bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neurite outgrowth activity of protease nexin‐1 on neuroblastoma cells requires thrombin inhibition
Authors: Gurwitz David , Cunningham Dennis D. 
Published: February 2005

AbstractProtease nexin‐1 (PN‐1) is a protein proteinase inhibitor recently shown to be identical with the glial‐derived neurite‐promoting factor or glial‐derived nexin. It has been shown to promote neurite outgrowth in neuroblastoma cells and in sympathetic neurons. The present experiments were designed to further test the hypothesis that this activity on neuroblastoma cells is due to its ability to complex and inhibit thrombin. It has been suggested that PN‐1 :thrombin complexes might mediate the neurite outgrowth activity of PN‐1. However, the present studies showed that such complexes, unlike free PN‐1, did not promote neurite outgrowth. The neurite outgrowth activity of PN‐1 was only detected in the presence of thrombin or serum (which contains thrombin). PN‐1 did not affect the rate or extent of neurite outgrowth that occurred when neuroblastoma cells were placed in serum‐free medium. Retraction of neurites by thrombin was indistinguishable in cells whose neurites had been extended in the presence or absence of PN‐1. The neurite‐promoting activity of PN‐1 was inhibited by an anti‐PN‐1 monoclonal antibody, which blocks its capacity to complex serine proteinases. The plasma thrombin inhibitor, antithrombin III, stimulated neurite outgrowth but only when its thrombin inhibitory activity was accelerated by heparin. The neurite outgrowth activity of both antithrombin III and PN‐1 corresponded to their inhibition of thrombin. Together, these observations show that PN‐1 promotes neurite outgrowth from neuroblastoma cells by inhibiting thrombin and suggest that this depends on the ability of thrombin to retract neurites.
Adenine nucleotides modulate phosphatidylcholine metabolism in aortic endothelial cells
Authors: Pirotton Sabine , Robaye Bernard , Lagneau Christian , Boeynaems Jean‐Marie 
Published: February 2005

AbstractATP and ADP, in concentrations ranging from 1—100 μM, increased the release of [3H]choline and [3H]phosphorylcholine (P‐choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP‐γS and ADPβS) and adenosine 5′‐(β,γ imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5‐(α,β methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 μM), the tumor promoter phorbol 12‐myristate 13‐acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 μM). Down‐regulation of protein kinase C, following a 24‐hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P‐choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).
Comparison between transport and degradation of leucine and glutamine by peripheral human lymphocytes exposed to concanavalin A
Authors: Koch Brigitte , Schröder Marie‐Theres , Schäfer Gertrud , Schauder Peter 
Published: February 2005

AbstractTransport and pathways of leucine and glutamine degradation were evaluated in resting human peripheral lymphocytes and compared with the changes induced by concanavalin A (ConA). Cells were incubated with [1‐14C]leucine (0.15 mM), [U‐14C]leucine (0.15 mM), or [U‐14C]glutamine (0.4 mM) after culture with or without 2, 5, 7, or 10 μg/ml ConA for 2, 18, or 24 hours, respectively. Initial rates of transport of leucine and glutamine were augmented 2.7‐fold and threefold by the mitogen. Leucine transamination, irreversible oxidation, and catabolism beyond isovaleryl‐CoA were increased by 90%, 20%, and 60%, respectively. Glu‐tamine utilization increased threefold; accumulation of glutamate, aspartate, and ammonia increased by 700%, 50%, and 100%, respectively, and 14CO2 production by about 400% in response to ConA. The results indicate that ConA stimulates to about the same extent transport of leucine and glutamine into lymphocytes. Glutamine is mainly channeled into catabolic pathways, while leucine remains largely preserved. It is suggested that these metabolic changes provide more leucine for incorporation into protein and more N‐ and C‐atoms required for the synthesis of macromolecules and energy from glutamine.
Propagation of fetal human RPE cells: Preservation of original culture morphology after serial passage
Authors: Song Mi‐Kyoung , Lui Ge Ming 
Published: February 2005

AbstractThe permissive effects of extracellular matrix (ECM) on in vitro growth and differentiation of fetal human retinal pigment epithelial (RPE) cells have been studied. Factors which enhanced the effect of ECM to support cell division were also examined, including growth factors, culture media, and serum requirement. Under the specific culture conditions we have defined, it is possible to propagate these RPE cells at low density (less than 20 cells/mm2) with excellent growth properties for greater than 72 doublings (fourteen passages) in serial culture. Later‐passaged cells maintained the morphological appearance of early‐passaged cultures. ECM produced by bovine corneal endothelial cells was by far the most predominant factor in promoting rapid cell proliferation and viability over repeated passaging. Basic fibroblast growth factor (bFGF) exerted a substantial effect on the rate of cell division at different serum concentrations on plastic dishes. In addition, this factor showed profound synergistic effect when RPE cells were maintained on ECM, both in the preservation of cell morphology and also in long term viability. Other growth factors, such as epidermal growth factor (EGF) and transforming growth factor‐beta (TGF‐B), were also tested, but EGF effects were less prominent than those observed with bFGF, and TGF‐B had an inhibitory effect at high concentrations. The ability to obtain a relatively large number of human RPE cells in vitro which preserve the appearance of early passage cells may provide useful opportunities to study the physiological properties and pathological alterations involving this important cell type.
Cell size and RNA content correlate with cell differentiation and proliferative capacity of rat keratinocytes
Authors: Poot Martin , Rizk‐Rabin Marthe , Hoehn Holger , Pavlovitch Jana Henriette 
Published: February 2005

AbstractKeratinocytes from rat skin were separated according to their size in a specially designed unit‐gravity sedimentation chamber. The fractions obtained with this technique showed clear morphological differences, and analysis of size distribution confirmed that size was the criterion for separation. Simultaneous DNA and RNA staining of the fractions with acridine orange and subsequent flow cytometric analysis enabled one to classify cells into resting, proliferating, and differentiating stages. Cell size was not directly correlated with proliferation in situ as determined with acridine orange flow cytometry, nor with proliferative capacity in culture as assayed by BrdU/Hoechst flow cytometry. The smallest cells, exhibiting low DNA and RNA content, which do not proliferate in vivo, required a prolonged period of serum stimulation in vitro to initiate RNA and DNA synthesis. Cells of intermediate size exhibited early RNA synthesis and maximal proliferative capacity, whereas the largest cell population displayed no RNA synthesis in culture and the least proliferative capacity. In conclusion, these results suggest that RNA synthesis early after serum stimulation, in addition to a specific, optimal cell size, correlates with the proliferative capacity of keratinocytes in cell culture.
New role for histamine in interleukin‐3‐induced proliferation of hematopoietic stem cells
Authors: Schneider Elke , Piquet‐Pellorce Claire , Dy Michel 
Published: February 2005

AbstractThis study investigated the effect of histamine generated by murine bone marrow cells in response to IL‐3 on one particular biological activity of this growth factor, i.e., triggering of cells forming colonies in spleen (CFU‐S) into S phase. Evidence is provided that i) IL‐3‐induced day‐8 CFU‐S cell cycling, evaluated by hydroxyurea suicide, is completely abrogated when the binding of histamine to its H2 receptors is blocked by the specific antagonist oxmetidine, whereas cetirizine, a H1 receptor antagonist, is ineffective; and ii) the entry of day‐8 CFU‐S intoS phase in response to IL‐3 is likevise abolished when the histamine synthesis promoted by the growth factor is prevented by α‐fluoromethylhistidine, a specific inhibitor of the histamine‐forming enzyme, histidine decarboxylase. Similar results are obtained with both drugs, when a progenitor‐enriched bone marrow population is used instead of total cells. Furthermore, i.v. injection of recombinant (r)IL‐3 results within 2 hr in a substantial increase in bone marrow cell histamine synthesis together with triggering of day‐8 CFU‐S into cycle, the latter being completely abolished by a simultaneous injection of the H2 histamine receptor antagonist oxmetidine. Thus, our findings support the notion that both in vitro and in vivo the proliferation of early CFU‐S in response to IL‐3 is modulated by histamine via its H2 receptors. This conclusion is also consistent with the observation that dimaprit, a specific agonist of these receptors not only enhances the sensitivity of day‐8 CFU‐S to HU after a 2 hr incubation with bone marrow cells but also increases, to the same extent as IL‐3, the number of colonies formed in irradiated spleens after a 5 hr pretreatment.
Relationship of c‐myc gene copy number and gene expression: Cellular effects of elevated c‐myc protein
Authors: Pallavicini M. G. , Rosette C. , Reitsma M. , Deteresa P. S. , Gray J. W. 
Published: February 2005

AbstractThe relationship between gene copy number and expression and cellular consequences of elevated levels of c‐myc protein has been investigated using recombinant Chinese hamster ovary cell lines transfected with DNA coding for the murine c‐myc gene. HC‐8 and LC‐5 recombinant cells carry approximately 800 and 50 copies of c‐myc sequences, respectively, under control of an inducible heat shock promoter. Multivariate flow cytometric analysis and clonogenic assays were used to measure the relationship among c‐myc expression, rate of DNA synthesis, and cell survival. Following heat exposure, maximally induced HC‐8 cells produced approximately tenfold more c‐myc protein than heated LC‐5 cells, suggesting a close relationship between gene copy number and level of expression. However, considerable heterogeneity in the level and time of c‐myc expression was observed following heat induction, even though the amounts of genomic c‐myc were reatively constant. Heterogeneity in gene expression was not attributable to variation in heat induction methodologies and/or cell cycle phase distributions. The presence of high levels of recombinant c‐myc protein was associated with a decreased rate of bromodeoxyuridine incorporation into DNA. High levels of c‐myc protein in HC‐8 cells were inversely correlated with cell survival postheating, suggesting that high levels of c‐myc protein are incompatible with cell survival.
Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors
Authors: Finesmith Tina H. , Broadley Kenneth N. , Davidson Jeffrey M. 
Published: February 2005

AbstractWound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of fibroblast‐like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of growth factors implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (21 and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin fibroblasts. Transforming growth factor‐b̃1 (TGF‐b̃1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic fibroblast growth factor (bFGF) alone had moderately stimulatory effects at low doses (0.1‐1.0 ng/ml), higher doses (≥ 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF‐b̃1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF‐b̃1 activity in highly contractile cells. Transforming growth factor‐α moderately enhanced contraction by cells from older granulation tissue. While both TGF‐b̃1 and bFGF enhanced wound repair, their differential effects on the fibroblast‐like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.
Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester
Authors: Bikfalvi A. , Alterio J. , Inyang A. L. , Dupuy E. , Laurent M. , Hartmann M. P. , Vigny L. , Raulais D. , Courtois Y. , Tobelem G. 
Published: February 2005

AbstractThe human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti‐bFGF antibody. Basic FGF‐like material from the HOME cell cytosol was bound onto a heparin‐Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti‐bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti‐acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20‐22 Kd. Northern blotting of nonstimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF‐like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.
Cell cycle regulation of an exogenous human poly(ADP‐ribose) polymerase cDNA introduced into murine cells
Authors: Bhatia Kishor , Kang Veronica H. , Notario Vicente , Haque Saikh J. , Cherney Barry W. , Smulson Mark E. , Stein Gary S. , Bustin Michael , Huppi Konrad 
Published: February 2005

AbstractWe have evaluated the regulation of expression of the poly(ADP‐ribose) polymerase gene during cell growth and replication. In a synchronized population of HeLa cells or in serum‐stimulated WI‐38 cells, steady‐state levels of the polymerase mRNA were highest at late S and S‐G2 phases and negligible in early S phase. Transcription did not solely account for the significant increase in the mRNA levels observed in late S phase by Northern analysis. The stability of the mRNA was dependent upon the percent proliferating cells in the culture. Accordingly, polymerase mRNA from cells in early exponential phase was significantly more stable than from cells in stationary phase of asynchronous growth. To clarify these observations, we utilized a novel heterologous expression system that involved murine 3T3 cells transfected with a human poly(ADP‐ribose) polymerase cDNA under the control of a non‐cell cycle‐specific promoter. Cells were synchronized, and a comparison was made of the endogenous (murine) and exogenous (human) polymerase mRNA levels. Both the endogenous and the exogenous mRNA were specifically stabilized by the same mechanisms and only during late S phase; therefore, we concluded that mRNA pools for the polymerase are regulated at the post‐transcriptional level. The heterologous expression system confirmed that the post‐transcriptional regulation system in the mouse cells can recognize and faithfully regulate the human cDNA in response to the murine cell cycle signals. More importantly, the presence of extra copies (human) of the polymerase gene did not provide an increased amount of the total polymerase mRNA or protein and, in fact, the sum of the endogenous and exogenous mRNA in the transfected cells was approximately the same as the level of endogenous transcript in the control cells. This suggested that there might be a limit to the amount of polymerase protein accumulating in the cellular pool and thus levels of poly(ADP‐ribose) polymerase may be autoregulated.
Plasminogen activator inhibitor type‐2 Is a major protein induced in human fibroblasts and SK‐MEL‐109 melanoma cells by tumor necrosis factor
Authors: Pytel Barbara A. , Peppel Karsten , Baglioni Corrado 
Published: February 2005

AbstractTumor necrosis factor (TNF) induces the synthesis of two proteins of Mr 42 and 36 kDa in human fibroblasts and SK‐MEL‐109 melanoma cells. To identify these proteins, a λgt10 cDNA library was prepared from the mRNA of TNF‐treated SK‐MEL‐109 cells. By screening this library, we found a cDNA that preferentially hybridized to TNF‐induced RNA. Hybrid‐selected mRNA was translated into a protein of 42 kDa; cDNA sequence analysis followed by a comparison with other known protein sequences identified this protein with plasminogen activator inhibitor, type‐2 (PAI‐2). After removal of TNF, PAI‐2 mRNA turned over rapidly, with an apparent half‐life of ∼ 2.5 h. Addition of dexamethasone increased the turnover of this mRNA, suggesting that the level of PAI‐2 mRNA could be regulated post‐transcriptionally by glucocorticoids. PAI‐2 was not secreted, but accumulated in fibroblasts continuously treated with TNF.
Pertussis toxin catalyzed ADP‐ribosylation of a 41 kDa G‐protein impairs insulin‐stimulated glucose metabolism in Bc3H‐1 myocytes
Authors: Moises Regina S. , Heidenreich Kim A. 
Published: February 2005

AbstractIn this study, we examined the effects of pertussis toxin (PT) on the ADP‐ribosylation of guanine nucleotide binding proteins (G‐proteins) and various insulinstimulated processes in cultured BC3H‐1 myocytes. Treatment of intact myocytes with 0.1 μg/ml PT for 24 hours resulted in the complete ribosylation of a 41 kDa protein. The 41 kDa PT substrate was immunoprecipitated with antibodies directed against a synthetic peptide corresponding to a unique sequence in the alpha subunit of Gi‐proteins. PT treatment of intact cells had no effect on insulin receptor binding or internalization. However, PT inhibited insulin‐stimulated glucose transport at all insulin‐concentrations tested (1–100ng/ml). Maximally stimulated glucose transport was reduced by 50% ± 15%. Insulin‐stimulated glucose oxidation was also decreased by 31% ± 8%. The toxin had no significant effect on the basal rates of glucose transport and glucose oxidation. The time course of PT‐induced inhibition on glucose transport correlated with the time course of the “in vivo” ADP‐ribosylation of the 41 kDa protein. The results suggest that a 41 kDa PT‐sensitive G‐protein, identical or very similar to Gi, is involved in the regulation of glucose metabolism by insulin in BC3H‐1 cells.
Butyrate blocks the accumulation of CDC2 mRNA in late G1 phase but inhibits both the early and late G1 progression in chemically transformed mouse fibroblasts BP‐A31
Authors: Charollais Renée‐HéLène , Buquet Christine , Mešter Jan 
Published: February 2005

AbstractSodium butyrate (6 mM) blocks the resumption of the cell division cycle in serum‐deprived chemically transformed Balb/c‐3T3 mouse fibroblasts (BP‐A31). The inhibition of G1 progression by sodium butyrate is not restricted to a specific mitogenic signaling pathway and is equally effective when tetradecanoyl phorbol acetate (TPA), insulin, or fetal calf serum (FCS) is used as inducer. The inhibitor acts in early as well as late G1 phase as indicated by experiments in which inhibitor was added and withdrawn at different times after restimulation of quiescent cells by FCS. At the gene expression level, sodium butyrate does not affect the inducibility of early cell cycle‐related genes (c‐myc, c‐jun) while blocking the induction of cdc 2 mRNA, a late G1 marker. We conclude that sodium butyrate does not interfere with the growth factor signaling pathways regulating the (early) cell cycle‐related gene expression. However, the presence of sodium butyrate early in G1 phase inhibits the cascade of events leading eventually to the expression of late G1‐characteristic genes such as cdc2. The antimitogenic activity of sodium butyrate may be related to its interference with an (unknown) process involved in the “mitogenic” cascade.