bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of specific proteases associated with the surface of human skin fibroblasts, and their modulation in pathology
Authors: Raynaud Françe , Bauvois Brigitte , Gerbaud Pascale , Evain‐Brion Danièle 
Published: February 2005

AbstractHuman skin fibroblasts were probed for cell surface protease activity. One activity removing dipeptides from the NH2‐terminal end of Gly‐Pro‐pNA was specifically inhibited by di‐isopropyl‐fluorophosphate (DFP), phenylmethanesulphonyl fluo‐ride (PMSF), and diprotin A, and thus was identified as dipeptidyl peptidase IV (DPP IV). A group of bestatin‐sensitive N‐exoaminopeptidase activities was also characterized when Ala‐, Leu‐, and Arg‐pNA were used as chromogenic substrates. Using human monoclonal antibodies anti‐CD 13 and anti‐CD 26 that recognized, respectively, an N‐Ala‐aminopeptidase and DPP IV, it was found that human dermal fibroblasts expressed the CD 13 and CD 26 antigen on their surface. In addition, both peptidases were specifically immunoprecipitated by monoclonal antibodies anti‐CD 13 and anti‐CD 26 from plasma membranes. Cell surface proteolytic activities were also investigated in human fibroblasts derived from dermatological and rheumatic diseases (i.e., psoriasis, rheumatoid arthritis, and lichen planus). It was found that these fibroblasts also expressed both types of proteinases initially identified on normal skin fibroblasts and that the levels of Ala‐aminopeptidase activities were similar in all cases. In contrast, the levels of Arg‐, Leu‐exoaminopeptidase, and DPP IV activities were significantly higher (up to 6.6‐fold) in the three pathological fibroblast populations than in their normal counterparts. These proteolytic enzymes, therefore, can potentially serve as markers in dermatological diseases. Taken together, our results suggest that skin fibroblast‐derived proteinases associated with both serine and N‐aminopeptidase activities may play an important role by participating in the extracellular events associated with fibroblast behaviour. © 1992 Wiley‐Liss, Inc.
Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH
Authors: Casciari Joseph J. , Sotirchos Stratis V. , Sutherland Robert M. 
Published: February 2005

AbstractTumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion. © 1992 Wiley‐Liss, Inc.
Fibroblast growth factor upregulates PGG/H synthase in rabbit microvascular endothelia cells by a glucocorticoid independent mechanism
Authors: Moatter Tariq , Gerritsen Mary E. 
Published: February 2005

AbstractThe present study was undertaken to determine the effects of acidic fibroblast growth factor (aFGF) on eicosanoid synthesis in microvessel endothelial cells derived from rabbit left ventricular muscle (RCME cells). We observed that aFGF increased AA conversion to PGE2 in a time‐ and dose‐dependent manner, and the stimulatory effect was abolished by actinomycin D and cycloheximide. Acidic FGF increased the recovery of PGG/H synthase activity following aspirin treatment, suggesting an action on de novo PGG/H synthase synthesis. Acidic FGF increased the incorporation of [35S] methionine into a 70 kD immunoreactive PGG/H synthase band. PGG/H synthase synthesis following aspirin treatment was also increased by transforming growth factor β, while epidermal growth factor basic FGF and platelet derived growth factor were without effect. In addition, the actions of aFGF on de novo PGG/H synthase were compared in several endothelial preparations. Acidic FGF treatment of aspirin treated endothelial cells from rabbit lung microvessels and small pulmonary artery and from human lung microvessels all showed an increase in PGG/H synthase recovery. In contrast, similar treatment of human umbilical vein endothelial cells was without effect. Pretreatment of RCME cells with dexamethasone (1 μM) did not alter the aFGF induction of PGG/H synthase activity. We conclude that aFGF stimulates PGE2 production by a mechanism that includes the de novo synthesis of PGG/H synthase. This mechanism appears to be distinct from previously described glucocorticoid sensitive translational controls of PG synthase synthesis by epidermal growth factor in smooth muscle and mesangial cells. © 1992 Wiley‐Liss, Inc.
A high level of cell surface phosphatidylinositol‐specific phospholipase C activity is characteristic of growth‐arrested 3T3 fibroblasts but not of transformed variants
Authors: Volwerk Johannes J. , Birrell G. Bruce , Hedberg Karen K. , Griffith O. Hayes 
Published: February 2005

AbstractConfluent monolayers of four contact‐inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol‐specific phospholipase C (ecto‐PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio‐labeled in the inositol group ([3H]‐lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto‐PLC activity, rapid uptake of [3H]‐lysoPI is accompanied by the simultaneous appearance of [3H]‐inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density‐dependent growth inhibition had levels of ecto‐PLC activity in the range of 50–800 pmol/min/106 cells (i.e., about 20–50 times greater than the activity observed for the transformed variants). The expression of ecto‐PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact‐inhibited mouse fibroblasts and the expression of inositol phospholipid‐specific phospholipase C activity at the external cell surface. © 1992 Wiley‐Liss, Inc.
Heterodimerization and transcriptional activation in vitro by NF‐κB proteins
Authors: Cohen Lucie , Hiscott John 
Published: February 2005

AbstractThe NF‐κB family of transcription proteins represents multiple DNA binding, rel related polypeptides that contribute to regulation of genes involved in immune responsiveness and inflammation, as well as activation of the HIV long terminal repeat. In this study multiple NF‐κB related polypeptides ranging from 85 to 45 kDa were examined for their capacity to interact with the PRDII regulatory element of interferon β and were shown to possess distinct intrinsic DNA binding affinities for this NF‐κB site and form multiple DNA binding homo‐ and heterodimer complexes in co‐renaturation experiments. Furthermore, using DNA templates containing two copies of the PRDII domain linked to the rabbit β globin gene the purified polypeptides specifically stimulated NF‐κB dependent transcription in an in vitro reconstitution assay as heterodimers but not as p50 homodimers. These experiments emphasize the role of NF‐κB dimerization as a distinct level of transcriptional control that may permit functional diversification of a limited number of regulatory proteins. © 1992 Wiley‐Liss, Inc.
Effect of osmolality on 86Rb+ uptake and release by Leishmania donovani
Authors: Blum J. J. 
Published: February 2005

AbstractPromastigotes from late‐log phase cultures of Leishmania donovani were washed and resuspended in Hanks' Balanced Salt Solution without glucose or phenyl red but with 20 mM (N‐[2‐hydroxyethyl] piperazine‐N'‐[2‐ethanesulfonic acid]) (HEPES) (HBSS−, 305 mOsm/kg). They were then added to a solution containing 86Rb such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular 86Rb was measured. Similarly, experiments were performed in which 86Rb was added to the cultures about 18 hr before collection, and the amount of 86Rb released from the washed cells was measured. Under iso‐osmotic conditions only about 1.3% of the intracellular 86Rb was released in 900 sec. This increased about 4‐fold if the osmolality was reduced from 305–153 mOsm/kg. This is much slower than the very rapid release of alanine in response to hypo‐osmotic stress, indicating that alanine release is not via a non‐specific pore. Reducing the temperature from 26°C to 3–4°C completely inhibits 86Rb release under iso‐osmotic conditions and largely inhibits it under hypo‐osmotic conditions. The rate of 86Rb release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either 86Rb uptake or release, but carbonylcyanide P‐trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of 86Rb release and, after about a 300 sec exposure, completely inhibited 86Rb uptake. Amiloride partially inhibited 86Rb release, but had no effect on uptake. A decrease in pH from 7.1–5.9 had little effect on 86Rb release under iso‐osmotic conditions and slightly increased the rate of release under hypo‐osmotic conditions, but it decreased the rate of uptake under both iso‐osmotic and hypo‐osmotic conditions. Cells taken from 3‐day stationary phase cultures released 86Rb more slowly under iso‐osmotic conditions than cells from late log phase cultures, but were more responsive to hypo‐osmotic stress than were log phase cells. These data appear to rule out an [Na‐K‐Cl] transporter or a [K‐Cl] cotransporter as the means of K+ release, but are consistent with the possiblity that a K+/H+ exchanger is present. The possibility that other carrier systems may be present is also discussed. © 1992 Wiley‐Liss, Inc.
Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells
Authors: Van Dolah Frances M. , Ramsdell John S. 
Published: February 2005

AbstractOkadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration‐dependent manner with a half‐maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40–60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2‐M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S‐phase. The absence of a change in the proportion of S‐phase cells indicates that G1 ‐specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti‐tubulin demonstrated that 10 nM okadaic acid‐treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid‐treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression. © 1992 Wiley‐Liss, Inc.
Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism
Authors: Waters Christopher M. , Overholser Knowles A. , Sorkin Alexander , Carpenter Graham 
Published: February 2005

AbstractThe E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21–32, 1989). To determine whether E5 affects EGF:receptor interactions, we analyzed the kinetics of 125I‐EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I‐EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy‐independent in both cell lines. The 125I‐EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy‐dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I‐EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy‐independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor. © 1992 Wiley‐Liss, Inc.
Interleukin‐1β induction of TNF‐α gene expression: Involvement of protein kinase C
Authors: Bethea John R. , Gillespie G. Yancey , Benveniste Etty N. 
Published: February 2005

AbstractIn the human astroglioma cell line CH235‐MG, interleukin‐1β (IL‐1β) induces transcriptional activation of the tumor necrosis factor‐alpha (TNF‐α) gene, resulting in expression of TNF‐α mRNA and biologically active TNF‐α protein. This study was undertaken to elucidate intracellular signaling pathways involved in IL‐1β induction of the TNF‐α gene. We demonstrated that the protein kinase C (PKC) activator 4β‐phorbol 12β‐myristate 13α‐acetate (PMA) in concert with Ca++ ionophore A23187 induced expression of TNF‐α mRNA and protein, whereas an inactive PMA analogue (αPMA) had no effect. Various cyclic nucleotide activators such as 8‐Bromo cAMP, cholera toxin, and forskolin had no effect on TNF‐α production. Two PKC inhibitors, H7 and staurosporine (SS), abrogated IL‐1β induced TNF‐α expression in a dose‐dependent fashion. Treatment of CH235‐MG cells with a high concentration of PMA (1 μM) for an extended period of time (48 h) caused a greater than 90% reduction in total PKC activity. Further strengthening a role for PKC in this cytokine response is the fact that IL‐1β was no longer able to induce TNF‐α expression in these PKC depleted cells. Last, IL‐1β treatment produced an increase of total PKC activity in CH235‐MG cells. Taken together, these data demonstrate that IL‐1β induces TNF‐α gene expression in CH235‐MG cells in a PKC‐dependent manner. © 1992 Wiley‐Liss, Inc.
Plasminogen activator regulation in osteoblasts: Parathyroid hormone inhibition of type‐1 plasminogen activator inhibitor and its mRNA
Authors: Fukumoto Seiji , Allan Elizabeth H. , Yee John A. , Gelehrter Thomas D. , Martin T. John 
Published: February 2005

AbstractIn order to determine the mechanism by which parathyroid hormone (PTH) stimulates plasminogen activator (PA) activity in rat osteoblasts, we investigated the effect of human PTH(1–34) [hPTH(1–34)] on the synthesis of mRNAs for tissue‐type PA (tPA), urokinase‐type PA (uPA), and PA inhibitor‐1 (PAI‐1), and on release of PA activity and PAI‐1 protein in both normal rat calvarial osteoblasts and UMR 106‐01 osteogenic sarcoma cells. hPTH(1–34) (0.25–25 nM) decreased PAI‐1 mRNA and protein, and increased PA activity in both cell types in a dose‐dependent manner with ED50 of about 1 nM for both responses. Forskolin and isobutylmethylxanthine also stimulated PA activity and decreased PAI‐1 protein and mRNA in both cell types. hPTH(1–34) did not show any consistent effect on tPA and uPA mRNA in calvarial osteoblasts, but a modest (two‐fold) increase of both mRNAs was observed in UMR 106‐01 cells treated with 25 nM hPTH(1–34). However, when protein synthesis was inhibited with 100 μM cycloheximide, the increase of tPA and uPA mRNA by hPTH(1–34) was enhanced in UMR 106‐01 cells and became evident in calvarial osteoblasts. Fibrin autography also revealed that hPTH(1–34) increases tPA and uPA activity, especially after cycloheximide treatment in UMR 106‐01 cells. These results strongly suggest that PTH increases PA activity predominantly by decreasing PAI‐1 protein production through a cyclic adenosine monophosphate (cAMP)‐dependent mechanism in rat osteoblasts. The reduction of PAI‐1 protein by PTH results in enhanced action of both tPA and uPA, and would contribute to the specific roles of these PAs in bone. © 1992 Wiley‐Liss, Inc.
Synergistic actions of epidermal growth factor‐urogastrone and vasopressin in cultured aortic A‐10 smooth muscle cells
Authors: Mokashi Shalini , Severson David L. , Hollenberg Morley D. 
Published: February 2005

AbstractIn cultured rat aorta‐derived A‐10 cells, epidermal growth factor‐urogastrone (EGF‐URO) acts synergistically with arginine vasopressin (AVP) to augment the AVP‐mediated release of 3H‐arachidonate (3H‐AA) from 3H‐AA prelabeled cells. On its own, EGF‐URO had no effect on AA release and had no effect on calcium influx or efflux either in the absence or presence of AVP. The synergistic action of EGF‐URO was not affected by actinomycin D, cycloheximide, indomethacin, by the diacylglycerol lipase inhibitor U‐57, 908, or by the tyrosine kinase inhibitors genistein (GS) and tyrphostin (TP). TP did, nonetheless, completely abrogate 3H‐thymidine incorporation triggered in the presence of EGF‐URO. Although EGF‐URO stimulated an increase in calpactin‐II (lipocortin‐I) phosphorylation in permeabilized cells, no such increase was detected in intact cells exposed to EGF‐URO either alone or in combination with AVP, under conditions where EGF‐URO augmented the action of AVP. The phospholipase A2 inhibitor, mepacrine, had no effect on AVP‐mediated AA release, but abolished the synergistic action of EGF‐URO. We conclude that in contrast with our previous results with gastric smooth muscle strips, wherein EGF‐URO acts via the diacylglycerol lipasemediated metabolism of diacylglycerol, and in keeping with observations with cultured mesangial cells, EGF‐URO acts synergistically with AVP in A‐10 cells via the activation of phospholipase A2. This synergistic action of EGF‐URO does not appear to be due to increased levels of cyclooxygenase and would appear not to require increased tyrosine kinase activity. © 1992 Wiley‐Liss, Inc.
Inhibition of angiogenesis in vitro and in ovo with an inhibitor of cellular protein kinases, MDL 27032
Authors: Wright Paul S. , Cross‐Doersen Doreen , Miller Jerry A. , Jones Winton D. , Bitonti Alan J. 
Published: February 2005

AbstractProtein kinase C (PKC) was implicated as an important positive regulator of angiogenesis by studies showing that tumor promoting phorbol esters, which activate PKC, stimulate angiogenesis both in vitro and in vivo. Therefore, inhibitors of PKC might be expected to block angiogenesis. MDL 27032 [4‐propyl‐5‐(4‐pyridinyl)‐2(3H)‐oxazolone], an inhibitor of cellular protein kinases, prevented capillary‐like tube formation by human umbilical vein endothelial cells (HUVEC) on basement membrane preparations, an in vitro model for angiogenic activity. MDL 27032 had an IC50 = 50 μM, whereas MDL 27044, the 4‐methyl analog of MDL 27032, was less effective (IC50 > 100 μM). This selectivity was reflected in the relative abilities of the two compounds to inhibit PKC and protein kinase A (PKA) activity prepared from HUVEC, and also to inhibit the basic fibroblast growth factor stimulated proliferation of HUVEC. MDL 27032 (0.3 μg/egg) also significantly inhibited neovascularization in yolk sac membranes of developing chick embryos, whereas MDL 27044 added at concentrations up to 3 μg/egg was not inhibitory when compared with vehicle treated controls. Adhesion of HUVEC to individual extracellular matrix proteins, including laminin, fibronectin, and fibrinogen, but not to the mixture of matrix components or collagen type I and IV, was inhibited after treatment with MDL 27032. These studies suggest that MDL 27032, may have potential as an anti‐angiogenic agent because it disrupts both formation of tube‐like structures by HUVEC on Matrigel and normal neovascularization in ovo. This inhibition may in part be due to altered cellular interactions with the extracellular matrix. © 1992 Wiley‐Liss, Inc.