bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thrombin increases proliferation and decreases fibrinolytic activity of kidney glomerular epithelial cells
Authors: He Ci‐Jiang , Rondeau Eric , Medcalf Robert L. , Lacave Roger , Schleuning Wolf‐Dieter , Sraer Jean‐Daniel 
Published: February 2005

AbstractHuman glomerular epithelial cells (GECs) in culture synthesize single‐chain, urokinase‐type plasminogen activator (SC‐uPA), tissue‐type plasminogen activator (t‐PA), and plasminogen activator inhibitor 1 (PAI‐1) and possess specific membrane‐binding sites for u‐PA. Using purified 125I‐alpha thrombin, we demonstrate here the presence of two populations of specific binding sites for thrombin on GECs (1.Kd = 4.3 ± 1.0 × 10−10 M, 5.4 ± 1.4 × 104 M sites per cell, 2. Kd = 1.6 ± 0.5 × 10−8 M, 7.9 ± 1.8 × 105 sites per cell). Purified human alpha thrombin promoted the proliferation of GECs and induced a time‐and dose‐dependent increase of SC‐uPA, t‐PA, and PAI‐1 antigens released by GECs. Thrombin‐mediated increase in antigen was paralleled by an increase in the levels of corresponding u‐PA and PAI‐1 messenger RNA. In contrast, thrombin decreased u‐PA activity in conditioned medium. This discrepancy between u‐PA antigen and u‐PA activity was explained by a limited proteolysis of SC‐uPA by thrombin, leading to a two‐chain form detected by immunoblotting and that could not be activated by plasmin. Thrombin also decreased the number of u‐PA binding sites on GECs (p < 0.05) without changing receptor affinity. Hirudin inhibited the binding and the cellular effects of thrombin, whereas thrombin inactivated by diisopropylfluorophosphate had no effect, indicating that both membrane binding and catalytic activity of thrombin were required. We conclude that thrombin, through specific membrane receptors, stimulates proliferation of GECs and decreases the fibrinolytic activity of GECs both at the cell surface and in the conditioned medium. These results suggest that thrombin could be involved in the pathogenesis of extracapillary proliferation and persistency of fibrin deposits in crescentic glomerulonephritis.
Bindings of the lectins Griffonia simplicifolia‐1 and pokeweed mitogen mark discrete stages of myoepithelial‐like differentiation of cell lines from the rat mammary gland
Authors: Rudland Philip S. , Hughes Christine M. 
Published: February 2005

AbstractIndividual single‐cell‐cloned cell lines of the different rat mammary (Rama) cell types have been tested for their ability to bind the lectins Griffonia simplicifolia‐1(GS‐1) and pokeweed mitogen (PWM) using fluorescent, histochemical, and radioactive assays. Myoepithelial‐like cell lines isolated from neonatal rat mammary glands and from nonmetastasizing tumors strongly bind GS‐1 and PWM, whereas the corresponding epithelial and fibroblastic cell lines do not. When the epithelial cell lines are grown on floating gels of polymerised rat tail collagen, the basally situated or peripheral cells are stained strongly with peroxidase‐conjugated lectins, whereas the apically or luminally situated cells are unstained. The capacity of cell lines intermediate in morphology between epithelial and myoepithelial‐like cells to bind to GS‐1 is as follows: Rama 25 epithelial < Rama 25–12 < Rama 25‐I1 < Rama 25–14 < Rama 29 myoepithelial‐like cells, the same order as for other markers of myoepithelial cells. Conjugated PWM, however, binds only to the myoepithelial‐like cell lines. Treatment of Rama 25 epithelial cells with agents that disrupt microtubules accelerates their conversion to elongated, myoepithelial‐like cells in culture. The binding of cells to GS‐1 is observed prior to, and that to PWM after, the major morphological change. It is suggested that the stepwise appearances of carbohydrate receptors for GS‐1 and PWM mark discrete stages in the differentiation of epithelial to myoepithelial‐like cells in culture, in the same way that they mark similar differentiation stages in ductal development in mammary glands of prepubertal rats.
IL‐3 specifically inhibits GM‐CSF binding to the higher affinity receptor
Authors: Taketazu Fumitoshi , Chiba Shigeru , Shibuya Kyoichi , Kuwaki Tomoaki , Tsumura Haruhiko , Miyazono Kohei , Miyagawa Kiyoshi , Takaku Fumimaro 
Published: February 2005

AbstractThe inhibition of binding between human granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and its receptor by human interleukin‐3 (IL‐3) was observed in myelogenous leukemia cell line KG‐1 which bore the receptors both for GM‐CSF and IL‐3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U‐937 or in gastric carcinoma cell line KATO III, both of which have apparent GM‐CSF receptor but an undetectable IL‐3 receptor. In KG‐1 cells, the cross‐inhibition was preferentially observed when the binding of GM‐CSF was performed under the high‐affinity binding condition; i.e., a low concentration of 125I‐GM‐CSF was incubated. Scatchard analysis of 125I‐GM‐CSF binding to KG‐1 cells in the absence and in the presence of unlabeled IL‐3 demonstrated that IL‐3 inhibited GM‐CSF binding to the higher‐affinity component of GM‐CSF receptor on KG‐1 cells. Moreover, a chemical cross‐linking study has revealed that the cross‐inhibition of the GM‐CSF binding observed in KG‐1 cells is specific for the β‐chain, Mr 135,000 binding protein which has been identified as a component forming the high‐affinity GM‐CSF receptor existng specifically on hemopoietic cells.
Attenuation of LPS‐induced neutrophil thromboxane B2 release and chemiluminescence
Authors: Zheng Hui , Crowley Jeffrey J. , Chan Jane C. , Raffin Thomas A. 
Published: February 2005

AbstractPolymorphonuclear leukocytes (PMN) may play a key role in acute lung injury and ARDS. The mechanisms of PMN‐mediated lung injury include the release of inflammatory mediators, such as oxygen free radicals which cause direct tissue injury, and arachidonic acid metabolites which cause pulmonary vasoconstriction and increased vascular permeability. The goals of this in vitro study were (1) to assess the effects of PMN‐activating agents (lipopolysaccharide, LPS; phorbol myristate acetate, PMA; tumor necrosis factor, TNF) on PMN thromboxane B2 (TXB2) release and oxygen free radical production and (2) to determine the effects of agents purported to suppress PMN activity (pentoxifylline, PTX; adenosine; dibutyryl cyclic AMP, DBcAMP; and terbutaline, TBN) on activator‐induced PMN TXB2 release and oxygen free radical production. PMN TXB2 release was determined by radioimmunoassay and oxygen free radical production was monitored by Chemiluminescence. Our results show that (1) LPS and PMA significantly increase PMN TXB2 release, whereas tumor necrosis factor (TNF) has no effect; (2) LPS and PMA significantly increase PMN Chemiluminescence; (3) DBcAMP and TBN significantly reduce LPS‐induced PMN TXB2 release whereas PTX and adenosine do not; (4) TBN significantly reduces PMA‐induced PMN TXB2 release whereas other agents do not; (5)All agents(PTX, adenosine, DBcAMP, and TBN) significantly reduce LPS‐induced PMN Chemiluminescence but none attenuate PMA‐induced PMN Chemiluminescence. We conclude that:LPS and PMA activate PMN manifested by TXB2 release and Chemiluminescence. Additionally, all the PMN suppressing agents do attenuate some PMN functions. Of interest, PTX, adenosine, DBcAMP, and TBN have different effects depending upon functional assay and activating agent. It will be important to investigate the mechanisms by which PMN suppressing agents alter signal transduction resulting in differential effects on PMN function.
Developmental patterns in the antioxidant defenses of the housefly, Musca domestica
Authors: Allen R. G. , Oberley L. W. , Elwell J. H. , Sohal R. S. 
Published: February 2005

AbstractThe activities of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione S‐transferases, GSSG reductase, thiol transferases, gamma glutamyl‐cysteine synthetase, and glucose‐6‐phosphate dehydrogenase, and the concentrations of H2O2 and reduced and oxidized glutathione were determined in the various developmental stages of houseflies. Housefly development was correlated with a progressive increase of cellular oxidizing equivalents and a loss of cellular reducing capacity. The loss of reducing equivalents appeared to result from a decrease in the activity of enzymes involved in glutathione and NADPH synthesis and a concomitant increase in glutathione‐oxidizing enzymes. Relatively little change was observed in SOD activity during housefly development; however, the electrophoretic pattern of MnSOD varied in a manner specific to developmental stage. A striking increase in H2O2 concentration occurred prior to pupation possibly due to changes in substrate catabolism. These results support the hypothesis that the cellular environment becomes progressively more oxidizing during development.
WS‐1 human fibroblasts contain distinct calcium and protein kinase C‐mediated pathways for activation of Na+/H+ exchange: Contrasting effects of thrombin and PMA
Authors: Hendey Bill , Mamrack Mark D. 
Published: February 2005

AbstractPMA and thrombin were examined for their ability to activate Na+/H+ exchange in growth‐arrested WS‐1 human fibroblasts. PMA or thrombin caused a cytoplasmic alkalinization that required extracellular sodium and was sensitive to 1 mM amiloride, suggesting that the rise in pH was mediated by the Na+/H+ exchanger. However, PMA and thrombin activated Na+/H+exchange by distinctly different mechanisms. The rate of cytoplasmic alkalinization caused by 30 nM PMA was slower than 10 nM thrombin. The PMA‐induced pH change was sensitive to the protein kinase inhibitors staurosporine (50 nM) and H‐7 (100 μM) No increase in intracellular calcium was observed after PMA treatment and the cytoplasmic alkalinization caused by PMA was not sensitive to the drug TMB8 (200 μM) or the intracellular calcium‐chelator BAPTA. In contrast, the thrombin‐induced rise in cytoplasmic pH was insensitive to 50 nM staurosporine and only partially reduced with 100 μM H‐7. The thrombin‐induced activation of Na+/H+ exchange was inhibited by 200 μM TMB8 or pretreatment with BAPTA. PMA caused translo‐cation of PKC activity from a cytoplasmic to membrane fraction whereas thrombin did not. Pretreatment with 50 nM staurosporine significantly reduced measurable PKC activity with or without PMA treatment. PMA and thrombin were also examined for their ability to induce DNA synthesis in growth‐arrested WS‐1 human fibroblasts. Unlike thrombin, PMA did not stimulate [3H]‐thymidine incorporation in cells serum‐deprived for 48 hours. In addition, PMA inhibited thrombin‐induced DNA synthesis when added at the same time or as late as 10 hours after thrombin addition. Therefore, thrombin and PMA activate Na+/H+ exchange by distinct pathways, but only the thrombin‐induced pathway correlates with a mitogenic response.
Membrane potential and amino acid transport in a mutant chinese hamster ovary cell line
Authors: Rotoli Bianca Maria , Bussolati Ovidio , Dall'asta Valeria , Gazzola Gian Carlo 
Published: February 2005

AbstractThe bioenergetics of amino acid transport system A was studied in two Chinese hamster ovary (CHO) cell lines, the parent line CHO‐PEOT/1 and CHY‐1, a mutant of the former exhibiting a low activity of the same transport system. The steady‐state transmembrane distribution ratio of the cationic amino acid L‐arginine (RARG) was employed as an indicator of membrane potential (Δψ). Evidence for the reliability of RARG to measure Δψ can be summarized as follows: (1) L‐arginine transmembrane distribution increased under conditions of cell hyperpolarization and decreased under conditions of cell depolarization; (2) L‐arginine distribution conformed closely to that expected for a probe of Δψ in conditions in which Δψ depends largely on the transmembrane potassium gradient; and (3) the value of Δψ obtained through a valinomycin null point experiment (−72.7 mV) was very similar to the value calculated from L‐arginine distribution using the Nernst equation (−73.4 mV). The transmembrane gradient of sodium electrochemical potential (Δ\documentclass{article}\pagestyle{empty}\begin{document}$ \tilde\mu $\end{document}Na), the driving force for the operation of system A, was slightly higher in the mutant cell line CHY‐1. In the same line, the intracellular level of the specific system A substrate MeAlB at steady state was also higher. Studies of the rheogenicity of system A in the two lines indicated that the depolarization associated with the entry of substrates of system A was proportional to the amount of amino acid taken up by the cells. Kinetic analysis showed that the low activity of system A in the mutant cell line was referrable to a decrease in transport Vmax. It is concluded that neither a decrease in energy available for the operation of system A nor a decreased efficiency of coupling of the system to Δψ is responsible for the defect observed in the mutant line.
Phorbol ester TPA inhibits the stimulation of bumetanide‐sensitive Na+/K+/Cl– transporter by different mitogens in quiescent BALB/c 3T3 mouse fibroblasts
Authors: Snyder David , Markus Miriam , Atlan Henri , Panet Rivka 
Published: February 2005

AbstractIn this study we examined the effect of the phorbol ester 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) on the bumetanide‐sensitive Na+/K+/Cl– transporter in quiescent BALB/c 3T3 cells. We have shown that exposure of quiescent BALB/c 3T3 cultures to phorbol ester did not inhibit the basal bumetanide‐sensitive Rb+ influx or efflux. In fact, at high concentration (100 ng/ml), TPA slightly stimulated the bumetanide‐sensitive Rb+ influx and efflux. However, when the quiescent cultures were stimulated by serum or by defined growth factors, the stimulated fraction of the bumetanide‐sensitive Rb+ influx was drastically inhibited by exposure of the cells to the phorbol ester TPA. Based on the above findings, we propose that activation of protein kinase C by the phorbol ester TPA does not inhibit the Na+/K+/Cl– cotransport activity; however it does suppress only the growth‐factors‐stimulated fraction of the cotransport in quiescent BALB/c 3T3 cells. These data propose that activation of kinase C has a regulatory feedback effect on the stimulation of the Na+/K+/Cl– cotransport activity by growth factors.
Multiple signal transduction pathways lead to extracellular ATP‐stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C–dependent and –independent pathways
Authors: Wang Ding‐Ji , Huang Ning‐Na , Gonzalez Fernando A. , Heppel Leon A. 
Published: February 2005

AbstractWe recently reported that extracellular ATP was mitogenic for Swiss 3T3, 3T6, and A431 cells (Huang et al.: Proc. Natl. Acad. Sci. USA, 86:7904–7908, 1989). Here we examined the possible involvement of activation of the protein kinase C (PKC) signal transduction pathway in the mechanism of action of extracellular ATP. A potent synergistic stimulation of DNA synthesis in quiescent cultures of 3T3 and 3T6 cells was observed when ATP was presented in combination with growth factors that activate PKC, such as bombesin, vasopressin, or tumor‐promoting phorbol esters. This finding suggests that ATP and these mitogens do not act through a common mechanism. In contrast, ATP was unable to show synergism with phorbol esters in A431 cells. We discovered striking differences when we examined the kinetics of formation of diacylglycerol (DAG) stimulated by ATP among these cell lines. Thus, ATP stimulated a sustained biphasic increase of DAG in A431 cells, but only a rapid transient increase of DAG formation was observed in 3T3 and 3T6 cells. The breakdown of phosphatidylcholine was stimulated by ATP in A431 cells; however, a significantly reduced effect was displayed in 3T6 cells. Furthermore, we found that the diacylglycerol‐kinase inhibitor, 1‐monooleoylglycerol, greatly potentiated ATP‐stimulated DNA synthesis in A431 cells. Finally, down‐regulation of PKC by long‐term exposure to phorbol dibutyrate (PDBu) prevented stimulation of DNA synthesis induced by bombesin, vasopressin, or phorbol esters in 3T3 or 3T6 cells, while it had no such effect on ATP‐stimulated mitogenesis in the presence of insulin or epidermal growth factor. On the other hand, PDBu‐mediated down‐regulation of PKC partially inhibited [3H]thymidine incorporation stimulated by ATP in A431 cells. Taken together, we conclude that a protein kinase C‐dependent pathway is partially involved in ATP‐stimulated DNA synthesis in A431 cells, but a protein kinase C‐independent pathway exists in 3T3 and 3T6 cells. Pertussis toxin (PTX) inhibited the sustained phase of DAG formation and the breakdown of phosphatidylcholine stimulated by ATP in A431 cells. This suggests involvement of a PTX‐sensitive G protein.
Regulation of myosin and overall protein degradation in mouse C2 skeletal myotubes
Authors: Gulve Eric A. , Mabuchi Katsuhide , Dice J. Fred 
Published: February 2005

AbstractWe compared the breakdown of total cellular protein with that of the contractile protein, myosin, in cultured C2 mouse skeletal myotubes. The degradation of long‐lived cellular proteins (which comprise the vast majority of myotube proteins) was inhibited by serum, insulin, and rat insulin‐like growth factor‐2. A physiological concentration of insulin was effective, but most of the effect of insulin occurred at concentrations well above the physiological range. IGF‐2 inhibited protein breakdown at concentrations well within the range of total IGF‐2 known to be present in the serum of fetal and neonatal rats. The breakdown of short‐lived proteins was not altered by insulin or serum. We measured myosin degradation using a monoclonal antibody directed against myosin heavy chain. The half‐life of myosin was 27 hours, and myosin breakdown was not altered by withdrawal of serum. Therefore, the enhanced protein degradation in response to serum withdrawal applies to certain proteins, but not to others.
Macrophage membrane glycoproteins that bind Griffonia simplicifolia I‐B4: Effect on cytotoxicity and protein secretion
Authors: Warfel Alwin H. , Zucker‐Franklin Dorothea , Zheng Zhaoyao Y. 
Published: February 2005

AbstractThioglycollate elicited peritoneal (TG‐Mϕs) but not resident peritoneal Mϕs (R‐Mϕs) were found to bind the lectin Griffonia simplicifolia isotype I‐B4 (GSI‐B4). This was demonstrated by ultrastructural studies and FACS analyses. Membranes from TG‐Mϕs were isolated, separated on SDS‐PAGE, electrotransferred onto nitrocellulose, and exposed to peroxidase‐labeled GSI‐B4. These procedures revealed two major membrane glycoproteins of molecular weights 180,000 and 94,000 daltons that bound the lectin GSI‐B4 which has a specificity for recognizing terminal α‐galactosyl residues. The presence of these epitopes on the two membrane glycoproteins was further substantiated by the fact that treatment of the membranes with α‐galactosidase destroyed their capacity to bind GSI‐B4 and that α‐D‐galactopyranoside but not N‐acetyl‐D‐glucosamine competitively inhibited GSI‐B4 from binding to the glycoproteins. Treatment of TG‐Mϕs with GSI‐B4 reduced the capacity of interferon (IFN) and lipopolysaccharide (LPS), or IFN alone, to induce Mϕ mediated cytotoxicity towards tumor cells by as much as 40%. GSI‐B4 also caused alterations in the pattern of biosynthetically 35S‐methionine labeled secreted proteins as early as 2 hours after contact with TG‐Mϕs. Out of 35 discernible proteins on fluorograms of SDS‐PAGE separated proteins, 5 were down‐regulated and 9 were enhanced. It is suggested that the two novel Mϕ membrane proteins may play a role in regulating the response of Mϕ subpopulations to their humoral and cellular environments.
A serum factor promotes collagenase synthesis by an osteoblastic cell line
Authors: Puccinelli Joseph M. , Omura Terry H. , Strege David W. , Jeffrey John J. , Partridge Nicola C. 
Published: February 2005

AbstractRegulation of the synthesis of collagenase was investigated in the osteoblastic cell line, UMR 106‐01. The cells were stained by the avidin‐biotin‐complex technique for the presence of the enzyme. By this method, it was possible to identify cells producing collagenase. Synthesis, but not secretion, was found to be constitutive in these cells with the enzyme located intracellularly in cytoplasmic vesicles and the Golgi apparatus. The amount of collagenase contained within UMR cells and the number of cells synthesizing the enzyme were proportional to the concentration of fetal bovine serum in the incubating medium. When serum was withdrawn from the osteosarcoma cells, the content of collagenase decreased with time and the enzyme became undetectable by 48 h of serum depletion. The decrease in collagenase content could be completely reversed by resupplying serum to the cells. The collagenase promoting activity of serum could not be eliminated by adsorption on activated charcoal but was retained by a dialysis membrane with a 12,000 mol wt cutoff. A range of bone‐seeking hormones or agents known to affect collagenase secretion was added to the medium in an attempt to mimic the effect of serum on collagenase accumulation. None of these agonists, including parathyroid hormone, could reproduce the effect of serum on these cells, although parathyroid hormone could act as a collagenase secreta‐gogue in the presence or absence of serum. It is concluded that fetal bovine serum contains a yet unidentified factor or factors greater than 12,000 mol wt responsible for the continued synthesis of collagenase by UMR 106‐01 cells.
Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast
Authors: LaFond J. , LeClerc M. , Brunette M. G. 
Published: February 2005

AbstractWe have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full‐term human placenta. These purified membranes were enriched 25‐fold in Na+/ K+‐adenosine triphosphatase (ATPase), 37‐fold in [3H]dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis‐Menten kinetics, with a Km Ca2+ of 0.18 ± 0.05 μM and Vmax of 0.93 ± 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration‐dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver‐Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP‐independent Ca2+ uptake was not sensitive to 10−6 M nitrendipine nor to 10−4 M verapamil. An ATP‐dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 ± 0.02 μM and Vmax 3.4 ± 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10−6 M calmodulin strongly enhanced the initial ATP‐dependent Ca2+ uptake. Finally, no Na+ /Ca2+ exchange process could be demonstrated.