular aggregates of cells were formed as the network underwent further retraction. Network regression was prevented when either dithiothreitol (DTT) or anti‐TG F‐β1 antibodies were added during the assay. The addition of exogenous TGF‐β1 promoted the regression of endothelial cells into the clusters. This response to TGF‐β1 was blocked by potent serine threonine protein kinase inhibitors, H‐7 and HA 100. TGF‐β1 was released from polymerized Matrigel by incubation with Dulbecco's modified eagle's medium (DMEM) in the absence of cells. The Matrigel‐conditioned DMEM inhibited endothelial DNA synthesis even in the presence of anti‐TGF‐β1 antibodies. These results suggest that TGF‐β1 and possibly other soluble factors from Matrigel may be important for differentitation and remodeling of endothelial cells in a capillary network with possible implications for wound healing and development. © 1994 Wiley‐Liss, Inc.

This article is a US Government work and as such, is in the public domain in the United States of America

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Differential effects of transforming growth factor‐β1 on distinct developmental stages of murine megakaryocytopoiesis
Authors: Jackson Heather , Williams Neil , Westcott Keith R. , Green Ralph
Published: February 2005

The effect of transforming growth factor‐β1 (TGFβ1) on three developmental stages of megakaryocytopoiesis was investigated. Using a murine bone marrow agar culture system, titrated doses of TGFβ1 were added to cultures assaying primitive high proliferative megakaryocyte progenitors, committed megakaryocyte precursors, and nondividing, endoreduplicating megakaryocytes. The growth of high proliferative megakaryocyte colony‐forming cells (HPP‐CFU‐Mk) that require the growth factors interleukins‐1, 3 and 6 (IL‐1 + IL‐3 + IL‐6) for colony detection was abrogated by the addition of 1 ng TGFβ1/ml. The sensitivity of committed megakaryocyte progenitors (colony‐forming unit‐megakaryocyte, CFU‐Mk) to TGFβ1 depended on the growth factor combination. TGFβ1 (1 ng/ml) completely inhibited megakaryocyte colony formation from CFU‐Mk only in cultures stimulated by low doses of IL‐3. TGFβ1 (> 10 ng/ml) could only marginally inhibit megakaryocyte colony forrmation generated in the presence of either high doses of IL‐3 or the combination of low dose IL‐3 + IL‐6. TGFβ1 inhibited both IL‐3‐dependent and IL‐6‐dependent megakaryocyte growth but tenfold higher doses of TGFβ1 were required to inhibit growth generated by the combination of IL‐3 + IL‐6. The data showed that the capacity of TGFβ1 to inhibit distinct differentiation stages of the megakaryocytopoietic lineage depended on the concentration and combination of growth factors involved. © 1994 Wiley‐Liss, Inc.
Induction of Bcl‐2 gene expression by intercellular information from hemopoietic supportive stromal cells to DA‐1 cells
Authors: Takashita Emi , Sugimoto Kenkichi , Adachi Yasuhiro , Mori Kazuhiro J.
Published: February 2005

When Interleukin‐3 (IL‐3) dependent DA‐1 cells were cultured on hemopoietic supportive stromal cells (MS‐5), DA‐1 cells survived and proliferated in the absence of detectable IL‐3. Although IL‐3 was not produced by the MS‐5 cells, their production of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was increased when they were co‐cultured with DA‐1 cells. This suggests that DA‐1 cells transmit signals to stromal cells that enhance growth factor(s) production. Expression of bcl‐2 by DA‐1 cells was induced when they were co‐cultured with MS‐5 cells, suggesting that DA‐1 cells express bcl‐2 strongly in response to a signal produced by MS‐5 cells. These data indicate the existence of a two‐way interaction between DA‐1 cells and hemopoietic supportive stromal cells. © 1994 Wiley‐Liss, Inc.
Endothelin secretion is regulated by cyclic AMP and phosphatase 2A in endothelial cells
Authors: Magnusson Andres , Halldorsson Haraldur , Thorgeirsson Gudmundur , Kjeld Matthias
Published: February 2005

Endothelin is a 21 amino acid peptide secreted by endothelial cells and is the most potent vasoconstrictor known. The present study examines regulatory mechanisms of endothelin secretion, focusing on the role of protein phosphorylation. Endothelin secretion was measured by radioimmunoassay in primary cultures of human umbilical vein endothelial cells. While treatment that raised cAMP levels reduced the basal endothelin secretion rate, agents that elevated cGMP had no effect. Downregulation or inhibition of protein kinase C resulted in decreased endothelin secretion, suggesting that protein kinase C regulates endothelin secretion in the opposite direction to cAMP dependent protein kinases Okadaic acid, at concentrations that selectively inhibit protein phosphatases 2A, reduced the endothelin secretion and the effects of okadaic acid and db‐cAMP were additive. Endothelin production was stimulated by fetal calf serum and by the protein kinase inhibitor 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine (H7), but was inhibited by the calmodulin antagonist trifluoperazine. The present findings that regulators of cAMP‐dependent protein kinases, protein kinase C, calmodulin, and protein phosphatase 2A all affect endothelin secretion suggest that endothelin secretion is controlled by phosphorylation/dephosphorylation of as yet unidentified regulatory proteins within the cell. © 1994 Wiley‐Liss, Inc.
c‐Ha‐rasEJ transfection of rat aortic smooth muscle cells induces epidermal growth factor responsiveness and characteristics of a malignant phenotype
Authors: Sadhu D. N. , Lundberg M. S. , Burghardt R. C. , Ramos K. S.
Published: February 2005

Although the role of several protooncogenes, including sis, myc, and myb in the regulation of growth and differentiation of vascular cells has been examined in some detail, limited information is available on the contribution of ras genes to these processes. In the present studies the influence of oncogenic ras transfection on the phenotypic expression of rat aortic smooth muscle cells (SMCs) was examined. Cultured rat aortic SMCs during early passage (P4) were transfected by lipofection with c‐Ha‐rasEJ in a pSV2 neo vector or with pSV2 neo vector alone. Stable transfectants were selected in G418 over a 6‐week period. Oncogene‐transfected cells (ras‐LF‐1) exhibited differences in morphology and growth pattern relative to vector controls (neo‐LF‐1), or naive SMCs, including the development of prominent processes and the appearance of focal cellular arrangements giving rise to latticelike structures. Southern analysis revealed multiple integration of oncogenic ras in ras LF‐1 cells. Transfection of c‐Ha‐rasEJ was associated with a twofold increase in p21 levels relative to pSV2 vector controls demonstrating that exogenous ras was expressed in these cells. Overexpression of ras p21 afforded SMCs a lower serum requirement for growth compared to vector controls, anchor‐age independent growth on soft agar, and acquisition of epidermal growth factor (EGF) responsiveness. Stimulation of serum‐deprived SMCs with 5% fetal bovine serum (FBS) increased steady‐state levels of c‐Ha‐ras mRNA in both ras‐LF‐1 and neo‐LF‐1 but ras induction was more pronounced in ras‐transfected cells. α‐smooth muscle (SM) actin gene expression was markedly reduced in ras‐transfected cells relative to vector controls. These results show that transfection of c‐Ha‐rasEJ into aortic SMCs induces an altered phenotypic state characterized by alterations in growth factor‐related signal transduction and tumorigenic potential. © 1994 Wiley‐Liss, Inc.
Osteogenic protein‐1, a bone morphogenic protein member of the TGF‐β superfamily, shares chemotactic but not fibrogenic properties with TGF‐β
Authors: Postlethwaite Arnold E. , Raghow Rajendra , Stricklin George , Ballou Leslie , Sampath T. Kuber
Published: February 2005

We have previously shown that recombinant human osteogenic protein‐1 (rhOP‐1), a bone morphogenetic protein member of the TGF‐β superfamily, can induce new bone formation when implanted with an appropriate carrier at subcutaneous sites in rats and can restore completely large diaphyseal segmental defects in laboratory animals. The role of OP‐1 in the early events of bone induction viz, chemotaxis of phagocytic leukocytes, and fibroblastic mesenchymal cells is currently unknown. In the present study, we examined the effect of rhOP‐1 on chemotaxis of phagocytic leukocytes (human neutrophils and monocytes) and fibroblastic mesenchymal cells (infant foreskin fibroblasts). Since OP‐1 is structurally related to TGF‐β1, we assessed the effects of OP‐1 on several other fibroblast functions (in addition to chemotaxis) known to be modulated by TGF‐β1. Our results demonstrated that rhOP‐1, like TGF‐β1, is a potent chemoattractant for human neutrophils, monocytes, and fibroblasts. However, in contrast to TGF‐β1, OP‐1 does not to stimulate fibroblast mitogenesis, matrix synthesis [collagen and hyaluronic acid (hyaluronan)], or production of tissue inhibitor of metalloproteinase (TIMP), i.e., fibroblast functions associated with fibrogenesis. These results clearly demonstrate a dichotomy between these two members of the TGF‐β superfamily with regard to fibrogenic effects on fibroblasts but a similarity in their chemotactic properties. © 1994 Wiley‐Liss, Inc.
Effect of membrane potential on surface Ca2+ receptor activation in rat osteoclasts
Authors: Shankar Vijai S. , Huang Christopher L.‐H. , Adebanjo Olugbenga , Simon Bruce , Alam A. S. M. Towhidul , Moonga Baljit S. , Pazianas Michael , Scott Roderick H. , Zaidi Mone
Published: February 2005

Osteoclasts are known to possess a divalent cation‐sensitive receptor, the Ca2+ receptor (CaR). The latter monitors changes in the local Ca2+ concentration generated as a result of hydroxyapatite dissolution. CaR activation elevates cytosolic [Ca2+] and thereby inhibits osteoclastic bone resorption. Recent studies have used Ni2+ as a surrogate CaR agonist to elicit changes in cytosolic [Ca2+]. This article examines the effects of membrane potential changes on the kinetics of the cytosolic [Ca2+] signal resulting from such Ni2+‐induced CaR activation. Membrane potential was altered through variations in the extracellular [K+] in combination with applications of the K+ ionophore, valinomycin. Membrane potential changes were confirmed by independent electrophysiological patch clamp studies of whole osteoclasts. The application of valinomycin produced a distinct, sustained elevation of cytosolic [Ca2+] in single fura 2‐loaded cells, a “primary” response. This response was independent of valinomycin concentration (between 5 nM to 5 μM) and persisted in Ca2+‐free, EGTA‐containing solutions. It also persisted both in high (105 mM) and low (5 mM) extracellular [K+]. A gradual “secondary” elevation of cytosolic [Ca2+] then followed with the continued application of valinomycin, but this was eliminated by sequestering the extracellular [Ca2+] or by increasing extracellular [K+] from 5 to 105 mM. In a separate set of experiments, the presence of 5 μM [valinomycin]‐([K+] = 5 mM) prolonged the cytosolic [Ca2+] signal elicited by 50 μM‐[Ni2+] application. These prolonged kinetics persisted in low extracellular [Ca2+] (zero‐added Ca2+), but reverted to a rapid time‐course in the presence of 105 mM‐[K+] or at higher [Ni2+] (500 μM and 5 mM). The experiments thus indicate that membrane voltage modifies the kinetics of CaR activation by Ni2+ and therefore suggests that the CaR is an integral protein in the osteoclast surface membrane. © 1995 Wiley‐Liss, Inc.
Chloride is required for receptor‐mediated divalent cation entry in mesangial cells
Authors: Kremer Sidney G. , Zeng Wenjia , Hurst Roger , Ning Terri , Whiteside Catharine , Skorecki Karl L.
Published: February 2005

Agonists which stimulate the inositol 1,4,5 trisphosphate ([1,4,5]‐IP3)‐dependent mobilization of Ca2+ from intracellular stores also stimulate entry of divalent cations across the cell membrane. Under appropriate experimental conditions, divalent cation entry across the cell membrane can be monitored as the rate at which the intracellular fluorescence of divalent cation indicators is quenched by the addition of Mn2+ to the extracellular medium. We report that addition of vasopressin to fura‐2‐loaded glomerular mesangial cells in culture markedly accelerated the rate at which Mn2+ quenched fura‐2 fluorescence at its Ca2+‐insensitive wavelength in the presence of extracellular NaCl, but that this quench response was attenuated when Cl− was removed from the extracellular medium by equimolar substitution with impermeant anions (gluconate, methanesulfonate, acetate, lactate). Similarly, loss of agonist‐induced quench also occurred when Cl− was substituted with gluconate in K+‐containing media. Addition of the Cl− channel inhibitor, 5‐nitro‐2‐(3‐phenylpropylaminobenzoic acid) (NPPB), also inhibited Mn2+‐induced quench of fura‐2 fluorescence following vasopressin addition. In contrast, in the presence of gramicidin to provide an alternate conductance pathway to accompany divalent cation entry, agonist‐dependent Mn2+ quench occurred even in the absence of extracellular Cl−, indicating that the requirement for Cl− was not the result of cotransport on a common transporter nor the result of Cl− serving as a necessary cofactor for divalent cation entry. A similar dependence on extracellular Cl− was observed for other Ca2+‐mobilizing agonists such as endothelin, as well as the intracellular Ca2+ ATPase inhibitor, thapsigargin. Extracellular Cl− dependence for agonist‐induced divalent cation entry was also reflected in a corresponding extracellular Cl− dependence for agonist‐induced mesangial cell contraction. It has been previously shown by ourselves (Kremer et al., 1992a, Am. J. Physiol., 262:F668–F678) and others that agonist‐stimulated calcium mobilization in mesangial cells is accompanied by inhibition of K+ conductance and increased Cl− conductance. Accordingly, we conclude that the current findings suggest that activation of Cl− conductance provides regulated charge compensation for receptor‐mediated divalent cation entry in response to Ca2+‐mobilizing vasoconstrictor agonists in mesangial cells. © 1995 Wiley‐Liss, Inc.
Mitotic cycle reactivation in terminally differentiated cells by adenovirus infection
Authors: Crescenzi Marco , Soddu Silvia , Tatò Franco
Published: February 2005

Different cell types (e.g., neurons, skeletal and heart myocytes, adipocytes, keratinocytes) undergo terminal differentiation, in which acquisition of specialized functions entails definitive withdrawal from the cell cycle. Such cells are distinct from quiescent (reversibly growth‐arrested) cells, such as contact‐inhibited fibroblasts. Terminally differentiated cells can not be induced to proliferate by means of growth factor stimulation or transduction of cellular oncogenes. An important first step toward defining the molecular basis for such unresponsiveness is to find a practical means to overcome the proliferative block. Furthermore, determining whether terminally differentiated, postmitotic cells still retain a potential competence for proliferation that can be reactivated would have important theoretical and practical implications. To address these questions, we exploited the properties of adenoviruses. These viruses can infect postmitotic cells and express E1A, a powerful activator of proliferation in reversibly growth‐arrested cells. We infected terminally differentiated skeletal muscle cells and adipocytes with human adenovirus type 5 or 12, obtaining full reentry into the cell cycle, including DNA synthesis, mitosis, cytokinesis, and extended proliferation. Similar results were obtained with established cell lines and primary cells belonging to several species, from quail to humans. Genetic analysis indicated that the smaller splice product of E1A, E1A 12S, is sufficient to induce cell cycle reactivation in otherwise permanently nonmitotic cells. These results demonstrate that terminally differentiated cells retain proliferative potential and establish adenovirus as a convenient and powerful means to force such cells to reenter the cell cycle. © 1995 Wiley‐Liss, Inc.
Expression of a voltage‐dependent potassium current precedes fusion of human muscle satelite cells (myoblasts)
Authors: Widmer H. , Hamann M. , Baroffio A. , Bijlenga P. , Bader C. R.
Published: February 2005

Using the whole‐cell recording patch clamp tehnique in clonal cultures of human muscle satellite cells (SC), we studied a voltage‐gated potassium current analogous to the delayed rectifier current (Ikdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed Ikdr In contrast, Ikdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum‐free medium (SFM) and within 24 h in differetiating medium. We belive that Ikdr may be a characteristic feature of fusion‐component SC and that it may be involved in the fusion process for the following resons: (1) after the transfer in differentiating medium, cultures of SC in which the expression of Ikdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; (2) in the latter “naive” SC, Ikdr was expressed during the first day in differentiating medium, before SC began to fuse; (3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express Ikdr; (4) TEA, at a concentration of 3 mM, reduces the amplitude of Ikdr by 55% and the fusion index by 55–67%. © 1995 Wiley‐Liss, Inc.
EGF receptor‐mediated signals are differentially modulated by concanavalin A
Authors: Hazan Rachel , Krushel Leslie , Crossin Kathryn L.
Published: February 2005

NIH 3T3 cells expressing hgh levels of the human epidermal growth factor (EGF) receptor were used to examine the effects of the lectin concanavalin A (Con A) on EGF‐mediated signaling events. Proliferation of NIH 3T3 cells expressing high levels of the human EGF receptor was inhibited in a dose‐dependent manner by Con A. At the same time, Con A also inhibited both dimerization and tyrosine phosphorylation of the EGF receptor. Tyrosine phosphorylation of the enzyme phospholiphase C‐γ, a substrate of the phosphorylated EGF receptor kinase, was also inhibited. In contrast, EGF‐stimulated changes in pH, calcium, and levels of inositol phosphates were unaffected by the presence of Con A. These results indicate that certain signals (changes in the levels of intracellular calcium, pH, and inositol phosphates) mediated by EGF binding to its receptor still occur when receptor dimerization and phosphorylation are dramatically decreased, suggesting that multiple independent signals are transmitted by the binding of EGF to its receptor. © 1995 Wiley‐Liss, Inc.
Contribution of response kinetics to the response pattern: Studies of responses to thyrotropin‐releasing hormone in Xenopus oocytes
Authors: Lipinsky Dafna , Gershengorn Marvin C. , Oron Yoram
Published: February 2005

In Xenopus oocytes injected with total rat pituitary GH3 cell RNA, thyrotropin‐releasing hormone (TRH) causes the activation of the inositol lipid transduction pathway and the induction of chloride conductance via calcium‐activated channels (Oron et al., 1987, Mol. Endocrinol., 1:918–925). This response exhibits characteristic prolonged latency (Oron et al., 1988, Proc. Natl. Acad. Sci. U.S.A., 85:3820–3824; Lipinsky et al., 1993, Pflugers Arch., 425:140–149). We examined the role of agonist diffusion in the extracellular medium in the generation of latency and the detemination of response amplitude. An increase in the viscosity of the medium markedly prolonged the latency and decreased the amplitude of the response. Moreover, an increase in the viscosity of the medium in the immediate vicinity of the oocyte had a major effect on both the latency and the amplitude of the response, which appeared to be a result of desensitization rather than restricted diffusion of chloride to the medium. Extrapolation to [TRH] ∞ yielded a diffusion‐dependent latency value of 0 and a diffusion‐independent latency value of 4 seconds. In low viscosity medium, at all TRH concentrations, diffusion contributed less than 2% to the latency of the response. This implied that events distal to ligand binding are responsible for a major part of latency. Analysis of the dependence of latency and amplitude of the response on [TRH] yielded Hill coefficients markedly smaller than unity, suggesting postreceptor negative modulation of the response. Preincubation of cells with a specific inhibitor of protein kinase C, chelerythrine, increased the Hill coefficients to unity and changed the shape of the Hill plot of response amplitudes. Our results suggest that at low agonist concentrations, even in a low viscosity medium, the prolonged latency allows negative effects on both latency and amplitude by a simultaneous activation of a protein kinase C. © 1995 Wiley‐Liss, Inc.
Effects of mild heat shock on glycogenesis and its regulation by insulin in cultured fetal hepatocytes
Authors: Zachayus J. L. , Plas C.
Published: February 2005

The effects of a mild heat shock were investigated using cultured 15‐day‐old fetal rat hepatocytes in which an acute glucocorticoid‐dependent glycogenic response to insulin was present. After exposure from 15 min to 2 h at 42.5°C, cell surface [125I]insulin binding progressively decreased down to 60% of the value shown in cells kept at 37°C, due to a decrease in the apparent number of insulin binding sites with little change in insulin receptor affinity. In parallel cultures, protein labeling with [35S]methionine exhibited stimulated synthesis of specific proteins, in particular, 73‐kDa Hsc (heat shock cognate) and 72‐kDa Hsp (heat shock protein). When cells were returned to 37°C after 2 h at 42.5°C, cell surface insulin binding showed a two‐third restoration within 3 h (insulin receptor half‐life = 13 h), with similar concomitant return of Hsps72,73 synthesis to preinduction levels. The rate of [14C]glucose incorporation into glycogen measured at 37°C after 1‐ to 2‐h heat treatment revealed a striking yet transient increase in basal glycogenesis (up to 5‐fold). At the same time, the glycogenesis stimulation by insulin was reduced (from 3.2 to 1.4—fold), whereas that induced by a glucose load was maintained. Induction of thermotolerance after a first heating was obtained for the heat shock‐dependent events except for the enhanced basal glycogenesis. In insulin‐unresponsive cells grown in the absence of glucocorticoids, heat shock decreased the glycogenic capacity without modifying the glucose load stimulation, supporting the hypothesis that insulin and thermal stimulation of glycogenesis share at least part of the same pathway. Inverse variations were observed between Hsps72,73 synthesis and both cell surface insulin receptor level and insulin glycogenic response in fetal hepatocytes experiencing heat stress. © 1995 Wiley‐Liss, Inc.
Suppression of tumorigenicity, but not invasion, in glioblastoma/HeLa cell hybrids
Authors: Ess Kevin , Chen Haiyan , Kier Ann , Brackenbury Robert
Published: February 2005

Somatic cell hybrids between SNB‐19 human glioblastoma cells and human D98OR HeLa parental cells were produced and analyzed for their ability to form tumors in nude mice and to invade reconstituted extracellular matrix (Matrigel). Whereas both the SNB‐19 and D98OR HeLa parental cells form tumors, four of six hybrid lines did not form tumors, even after periods up to six months, suggesting that each cell type can complement the tumorigenicity of the other. SNB‐19 cells showed high rates of Matrigel invasion at all cell densities examined, whereas D98OR HeLa cells showed lower rates of invasion that were further reduced at high cell density. All six hybrid cell lines displayed a combination of these properties: at low cell density, the hybrids showed high rates of invasion, similar to the SNB‐19 cells, but the invasion rate diminished at higher cell densities, similar to the D98OR HeLa cells. Taken together, these results provide new experimental evidence that several distinct genetic changes are involved in generating the tumorigenic and invasive phenotype of glioblastoma cells. © 1995 Wiley‐Liss, Inc.
Transcriptional and posttranscriptional regulation of urokinase‐type plasminogen activator expression in endothelial cells by basic fibroblast growth factor
Authors: Gualandris Anna , Presta Marco
Published: February 2005

The mechanism of induction of urokinase‐type plasminogen activator (uPA) by basic fibroblast growth factor (bFGF) was explored in fetal bovine aortic endothelial GM 7373 cells. A three‐ to four‐fold increase in the steady‐state levels of uPA mRNA was observed after 6 h of incubation of the cell cultures with bFGF. Accordingly, nuclear run‐on experiments showed a 2–2.4‐fold increase in the rate of uPA gene transcription during the first 4 h of treatment with the growth factor. bFGF did not affect uPA mRNA stability, as evaluated by chase experiments with the mRNA synthesis inhibitor actinomycin D. Upregulation of uPA mRNA was followed by a delayed increase in uPA protein synthesis paralleled by an increase in secreted and cell‐associated uPA activity. Twelve h were required before accumulated uPA mRNA was translated into the corresponding protein. During this time interval, the continuous presence of biologically active bFGF in the extracellular environment represented an absolute requirement for uPA mRNA translation. Substitution of residues Lys‐27, Lys‐30, and Arg‐31 to glutamine residues in the bFGF molecule resulted in a mutant (M1Q‐bFGF) that caused uPA mRNA accumulation in the absence of a significant increase in cell‐associated uPA activity. M1Q‐bFGF also induced an increase in cell‐associated uPA activity only when added to the cell cultures in the presence of soluble heparin. These results provide evidence that bFGF can affect uPA expression in endothelial GM 7373 cells both at transcriptional and posttranscriptional/translational levels. They also show the possibility to dissociate upregulation of uPA mRNA from upregulation of uPA activity by mutagenesis of the bFGF molecule. © 1995 Wiley‐Liss, Inc.
Impaired alveolar macrophage function in smoke inhalation injury
Authors: Herlihy J. P. , Vermeulen M. W. , Joseph P. M. , Hales C. A.
Published: February 2005

The high incidence of both bacterial pneumonia and the adult respiratory distress syndrome (ARDS) associated with smoke inhalation injury (SII) may result, at least in part, from smoke‐induced injury to the alveolar macrophage (AM). Specifically, we hypothesized that AM antimicrobial function, ability to phagocytose apoptotic PMNs, and capacity to prevent apoptosis in PMNs are impaired by smoke. To test these hypotheses, AMs were harvested by bronchoalveolar lavage from sheep before and after the animal was exposed to cotton smoke. The two populations of AMs were incubated with Pseudomonas aeruginosa (PSA) in vitro. Normal AMs (NAMs) phagocytosed a mean of 99 ± 11% of the PSA placed in their wells, whereas smoke‐exposed AMs (SAMs) ingested only 60 ± 8%. NAMs killed 80 ± 8% of PSA ingested, whereas SAMs killed only 56 ± 16% (P < 0.05). When sheep PMNs, allowed to undergo apoptosis, were incubated with the two AM populations, 66 ± 3% of the NAMs and 40 ± 6% of the SAMs demonstrated phagocytosis of these apoptotic PMNs (P < 0.05). Fresh sheep PMNs were incubated in unconditioned media, NAM and SAM‐conditioned media, and followed over 48 hr for the development of apoptosis and maintenance of viability. The NAM‐conditioned media markedly prevented apoptosis and augmented PMN survival relative to the unconditioned and SAM‐conditioned media (P < 0.05). The poor antimicrobial function known to be characteristic of apoptotic PMNs, together with the directly impaired antimicrobial function of AMs, may contribute to the infectious complications of Sll. If the PMNs recruited to the lung in Sll are not properly supported by the AMs following smoke injury, large numbers may undergo apoptosis. If not properly disposed of by these SAMs, the apoptotic PMNs could eventually lyse, releasing tissue toxins, resulting in escalation of lung injury and leading to ARDS. © 1995 Wiley‐Liss, Inc.
Three patterns of cytokine expression potentially involved in epithelial‐fibroblast interactions of human ocular surface
Authors: Li De‐Quan , Tseng Scheffer C. G.
Published: February 2005

Signals transmitted from mesenchyme to epithelia or vice versa constitute the basis of reciprocal epithelial‐mesenchymal interactions. As a first step toward understanding epithelial‐mesenchymal interactions on the ocular surface where the transit amplifying cell‐containing corneal epithelium is anatomically separated from the stem cell‐containing limbal epithelium, we sought to characterize the expression patterns of cytokines and their receptors by primary epithelial and early‐passaged fibroblast cultures of human cornea and limbus. Northern hybridization with oligonucleotide and cDNA probes to a total of 25 cytokines and 12 of their receptors revealed that the positively expressed cytokines could be divided into the following four patterns. Type I: TGF‐α, IL‐β, and PDGF‐B were expressed exclusively by epithelial cells but their respective receptors EGFR and IL‐1R were predominantly and PDGFR‐β was exclusively expressed by fibroblasts. Type II: IGF‐I, TGF‐β1, β2, LIF, and bFGR, and their receptors were expressed by both epithelial cells and fibroblasts. FGFR‐1 (flg) and FGFR‐2 (bek) were expressed more by fibroblasts and bFGF was expressed more by corneal than limbal epithelial cells. Type III: keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) were expressed exclusively by fibroblasts and their respective receptors, KGFR and c‐met, were predominantly expressed by epithelial cells. Combined with RT‐PCR, the quantity of KGF and KGFR transcripts was highest in limbal fibroblasts and epithelial cells, respectively. In contrast, the quantity of HGF and HGFR (c‐met) transcripts was highest in corneal fibroblasts and epithelial cells, respectively. Type IV: M‐CSF and IL‐8 were expressed by fibroblasts and/or epithelial cells but their receptors were not expressed by epithelial cells nor fibroblasts, but by immune or inflammatory cells. In addition to these potential paracrine actions, autocrine actions mediated by TGF‐α/EGFR, IL‐β/IL1‐R, and bFGF/FGFR‐1 were more expressed by corneal than limbal epithelial cells. Immunofluorescence staining on human corneoscleral cryosections confirmed that EGFR and bFGF were not expressed by the limbal basal epithelium, but expressed strongly by the corneal epithelium, a pattern consistent with Northern hybridization. These results indicate that ocular surface epithelial cells and fibroblasts can express a myriad of cytokines, among which the first three patterns constitute the network of potential epithelial‐mesenchymal cytokine dialogues. The difference of certain cytokine expression between corneal and limbal regions suggests that this network participates in normal epithelial growth and differentiation, and plays an important role in wound healing. © 1995 Wiley‐Liss, Inc.
Bafilomycin A1 inhibits lysosomal, phagosomal, and plasma membrane H+‐ATPase and induces lysosomal enzyme secretion in macrophages
Authors: Tapper Hans , Sundler Roger
Published: February 2005

Bafilomycin A1, a specific inhibitor of H+‐ATPases of the vacuolar type, was in the present study shown, at similar concentrations, to induce secretion of lysosomal enzyme and to elevate lysosomal pH in mouse macrophages. These results lend support to the previous suggestion of a triggering role for an increase in lysosomal pH and a permissive role for cytosolic pH in the exocytosis of macrophage lysosomal enzyme. Vacuolar H+‐ATPases are present in the macrophage plasma membrane as well as in intracellular membranes, for example, those of the lysosomal and phagosomal compartments. Phagosomal acidification was shown to be achieved in part by a mechanism with a similar sensitivity to bafilomycin A1 as lysosomal H+ transport and in part by an early, bafilomycin A1‐insensitive mechanism. We found a lesser sensitivity towards bafilomycin A1 of the lysosomal and phagosomal H+‐ATPase than that localized in the plasma membrane, indicating differences among H+‐ATPases at the subcellular level. Also, by attempts to mobilize lysosomal H+‐ATPase to the plasma membrane, support was obtained for the notion that subcellular H+‐ATPase populations differ and thus possibly could be differentially regulated. © 1995 Wiley‐Liss, Inc.
Na+ transport processes in isolated guinea pig nasal gland acinar cells
Authors: Ikeda Katsuhisa , Ishigaki Motoaki , Wu Dazheng , Sunose Hiroshi , Takasaka Tomonori
Published: February 2005

In the dispersed acinar cells of the submucosal nasal gland in the guinea pig, intracellular Na+ concentration ([Na+]i) was measured with a microfluorimetric imaging method and the cytosolic indicator dye, sodium‐binding benzofuran isophthalate, under HCO3−‐free conditions. In the unstimulated condition, the [Na+]i was averaged to 12.8 ± 5.2 mM. Addition of 100 μM ouabain or removal of external K+ caused an increase in [Na+]i. Replacement of external Cl− with NO3− or addition of 0.5 mM furosemide reversibly decreased the [Na+]i. The recovery process from the reduced [Na+]i was inhibited by removal of either K+ or Cl− in the bath solution. These findings indicate the presence of a continuous influx of Na+ coupled with K+ and Cl− movement. Application of acetylcholine (ACh, 1 μM) caused an increase in [Na+]i by about 15–20 mM, which was completely inhibited by addition of 10 μM atropine. Increased cytosolic Na+ induced by ACh was extruded by the Na+‐K+ pump. Removal of external Cl− and addition of 50 μM dimethylamiloride inhibited ACh‐induced increase in [Na+]i by about 66% and 19%, respectively. In both unstimulated and stimulated state, Na+‐K+ pump, Na‐K‐Cl cotransport, and Na+‐H+ exchange play a critical role in maintaining intracellular electrolyte environment and in controlling a continuous secretion of nasal fluids. © 1995 Wiley‐Liss, Inc.
Biphasic effect of 1,25‐dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation
Authors: Bollag Wendy B. , Ducote Janet , Harmon Charles S.
Published: February 2005

1,25‐Dihydroxyvitamin D3 [1,25(OH)2D3] has been proposed as a physiologic regulator of keratinocyte growth and differentiation. Utilizing a proliferative serum‐free culture system, we have found that at physiologic (picomolar) concentrations this hormone stimulated proliferation of primary mouse epidermal keratinocytes; at higher (nanomolar to micromolar) doses, growth was inhibited by 1,25(OH)2D3. We investigated the nature of the signal transduction mechanism underlying the response to 1,25(OH)2D3 and observed little or no effect of either low or high concentrations of the hormone on cytosolic calcium levels or Fos expression. Furthermore, the protein kinase C inhibitor, Ro 31‐7549, had very little effect on the growth inhibition induced by a high dose (1 μM) of 1,25(OH)2D3. This lack of rapid signal transduction events was consistent with the inability of a short (4‐hour) exposure to 1,25(OH)2D3 to initiate a complete growth‐inhibitory response as measured using [3H]thymidine incorporation. Our results indicate that physiologic concentrations of 1,25(OH)2D3 are required for optimal keratinocyte growth. Furthermore, we found no evidence of rapid effects of 1,25(OH)2D3 and suggest that in mouse epidermal keratinocytes, the response to this hormone is mediated by a slow transduction pathway, such as that activated by the intracellular 1,25(OH)2D3 receptor (VDR). © 1995 Wiley‐Liss, Inc.
Hematopoietic‐promoting activity of the murine stromal cell line MS‐5 is not related to the expression of the major hematopoietic cytokines
Authors: Kobari Ladan , Dubart Anne , Le Pesteur Françoise , Vainchenker William , Sainteny Françoise
Published: February 2005

As an approach for characterizing the molecules involved in the proliferation and differentiation of hematopoietic stem cells we have compared the ability of four murine stromal cell lines, MS‐5, MS‐K, both derived from Dexter cultures, BMS1 and BMS2 both derived from Whitlock‐Witte cultures, to sustain murine long term hematopoiesis and to express the major hematopoietic cytokine genes. As opposed to the three other cell lines, MS‐5 supports the maintenance of stem cells for up to 4–5 weeks. However, reconstituting stem cell output was reduced while clonogenic cell (day 12 and day 8 spleen colony‐forming units, granulo‐macrophagic, and erythroid progenitor cells) output was markedly increased. This hematopoietic‐promoting activity is at least in part mediated by soluble molecules since medium conditioned with MS‐5 cells was able to partially complement the nonsupportive cell line BMS1. The comparative study of the cytokine gene expression in MS‐5 and in the nonsupportive cell lines included Northern blot and reverse transcriptase‐polymerase chain reaction analysis of messenger RNA for interleukin‐1, ‐3, ‐6, granulo‐macrophagic‐colony‐stimulating factor (GM‐CSF), granulocyte‐CSF, macrophage‐CSF, stem cell factor, transforming growth factor‐beta, tumor necrosis factor‐alpha, macrophage inflammatory protein‐1 alpha, and leukemia inhibitory factor. None of these molecules or their association were found to clearly confer to the MS‐5 cell line its hematopoietic‐promoting activity raising the possibility that uncharacterized molecule(s) would be involved in the proliferation and differentiation of stem cells. © 1995 Wiley‐Liss, Inc.
Regulation of myelin basic protein gene transcription: Identification of a distal cis‐acting regulatory element
Authors: Haque Nasreen S. , Haas Susan , Knobler Robert L. , Khalili Kamel
Published: February 2005

The myelin basic protein (MBP) gene contains sequences located upstream of its transcription start site which play a key role in glial‐specific transcription of the MBP promoter. Earlier analysis of the 320 bp upstream regulatory sequence of MBP has revealed multiple cis‐acting regulatory motifs which differentially regulate transcription of a heterologous promoter fused to a reporter gene in glial and nonglial cells. In the present study, we have focused on a region designated MB3, which is located between −93 to −130 nucleotides with respect to the RNA start site, and contains a binding site for the NF1/CTF family of transcription activators. Results from DNase I footprint protection analysis of nuclear proteins prepared from mouse brain revealed a major region within the MB3 regulatory element that specifically interacts with the proteins derived from mouse brain at various stages of brain development. Using synthetic oligonucleotides spanning the protected region, we show that the double‐stranded MB3 sequence interacts with nuclear proteins from mouse brain and forms specific major C1 and a minor C2 complex. Methylation interference experiments have allowed the identification of the G‐residues within nucleotides −100 to −108, named MB3a, which are distinct from the NF1/CTF of MB3 that contact with nuclear proteins to form the major C1 complex. Results from band shift studies revealed assembly of the C1 complex upon incubation of MB3 DNA with the nuclear proteins from various cells of glial origin. Site‐directed mutagenesis experiments revealed that the identified G‐residues for DNA‐protein interaction are important to confer transcriptional activity to this domain in transiently transfected glial cells. © 1995 Wiley‐Liss, Inc.
Carboxyl‐terminal truncation of leucine76 converts heparin‐binding EGF‐like growth factor from a heparin‐enhancible to a heparin‐suppressible growth factor
Authors: Cook Paul W. , Damm Deborah , Garrick Brett L. , Wood Kathleen M. , Karkaria Cyrus E. , Higashiyama Shigeki , Klagsbrun Michael , Abraham Judith A.
Published: February 2005

Previous studies have indicated that heparin differentially regulates heparin‐binding EGF‐like growth factor (HB‐EGF) and amphiregulin (AR) mitogenic activity. To further explore this phenomenon, these mitogens were compared under identical cell culture conditions in two different assays. The results of our present investigation demonstrated that AR‐mediated mitogenic activity in the murine AKR‐2B fibroblast‐like cell line was inhibited by heparin, while HB‐EGF activity was enhanced. However, the absolute effect of heparin appeared to be cell type specific since HB‐EGF mitogenic activity was not dramatically affected by coincubation with heparin when tested on human dermal fibroblasts. Several studies have indicated that mutation of a conserved leucine in the carboxyl‐terminal region of both EGF and transforming growth factor‐α results in decreased affinity for EGF receptors. Since this leucine is present in the analogous position of HB‐EGF, but absent in AR, we examined the effect of deleting this residue by carboxyl‐terminal truncation of HB‐EGF. Analysis of recombinant forms of HB‐EGF demonstrated that HB‐EGF can be converted to a heparin‐inhibited growth factor if the putative mature form of the protein is truncated by two residues (leucine76 and proline77) at the carboxyl terminus. Further analysis demonstrated that only leucine76 appears to be required for heparin‐dependent enhancement of HB‐EGF‐mediated mitogenic activity, indicating that this amino acid may play a pivotal role in controlling the response of HB‐EGF to heparin or related glycosaminoglycan sulfates. Our results also suggest that expression of different HB‐EGF forms in vivo could result in the production of HB‐EGFs with divergent responses to sulfated glycosaminoglycans and proteoglycans. © 1995 Wiley‐Liss, Inc.
Effect of lysophospholipids on signaling in the human Jurkat T cell line
Authors: Xu Yan , Casey Graham , Mills Gordon B.
Published: February 2005

Lysophospholipids have recently been demonstrated to induce activation and proliferation of fibroblasts and other cell lineages by interacting with high affinity cell surface receptors leading to specific intracellular signaling events. Platelet activation, likely at the site of injury or inflammation, results in increased production of lysophospholipids suggesting a possible source of lysophospholipids. We have recently demonstrated that high concentrations of lysophospholipids are present in ascites and plasma from ovarian cancer patients, suggesting that physiologically produced lysophospholipids could interact with cells present in these fluids, including lymphocytes, and alter their function. We demonstrate herein that lysophosphatidic acid (LPA), lysophosphatidylserine (LPS), and sphingosylphosphorylcholine (SPC) activate the Jurkat T cell line. Each of the lysophospholipids induced a transient increase in cytosolic free calcium ([Ca2+]i) in Jurkat cells. Increases in [Ca2+]i were cross‐desensitized by LPA, LPS and SPC, suggesting that the lysophospholipids share the same receptor(s) or that their downstream signaling pathways converge or interact. Lysophosphatidylgycerol (LPG), a competitive inhibitor of the putative LPA receptor, inhibited the calcium releasing activity of LPA, but not that of LPS and SPC, suggesting that these lysophospholipids interact with different receptors and that desensitization is due to interactions in downstream signaling pathways. The ability of the lysophospholipids to induce increases in [Ca2+]i was attenuated, but not completely blocked, by increases in [Ca2+]i induced by activation of the thrombin receptor. In contrast, increases in [Ca2+]i induced by the lysophospholipids and cross‐linking the CD3 component of the T cell receptor complex with the UCHT1 antibody did not undergo heterologous desensitization. Strikingly, LPA is sufficient to stimulate proliferation of Jurkat cells in serum‐free medium or in synergy with low concentrations of fetal bovine serum. In addition, LPA also increased the production of the T cell growth factor, interleukin 2 (IL‐2), by Jurkat cells treated with phorbol esters. LPS, in contrast, inhibited Jurkat proliferation while increasing IL‐2 production and SPC inhibited both processes. Thus, although all three lysophospholipids were sufficient to induce a transient increase in [Ca2+]i in Jurkat cells, they induced markedly different physiological consequences. © 1995 Wiley‐Liss, Inc.
P‐glycoprotein stability is affected by serum deprivation and high cell density in multidrug‐resistant cells
Authors: Muller Catherine , Laurent Guy , Ling Victor
Published: February 2005

The control of P‐glycoprotein (Pgp) expression in multidrug‐resistant cells (MDR) is complex and may be regulated at different levels. We have investigated Pgp stability in four different human and hamster MDR cell lines. Using a pulse‐chase procedure we show that Pgp half‐life is between 14 and 17 h in all these cell lines when they are growing exponentially. However, in the presence of a low level of serum, Pgp half‐life is increased four to sixfold. A similar effect is observed when the cell cultures are maintained in high cell density. The increased Pgp stability appears to be differently regulated as serum deprivation results in a general enhanced degradation of total cytoplasmic and membrane proteins. Moreover, the observed serum effect suggests the involvement of growth factors in the control of Pgp stability. These findings suggest that protein stability may be an important factor in the regulation of Pgp expression. © 1995 Wiley‐Liss, Inc.