bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of plasminogen, plasmin, and plasminogen activators in the migration of fibroblasts into plasma clots
Authors: Knox Peter , Crooks Sheila , Scaife Michael C. , Patel Sandhiya 
Published: February 2005

AbstractHuman diploid fibroblasts were seeded onto or into plasma clots and different aspects of cell adhesion and migration were measured. The roles of plasminogen activators and plasmin were studied by either the removal of plasminogen from plasma prior to clotting or by the addition of 10 mM ϵ‐aminocaproic acid, which brings about an inhibition of plasmin in this system. When cells were seeded onto the surface of plasma clots, rates of attachment, spreading, and migration were unaffected by plasminogen depletion or plasmin inhibition. In contrast, when cells were seeded into plasma clots, then, although the rates of cells spreading were unaffected, cell migration was abolished by plasminogen depletion or by plasmin inhibition. When cells were seeded onto the surface of plasma clots and the rate of migration into the clots was measured, there was an absolute requirement for plasmin activity; while fibroblasts migrated rapidly into the fibrin lattice of control clots, in the case of plasminogen‐depleted clots, cells failed to penetrate the lattice. Focussing through a plasma clot revealed that fibroblasts do not migrate through the fibrin lattice but instead, localized areas of fibrinolysis are generated and cells migrate over the surface of the area of lysis.
Phorbol ester induces cultured endothelial cells to invade a fibrin matrix in the presence of fibrinolytic inhibitors
Authors: Montesano R. , Pepper M. S. , Vassalli J.‐D. , Orci L. 
Published: February 2005

AbstractWe have previously shown that the tumor promoter 4β‐phorbol 12‐myristate 13‐acetate (PMA) induces capillary endothelial cells grown to confluency on the surface of three‐dimensional collagen gels to invade the underlying matrix and to form capillarylike tubular structures, a phenomenon mimicking angiogenic processes that occur in vivo (Montesano and Orci: Cell, 42:469–477, 1985). Since angiogenesis frequently occurs within a fibrin‐rich extracellular matrix, we have examined the ability of PMA‐treated endothelial cells to invade fibrin gels. Control endothelial cells grown on fibrin gels formed a confluent monolayer on the gel surface and did not invade the underlying matrix. Treatment of the cultures with PMA resulted in a progressive lysis of the substrate without invasion of the fibrin matrix. However, if the cells were treated with PMA either in the presence of fibrinolytic inhibitors (Trasylol, ϵ‐aminocaproic acid) or in the absence of detectable plasminogen, dissolution of the substrate was prevented, and the endothelial cells invaded the fibrin gel, forming vessellike tubular structures similar to those previously observed with collagen gels. These results demonstrate that the invasive and morphogenetic events induced by PMA do not necessarily require an interaction between endothelial cells and collagen fibrils but can also occur with other biologically relevant substrata. They also suggest (1) that invasion may occur via a plasmin‐independent mechanism and (2) that in vivo, neutralization of excess proteolytic activity may play an important permissive role in angiogenesis and other invasive processes by preventing uncontrolled matrix degradation.
Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: Modulation by thyrotropin
Authors: Chambard Marianne , Mauchamp Jean , Chabaud Odile 
Published: February 2005

AbstractIn the thyroid glands, thyroglobulin (Tg) is specifically synthesized by follicular cells and then secreted into the apical lumen where it is concentrated and used as a substrate for thyroid hormone synthesis. The presence of Tg in the circulation has been reported in normal and pathological situations. To determine the domains of the plasma membrane, apical and/or basolateral, involved in Tg secretion, porcine thyroid epithelial cells were cultured as a monolayer on the porous bottom of a culture chamber in which both apical and basal media are independently accessible. Control experiments using labeled Tg ascertained the tightness of the monolayer and showed that within 48 h only 0.2–0.5% of the Tg introduced in the apical medium was transferred through the cell layer into the basal compartment. For kinetic studies of Tg synthesis and secretion, monolayers were cultured for up to 72 h in the presence of 35S‐methionine and with or without 100 μU/ml thyrotropin (TSH) in the basal medium. Labeled Tg was measured by double immunoprecipitation and by fluorography of polyacrylamide gel electrophoresis. We showed that 80–95% of total secreted Tg was recovered in the apical medium. The remainder was secreted through the basolateral membranes in the basal medium. The amount of Tg secreted into the apical compartment was stimulated two‐ to threefold by TSH whereas no TSH effect was observed on secretion in the basal compartment. Moreover, measuring apical and basal volumes, we observed a net water flow from the apical to the basal side. It was stimulated threefold by TSH, increasing the Tg concentration in the apical compartment of the stimulated cell layer. During the culture time, the amount of Tg synthesized and secreted was increased by TSH, as was the Tg mRNA content, as determined by the dot‐blot hybridization method.
Cyclic activity of the receptors of cobalamin bound to transcobalamin II
Authors: Hall Charles A. , Colligan Pamela D. , Begley James A. 
Published: February 2005

AbstractThe activity of receptors specific for human transcobalamin II‐Cobalamin (TC II‐Cbl) were measured in virus‐transformed lymphoblasts, hepatocytes (hepatoma) and diploid fibrolasts. In all three types of human cells the receptor activity increased as cells went from a resting phase to the most actively dividing phase. Receptor activity declined as cell division slowed. The changes in activity of lymphoblasts and hepatocytes were produced by changes in receptor numbe and not by changes in affinity between receptors and TC II‐Cbl. The basis of the change in fibroblasts was not clear. The Cbl‐dependent methionine synthetase activity of fibroblasts, in contrast, tended to be greatest when the cultures were confluent and replication had slowed. As the fibroblasts became senescent the receptor activity for TC II‐Cbl declined and the fluctuations with the phase of the cell were blunted. However, the release of apo TC II from the cells was maintained. These observations must be taken into consideration when the respective cells are used as models. Even more important are the implications of the observations of the changes in receptor activity for TC II‐Cbl for the regulation of the entry of Cbl into cells.
Neutrophil C3bi receptors: Formation of membrane clusters during cell triggering requires intracellular granules
Authors: Petty Howard R. , Francis Joseph W. , Todd Robert F. , Petrequin Pamela , Boxer Laurence A. 
Published: February 2005

AbstractVideo‐intensification fluorescence microscopy has been used to study the cell surface distribution of the complement receptor (CR) for C3bi (CR3) on human neutrophils. Fluorescin‐ or rhodamine‐labeled monoclonal IgG or Fab fragments of antireceptor antibody were used as probes of receptor localization. C3bi receptors are uniformly distributed on untreated cells. Glass coverslips were coated with lipopolysaccharide (LPS) and serum was added; the serum deposits complement components, including C3bi, on the surface. When neutrophils were adherent to these coverslips, receptors were found in large clusters, and a fraction of the fluorescence remained uniform. Double‐labeling studies were conducted by first labeling with anti‐CR3 followed by attachment to LPS/serum‐treated slides. This, in turn, was followed by labeling with the antibody conjugated to a second fluorophore. These studies revealed that the CR3 clusters were predominantly new antigenic sites exposed after attachment to the LPS/serum‐treated slides. To determine the contribution of granule‐associated CR3, we have studied neutrophils defective in receptor up‐regulation, neutrophil cytoplasts, and a stimulator of granule release, A23187. Neutrophils from a patient with specific granule deficiency were found to be defective in granular CR3 and did not form clusters on C3‐modified surfaces. The patient's neutrophils were defective in CR3 up‐regulation and enzyme release as shown by fluorescence flow cytometry and gelatinase release, respectively. Cytoplasts also failed to show CR3 clusters on LPS/serum‐treated coverslips. Furthermore, neutrophils treated with A23187 demonstrated numerous CR3 clusters. We suggest that formation of CR3 membrane domains during immune recognition requires the participation of intracellular granules. We speculate that these domains are formed by fusion of CR3‐bearing granules at local sites of adhesion.
Regulation of glutathione levels in mouse spleen lymphocytes by transport of cysteine
Authors: Ishii Tetsuro , Sugita Yoshiki , Bannai Shiro 
Published: February 2005

AbstractCysteine and cystine transport activities of resting and activated mouse spleen lymphocytes were characterized in order to examine the contributions of cysteine and cystine to intracellular glutathione contents. Following stimulation with lipopolysaccharide, the lymphocytes markedly increased their capacity to transport cysteine. The uptake of cysteine was mediated mainly by the ASC system (Na+‐dependent neutral amino acid transport system especially reactive with alanine, serine, and cysteine). On the other hand, both the resting and the activated lymphocytes had extremely low cystine transport activities. Because of the instability of cysteine, the culture media usually contained cystine but not cysteine. Therefore, both the resting and the activated lymphocytes rapidly decreased their glutathione contents owing to their poor capacities to take up cystine. The effects of freshly added cysteine on the cellular glutathione contents were examined in the presence of bath‐ocuproinedisulfonate, a nontoxic copper‐specific chelator that inhibits autoxidation of cysteine. Cysteine added at 25–400 μM only partially prevented the rapid decrease of the glutathione contents in fresh resting lymphocytes. In the lipopolysaccharide‐activated cells, however, cysteine enhanced the cellular glutathione contents in a dose‐dependent manner. These results indicate that the enhanced activity of the ASC system increases the level of intracellular glutathione in the presence of cysteine.
Heparin potentiates the action of plasma membrane‐associated growth stimulatory activity
Authors: Nagasaki Takayuki , Lieberman Michael A. 
Published: February 2005

AbstractPlasma membranes prepared from mouse liver have been previously shown to contain growth stimulatory activity as determined with cultured mouse fibroblasts. This growth stimulatory activity, termed plasma membrane‐associated growth stimulatory activity (PMGA), is highly mitogenic in the presence of platelet‐poor plasma. We now demonstrate that the growth stimulatory action of PMGA is dramatically enhanced by the addition of heparin. The half‐maximal effect of heparin was observed at 1–3 μg/ml. The synergistic effect was seen in two distinct assays; the stimulation of DNA synthesis in quiescent cells, and an increase of cell number over a 3‐day culture period. Heparin, by itself, does not have any measurable influence on the growth of fibroblasts. The action of heparin is not unique to this glycosaminoglycan, as several other highly sulfated polysaccharides, including dextran sulfate, pentosan polysulfate, and fucoidan, also exhibited the highly synergistic effect. Among other glycosaminoglycans examined, chondroitin sulfate B and heparan sulfate had a small, but significant, effect on enhancing the growth stimulatory action of PMGA. Chondroitin sulfate A, chondroitin sulfate C, hyaluronic acid dextran, and poly‐L‐glutamic acid, however, had no detectable effect. Further experiments suggested that the effect of heparin is twofold, namely, both a potentiation of growth stimulatory activity and a protection of PMGA activity. The data presented here suggest that the association of various cell surface components, such as PMGA and specific proteoglycans, can modulate the growth potential of a cell.
Quantitation and tissue localization of the cellular retinoic acid‐binding protein
Authors: Eriksson Ulf , Hansson Eva , Nordlinder Hans , Busch Christer , Sundelin Johan , Peterson Per A. 
Published: February 2005

AbstractThe distribution of the cellular retinoic acid‐binding protein (CRABP) in some rat tissues has been determined, and the protein has been localized by immunocytochemical techniques in sections from rat testis. In the testis CRABP was found in the seminiferous tubuli with Sertoli cells and the spermatogonia most intensely stained. All other cells of the germinal epithelium appeared largely devoid of CRABP. By use of an enzyme‐linked immunosorbent assay CRABP was quantitatively estimated in several tissues and the highest levels were found in testis and eye. Comparisons of the tissue levels of CRABP and of the cellular retinol‐binding protein (CRBP) did not reveal any apparent correlation.
Effects of polyamine depletion on serum stimulation of quiescent 3T3 murine fibroblast cells
Authors: Schaefer Eugene L. , Seidenfeld Jerome 
Published: February 2005

AbstractNumerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine‐dependent steps within this defined sequence, mouse Balb/c‐3T3 fibroblasts were partially depleted of polyamines by treatment with DL‐α‐difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]‐thymidine, polyamine‐depleted cells had labeling indices similar to quiescent cells if they were serum‐stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine‐dependent steps occur during the pre‐commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post‐commitment), polyamine‐depleted quiescent cells were serum‐stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre‐commitment events. However, polyamine‐deficient quiescent cells serumstimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the precommitment period.
Messenger RNAs coding for enzymes of polyamine biosynthesis are induced during the G0‐G1 transition but not during traverse of the normal G1 phase
Authors: Stimac Eva , Morris David R. 
Published: February 2005

AbstractThe events occurring during emergence of cells from quiescence ( “G0”) are not necessarily identical to those in the G1 phase of continously dividing cells. Cellular levels of the mRNAs coding for ornithine decarboxylase (ODC) and S‐adenosyl‐methionine decarboxylase (SDC), key enzymes in polyamine synthesis, increased maximally within 5 h after addition of serum to resting 3T3 cells, following a kinetic course similar to that of c‐myc mRNA. In a pure early G1 population of cells, prepared by centrifugal elutriation of growing fibroblasts, the levels of ODC and SDC mRNAs were not significantly lower than in other phases of the cell cycle and approximated serum‐induced levels rater than the reduced values found in serum‐starved cells. Thus, we conclude that the mRNAs coding for the polyamine biosynthetic enzymes, like c‐myc, are growth controlled, but not regulated during traverse of a normal cell cycle.
Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formation: Its relationship to type β transforming growth factor (TGF‐β)
Authors: Mitjavila M. T. , Vinci G. , Villeval J. L. , Kieffer N. , Henri A. , Testa U. , Breton‐Gorius J. , Vainchenker W. 
Published: February 2005

AbstractWhole blood serum (WBS) and platelet‐poor plasma‐derived serum (PDS) from the same normal subject were compared for their abilities to support human megakaryocyte (MK) colony formation. In all cases, PDS promoted the growth of a higher number (20–50%) of MK colonies than did WBS. Increasing amounts of WBS decreased the number of colonies, whereas increasing concentration of PDS had no marked effects. Crude platelet extracts or platelet secretory products from thrombin‐activated platelets also elicited an inhibition of MK colony formation in a dose‐dependent manner. A complete inhibition was found for a dose equivalent to 1.109 platelets/ml and a 50% inhibition in a range of 1.107–1.108 platelets/ml. These platelet products were also inhibitory for erythroid progenitor growth. Platelets from two patients with gray platelet syndrome elicited only a minor inhibition of MK growth, suggesting that the platelet alpha granule is the origin of this inhibition. When platelet extracts were acid‐treated, the biological activity of the inhibitor on CFU‐MK and CFU‐E growth was 20–50‐fold higher. In addition, a potent stimulatory activity on the growth of day 7 CFU‐GM was observed. The enhancement of biological activities by acid treatment suggests that type β transforming growth factor (TGF‐β) could be involved in this platelet inhibitory activity. The homogeneous native TGF‐β (from 1 pg to 1 ng/ml) produced the same effects previously induced by platelet products. It totally inhibited CFU‐MK growth (at a 500 pg/ml), it inhibited CFU‐E growth, and it stimulated growth of day 7 CFU‐GM in the presence of a colony‐stimulating factor. The inhibition of CFU‐MK growth was also observed on purified progenitors. In conclusion, these results suggest that TGF‐β may be implicated in negative autocrine regulation of megakaryopoiesis. However, since this molecule has ubiquitous biological activities, its physiologic relevance as a normal regulator of megakaryopoiesis requires further investigation.
Plasma membrane fluidity measurements in intact endothelial cells: Effect of hyperoxia on fluorescence anisotropies of 1‐[4‐(trimethylamino)phenyl]‐6‐phenyl hexa‐1,3,5‐triene
Authors: Sheridan N. P. , Block E. R. 
Published: February 2005

AbstractFluorescence anisotropy measurements are widely used as sensitive indicators of cell membrane fluidity. 1‐[4‐(trimethylamino)phenyl]‐6‐phenyl hexa‐1,3,5‐triene (TMA‐DPH) is a cationic fluorescent aromatic hydrocarbon that anchors at the lipid‐water interface of membrane lipid bilayers. Its uptake into porcine pulmonary artery and aortic endothelial cells was monitored and the probe remained specifically localized on the cell surface for at least 4 h. It can therefore be recommended for use for specific plasma membrane lipid fluidity measurements in these cells. The effect of hyperoxia on plasma membrane fluidity was measured by using TMA‐DPH. In both cell types, hyperoxic damage resulted in decreases in plasma membrane fluidity. Recovery was achieved 48 h after a 42‐h hyperoxic exposure. These results indicate that TMA‐DPH is a sensitive probe of plasma membrane lipid domains of pulmonary artery and aortic endothelial cells and that hyperoxia causes reversible changes in the physical state of superficial lipid domains of the plasma membrane of these cells.
Joint oxygen‐glucose deprivation as the cause of necrosis in a tumor analog
Authors: Hlatky Lynn , Sachs Rainer K. , Alpen Edward L. 
Published: February 2005

AbstractThe sandwich system was recently developed as an in vitro tumor analog. Like spheroids, sandwiches are organized, multicellular systems in which the interplay between diffusion and consumption leads to the formation of spatial gradients; a necrotic center and a viable cell border subsequently develop. Using sandwiches of the 9L and V79 cell lines, the effects of oxygen and glucose deprivation on the onset and formation of necrosis were investigated. The data indicate that in sandwiches necrosis is a result of a shortage of both substances. Complementary cell monolayer experiments to determine a number of consumption parameters were performed. On the basis of the data, we propose a joint oxygen‐glucose deprivation model for V79 cell necrosis. It is assumed a cell dies when oxygen deprivation in conjunction with glucose deprivation lowers the cell's ATP production rate below a critical value. Interactions of the concentrations and consumptions of oxygen and glucose are analyzed theoretically; concentration profiles are obtained by numerically solving coupled non‐linear integral equations arising from the diffusion equation. The predicted viable border widths are in good agreement with the observed values.
Localization of protease nexin‐1 on the fibroblast extracellular matrix
Authors: Farrell David H. , Wagner Steven L. , Yuan Robert H. , Cunningham Dennis D. 
Published: February 2005

AbstractProtease nexin‐1 (PN‐1) is a protease inhibitor that is secreted by fibroblasts and several other cultured cells. PN‐1 forms complexes with certain serine proteases in the extracellular environment including thrombin, urokinase, and plasmin. The complexes then bind to the cells and are rapidly internalized and degraded. This report demonstrates that PN‐1 is present on the surface of fibroblasts, bound to the extracellular matrix. Immunofluorescent studies showed that PN‐1 colocalized with fibronectin on both intact cells and in preparations of extracellular matrix made from these cells. In contrast, PN‐1 did not colocalize with the epidermal growth factor receptor, a plasma membrane marker. An enzyme‐lined immunosorbent assay was developed which showed that the extracellular matrix contained at least 60–80% of the cellular immunoreactive PN‐1. Extraction of the matrix with 2 M NaCl removed PN‐1 in a form which reacted with 125l‐thrombin to form complexes which were immunoprecipitated by anti‐PN‐1 lgG and were of identical size as complexes made from soluble PN‐1 and 125l‐thrombin. These data indicate that in addition to its role as a soluble protease inhibitor, PN‐1 is also a component of the extracellular matrix and might control its proteolysis.