bluuebunny/crossref_metadata_2025 huggingface dataset:
Middle ear cell line that maintains vectorial electrolyte transport
Authors: Herman P. , Cassigena R. , Friedlander G. , Soler P. , Grodet A. , Tran Ba Huy P. , Amiel C.
Published: February 2005

The middle ear epithelium plays a major role in keeping the temporal bone cavities fluid‐free and air‐filled, which is a mandatory condition to allow optimum transmission of the sound vibrations from the tympanic membrane to the inner ear. Previous works have recently established the absorptive function of the middle ear epithelium, using primary cultures derived from Mongolian gerbil (Meriones unguiculatus). Because of the paucity of cells as obtained by enzymatic digestion, we developed a middle ear cell line (MESV) using wild‐type SV40 infection of primary culture of Mongolian gerbil's middle ear epithelial cells. Transformation was attested by nuclear expression of SV40 large T antigen, prolonged in vitro passages (presently beyond 50 passages), and tumor‐inducing ability when subcutaneously injected in athymic mice. Transport properties were evaluated after the fifteenth passage. MESV cells retained most cardinal properties of the original middle ear epithelial cells: cell polarization was evidenced by the presence of mature junctional complexes that separate the cell membrane in two distinct domains, with apical microvilli at the luminal side, and by vectorial sodium transport responsible for the transepithelial lumen‐negative potential difference (−9.3 ± 0.14 mV in culture conditions (n=9), −2.1 ± 0.25 mV after overnight growth factors and serum deprivation). Short‐circuit current was, like in primary cultures, mainly related to a sodium transport occuring through amiloride‐sensitive apical sodium channels, since apical addition of amiloride (10−5 M) reduced Isc from 7.0 = 1.4 to 0.6 ± 0.1 μA/cm2 (P < 0.01, n = 6). Cellular cAMP content was increased by isoproterenol and prostaglandin E2 from 40.5 ± 5.6 to 258.5 ± 17.3 and 55.6 ± 6.2 pmol/mg protein per 5 min, respectively (P < 0.05, n = 10). Isoproterenol and prostaglandin E2 increased Isc with very similar maximal effects: isoproterenol (10−4 M) increased Isc from 5.73 ± 0.31 to 12.77 ± 0.39 μA/cm2, while prostaglandin E2 increased Isc from 5.47 ± 0.21 to 12.87 ± 0.42 (n = 3). Since amiloride (10−5 M) abolished this stimulation, this may be related to an increase of the electrogenic sodium transepithelial transport. The MESV cell line could provide an interesting tool as a model of middle ear epithelial cells for the study of pathophysiological modulations of ion transport. © 1993 Wiley‐Liss, Inc.
Ca2+ dependence of inositol 1,4,5‐trisphosphate‐induced Ca2+ release in renal epithelial LLC‐PK1 cells
Authors: Tshipamba M. , De Smedt H. , Missiaen L. , Himpens B. , Van Den Bosch L. , Borghgraef R.
Published: February 2005

We have studied arginine vasopressin (AVP)‐, thapsigargin‐ and inositol 1,4,5‐trisphosphate (InsP3)‐mediated Ca2+ release in renal epithelial LLC‐PK1 cells. AVP‐induced changes in the intracellular free calcium concentration ([Ca2+]i) were studied in indo‐1 loaded single cells by confocal laser cytometry. AVP‐mediated Ca2+ mobilization was also observed in the absence of extracellular Ca2+, but was completely abolished after depletion of the intracellular Ca2+ stores by 2 μM thapsigargin. Using 45Ca2+ fluxes in saponin‐permeabilized cell monolayers, we have analysed how InsP3 affected the Ca2+ content of nonmitochondrial Ca2+ pools in different loading and release conditions. Less than 10% of the Ca2+ was taken up in a thapsigargin‐insensitive pool when loading was performed in a medium containing 0.1 μM Ca2+. The thapsigargin‐insensitive compartment amounted to 35% in the presence of 110 μM Ca2+, but Ca2+ sequestered in this pool could not be released by InsP3. The thapsigargin‐sensitive Ca2+ pool, in contrast, was nearly completely InsP3 sensitive. A submaximal [InsP3], however, released only a fraction of the sequestered Ca2+. This fraction was dependent on the cytosolic as well as on the luminal [Ca2+]. The cytosolic free [Ca2+] affected the InsP3‐induced Ca2+ release in a biphasic way. Maximal sensitivity toward InsP3 was found at a free cytosolic [Ca2+] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca2+] decreased the InsP3 sensitivity. Other divalent cations or La3+ did not provoke similar inhibitory effects on InsP3‐induced Ca2+ release. The luminal free [Ca2+] was manipulated by varying the time of incubation of Ca2+ ‐loaded cells in an EGTA‐containing medium. Reduction of the Ca2+ content to one‐third of its initial value resulted in a fivefold decrease in the InsP3 sensitivity of the Ca2+ release. © 1993 Wiley‐Liss, Inc.
Neutrophil adhesion to endothelial cells impairs the effects of catalase and glutathione in preventing endothelial injury
Authors: Siflinger‐Birnboim Alma , Malik Asrar B.
Published: February 2005

We studied the effects of the CuZn superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) on endothelial permeability to 125I‐albumin after activation of neutrophils (PMN) with phorbol 12‐myristate‐13‐acetate (PMA; 10−8M). PMN were either in direct contact with the endothelial cell monolayer grown on a porous gelatin‐coated microporous 10‐μm‐thick polycarbonate filter (upright system) or separated from the endothelium by a similar filter (inverted system). Transendothelial 125I‐albumin clearance rates were measured as an index of endothelial permeability. In the absence of antioxidants, activation of PMN increased transendothelial 125I‐albumin clearnace rates in both systems from 0.041 ± 0.006 μl/min (baseline) to 0.262 ± 0.18 μl/min (upright system) and from 0.063 ± 0.02 μl/min to 0.244 ± 0.06 μl/min (inverted system). PMA induced 80–90% of PMN to adhere to either gelatin‐coated filters or to endothelial cells, from the basal PMN adhesion value of 5.3 ± 2.2% and 4.3 ± 1.1%, respectively. SOD, which dismutates superoxide anion to hydrogen peroxide (H2O2), did not alter the transendothelial 125I‐albumin clearance rates in either systm at any concerntration from 10–300 U/ml. CAT (100–1,000 U/ml) and GSH (0.5–10 mM), which remove the H2O2 generated during PMN activation, did not alter the increase in transendothelial 125I‐clearance rates after PMN activation in the upright system, but both agents prvented the increase in transendothelial 125I‐clearance rates in the inverted system. We conclude that PMN activation with PMA causes endothelial injury irrespective of PMN contact to the endothelial monolayer. Moreover, H2O2, a release product of PMN activation, is a critical mediator of PMN‐dependent endothelial injury. Finally, the results indicate that CAT and GSH prevent endothelial injury only in the absence of direct PMN contact with endothelial cells, suggesting that antioxidants such as GSH and CAT are excluded from sites of PMN‐endothelial contact and thus are ineffective antioxidants. © 1993 Wiley‐Liss, Inc.
Interactive effects of nutrients and hormones on the expression of insulin‐like growth factor binding protein‐1 (IGFBP‐1) mRNA and peptide, and IGF I release from isolated adult rat hepatocytes
Authors: Arany E. , Strain A. J. , Hube M. J. , Phillips I. D. , Hill D. J.
Published: February 2005

Isolated adult rat hepatocytes were used to investigate and compare the actions of glucose or amino acids and insulin, glucagon, growth hormone, and dexamethasone on the expression of insulin‐like growth factor binding protein (IGFBP) mRNA, or the release of IGFBP and IGF peptides in vitro. Ligand blot analysis of culture medium conditioned for 24 h by monolayers of hepatocytes in the presence of 6.5 mM glucose revealed two species of IGFBPs, and abundant form of 30‐32 kDa and a minor species of 22‐24 kDa. Western blotting showed that two IGFBPs of 29‐30 and 32 kDa were recognized by antiserum against hIGFBP‐1, whereas hepatocytes contained a 1.6 kb transcript on Northern blot with a rat IGFBP‐1 cDNA. Insulin‐like growth factor BP‐2 mRNA was not detected in hepatocytes and IGFBP‐2 immunoreactive peptide not present in conditioned medium. The release of IGFBP‐1, determined by ligand blot, was independent of gucose concentration over the range of 2.7 mM–11.1 mM, but IGFBP‐1 mRNA was decreased following incubation with 6.5 mM gucose compared with 2.7 mM glucose. The release of IGFBP‐1 by hepatocytes was inhibited by insulin (10nM–1μM), as was mRNA abundance. However, these effects of insulin on IGFBP‐1 diminished with increasing glucose concentration. Increasing concentrations of total amino acids increased IGFBP‐1 release as did dexamethasone (100 pM–100nM), whereas growth hormone and gucagon were without effect. The release of IGF I was increased by insulin, growth hormone and dexamethasone but was decreased by glucagon and amino acids, whereas changes in glucose concentration had no effect. The results show that isolated adult rat hepatocytes release IGF I and IGFBP‐1 under the interactive control of nutrients and hormones involved in metabolic homeostasis. © 1993 Wiley‐Liss, Inc.
Growth promotion of transfected hepatoma cells by liver fatty acid binding protein
Authors: Keler Tibor , Sorof Sam
Published: February 2005

Former studies have linked hepatocyte growth with liver fatty acid binding protein (L‐FABP) of rat liver cytosol. In search for the roles of L‐FABP in hepatocytes, we previously stably transfected rat L‐FABP sense and antisense cDNAs into rat hepatoma HTC cells that do not contain L‐FABP RNA or protein, thereby providing a zero‐background, homologous cell model of L‐FABP‐expression suitable for controlled studies of its intracellular functions in hepatocyte‐derived cells. The present study demonstrates the abilities of L‐FABP to promote DNA synthesis and cell growth, preserve cell morphology, extend survival, and act cooperatively with unsaturated fatty acids in the transfected hepatoma cells in the absence of serum. Following removal of serum, the three control L‐FABP‐nonexpressing cell lines increased in cell lines increased in cell number for 24 hr and thereafter declined, whereas the three L‐FABP‐expressing cell lines exhibited a 39% higher rate of DNA synthesis per cell at 24 hr and grew in cell number for 48 hr. As a result, at 72 hr there were 2.5‐fold (avg.) as many L‐FABP‐expressing cells than L‐FABP‐nonexpressing cells. In addition, the L‐FABP‐expressing cells retained their original polygonal morphology at 48 hr, when in contrast most of the control nonexpressing cells were spherical in shape with membrane blebs. In an effort to identify the agonists that collaborate with L‐FABP in the growth promotion and preservation of cell morphology, various free fatty acids were examined at 48 hr for their ability to elminate the differences in behavior of the two cell types in the serum‐free medium. The unsaturated fatty acids, oleic acid (18:1 ω9), linoleic acid (18:2ω6), α‐linolenic acid (18: 3ω3), and arachidonic acid (20:4ω6), at 1 μM markedly elevated the level of DNA synthesis in the more depressed control L‐FABP‐nonexpressing cells and moderately raised it in the less depressed L‐FABP‐expressing cells. In accord, the control L‐FABP‐nonexpressing cells needed 10−6–10−5 M linoleic acid to achieve the extent of DNA synthesis attained by the expressing cells in the absence of added fatty acid. At 10 μM linoleic acid, their levels of DNA synthesis were equal. In contrast, five saturated fatty acids had no detectable effect on DNA synthesis. In addition, linoleic acid at 1 μM, but not the saturated fatty acid palmitic acid (16:0), prevented the above morphological alterations in the control L‐FABP‐nonexpressing cells observed in the absence of serum, thereby retaining their original polygonal morphology and that of the expressing cells. The findings are consistent with the concept that L‐FABP improves the efficacy of the utilization of unsaturated fatty acid ligands of L‐FABP in the formation, integrity, and fluidity of cell membranes that are involved in cell growth, morphology, and survival. © 1993 Wiley‐Liss, Inc.
Basic fibroblast growth factor and transforming growth factor beta‐1: Synergistic mediators of angiogenesis in vitro
Authors: Gajdusek Corinne M. , Luo Zhengyu , Mayberg Marc R.
Published: February 2005

We investigated the relative roles of basic fibroblast growth factor (bFGF) and transforming growth factor beta‐1 (TGF‐b) on bovine aortic endothelial cell mitogenesis and morphogenesis using two‐dimensional Petri dish cultures and a threedimensional hydrated collagen gel. bFGF alone stimulated endothelial cell proliferation with an EC50 of 0.5 ng/ml. At bFGF levels greater than 2.5 ng/ml, morphologic alterations in confluent monolayers predominated; cells changed from a cobblestone morphology to an elongated cell pattern and showed enhanced migration into a denuded area of a Petri dish. In the three‐dimensional model, exposure of endothelial cell monolayers to high bFGF levels stimulated minor cell migration directly under the monolayer but no invasion into the gel matrix. In combination with bFGF, heparin potentiated morphogenic changes, but not mitogenesis. bFGF, modification of the antiproliferative effect of TGF‐b in confluent cultures was evidenced by induction of endothelial cell sprouting in response to 0.5 ng/ml TGF‐b and 10–20 ng/ml bFGF in two‐dimensional cultures. On collagen gels, endothelial cells migrated into the deep layers of the gel in a dose‐dependent manner: invasion was maximal at 0.3–0.7 ng/ml TGF‐b with decreased invasion at higher concentrations. The optimal collagen concentration that supported cell invasion was 0.075% collagen with the number of invading cells decreasing with increasing collagen gel density. By scanning electron microscopy, invading endothelial cells assumed a fibroblast‐like appearance with slender cell extensions. We concluded that bFGF and TGF‐b had independent effects on endothelial cell morphology and mitogenesis in culture. In combination at specific doses, these agents stimulated sprouting in the two‐dimensional model and cell invasion in a collagen gel model. Morphogenic changes may be the primary event in determining angiogenesis. © 1993 Wiley‐Liss, Inc.
Effect of glutamine on heat‐shock‐induced mRNA and stress proteins
Authors: Nissim Itzhak , States Beatrice , Hardy Mattie , Pleasure Jeanette , Nissim Ilana
Published: February 2005

Our aim was to delineate the effect of glutamine on the level of heat shockinducible mRNA and synthesis of stress protein(s) in cultured kidney cells. Experiments were carried out using opossum kidney (OK) cells. The induction of HSP70 mRNA as well as the synthesis of 72,73 kDa stress proteins was evaluated in cell monolayers exposed to 45°C for 15 minutes followed by a recovery period at 37°C for 3 hours. Incubations were performed in Krebs buffer supplemented with 0, 2, 5, or 10 mM glutamine. A separate series of experiments was performed in the presence of glutamine metabolites, such as NH4CI, glutamate, or aspartate. Glutamine without preincubation at 37°C remarkably increased the steady‐state level of HSP70 mRNA as well as the production of 72,73 kDa stress proteins in a dose‐dependent manner. The production of stress protein(s) in the presence of glutamine was associated with decreased percent LDH efflux, suggesting cytoprotective action of glutamine in cultured kidney cells. However, when OK cells were preincubated for 1 hour at 37°C with 10 mM glutamine, there was an approximately fourfold decline in level of HSP70 mRNA compared with experiments in the presence of 10 mM glutamine without preincubation. In addition, metabolites of glutamine, i.e., ammonia and glutamate decreased the level of heat‐inducible HSP70 mRNA. Furthermore, aspartate or NH4CI had little effect on LDH release compared with heat shock experiments, without addition of amino acids. These observations suggest that metabolites of glutamine may blunt the steady‐state level of glutamate or HSP70 mRNA. The decreased level of HSP70 mRNA in the presence of NH4CI may explain the role of ammonia in renal injury and brain toxicity, as well as glutamate excitotoxicity. © 1993 Wiley‐Liss, Inc.
Tissue inhibitor of metalloproteinases‐2 inhibits bFGF‐induced human microvascular endothelial cell proliferation
Authors: Murphy Anne N. , Unsworth Edward J. , Stetler‐Stevenson William G.
Published: February 2005

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2), a protease inhibitor that binds to the latent and active forms of 72 kDa type IV collagenase (gelatinase A), was found to inhibit the in vitro proliferation of human microvascular endothelial (HME) cells stimulated with bFGF and 5% serum. The maximal inhibitory effect of TIMP‐2 on incorporation of 3H‐thymidine was evident 24 hours after bFGF stimulation of these cells and ranged between 45 and 60%. The half‐maximal effective concentration of TIMP‐2 was 107 ± 12 nM (S.D.). In contrast, TIMP‐1 was not found to slow the growth of HME cells. The inhibition of cell proliferation observed with TIMP‐2 was not mimicked by addition to the culture medium of BB94, a general matrix metalloproteinase inhibitor, nor antibodies to the 72 kDa type IV collagenase. In addition to growth, two other cell functions associated with the angiogenic process were tested for sensitivity to TIMP‐2. Cell adhesion to tissue culture platic was slightly stimulated by TIMP‐2, and cell migration was inhibited with short‐term exposure to TIMP‐2, but neither process was affected by longer‐term exposure. The ability of TIMP‐2 to inhibit cultured endothelial cell proliferation independent of protease inhibitory activity suggests that TIMP‐2 may have additional actions which may limit neovascularization associated with solid tumor growth and metastasis in vivo. © 1993 Wiley‐Liss, Inc.
Differential expression of P‐glycoprotein genes in primary rat hepatocyte culture
Authors: Lee Chow Hwee , Bradley Grace , Zhang Jian‐Ting , Ling Victor
Published: February 2005

The multidrug resistance (MDR)–associated protein, P‐glycoprotein (Pgp), is expressed on the bile canalicular surface of hepatocytes, where it is thought to function in the detoxification of xenobiotics or in the transport of specific metabolites. Several studies have shown that Pgp expression in rat liver can be perturbed in vivo and in vitro; however, it is not known which of the 3 Pgp genes (class I, II, or III) are involved. In rodents, the class I and II Pgp genes have been shown to mediate MDR while the class III gene apparently does not. In this eport, we have used gene‐specific probes generated from the 3′‐untranslated regions of the three rat Pgp genes (Deuchars et al.: Biochim. Biophys. Acta, 1130:157–165, 1992) to investigate Pgp gene expression in primary rat hepatocytes. We observed that the class II Pgp mRNA, the least abundant in the intact liver, is dramatically increased in culture over a 48 h period, while the class I Pgp showed only a modest increase in mRNA level. In contrast, the class III Pgp mRNA, which is the most abundant in the intact liver, exhibited a gradual decline. In rat liver hepatocytes, different culture conditions, as well as drugs such as cytochalasin D and colchicine, appear to affect the level of the class II Pgp gene expression. Moreover, under all these conditions, there is a strong correlation between the level of the class II Pgp and cytoskeletal (actin and tubulin) mRNAs. Thus, there may be a common mechanism regulating the expression of cytoskeletal protein genes and the class II Pgp gene. These findings have implications for our understanding of the regulation of Pgp gene expression in normal and malignant tissues. © 1993 Wiley‐Liss, Inc.
Membrane‐anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan‐4–like heparan sulfate proteoglycan and a distinct chondroitin sulfate form
Authors: Yeaman Charles , Rapraeger Alan C.
Published: February 2005

Proteoglycan accumulation by thioglycollate‐elicited mouse peritoneal macrophages and a panel of murine monocyte‐macrophage cell lines has been examined to determine whether these cells express plasma membrane‐anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre‐monocytes and is associated predominantly with the cell layer of mature macrophage‐like cells. In the P388D1 cell line, the cell‐associated chondroitin sulfate is largely present as a plasma membrane‐anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell‐associated heparan sulfate is composed of a proteoglycan fraction and protein‐free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan‐4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co‐migrates with syndecan‐4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage‐like cell lines accumulate similar amounts of syndecan‐4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage‐like cells. Syndecan‐1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells. © 1993 Wiley‐Liss, Inc.
Long‐term effects of tumor necrosis factor on LLC‐PK1 transepithelial resistance
Authors: Marano C. W. , Laughlin K. V. , Russo L. M. , Soler A. Peralta , Mullin J. M.
Published: February 2005

Renal epitheliaL LLC‐PK1 cell sheets incubated with tumor necrosis factor (TNF) undergo an acute, spontaneous, and rapidly reversible decrease in transepithelial resistance (TER). (Mullin et al., 1992). However, 24 to 72 h following TNF exposure, TER across the cell sheet increases 2‐fold. This later effect of TNF is also reversible, albeit slowly. The TER of TNF‐treated cell sheets then declines toward initia levels between 72 and 144 h following exposure to the cytokine. Whereas the long‐term increase in TER following TNF exposure is not associated with a decreased transepithelial 14C‐mannitol flux (size selectivity), the charge (anionic) selectivity of the LLC‐PK1 tight junction is decreased. Basal‐lateral (ouabain and bumetanide‐insensitive) Rb+ and apical Na+‐dependent alpha‐methylglucoside (AMG) uptake into the cell are both reduced in cultures exposed to TNF 24 h earlier. Correspondingly, this long‐term effect on TER is accompanied by a 30% decrease in short circuit current(iSCC). Along with an observed increase in basallateral methylamino‐isobutyric acid (MeAIB) influx into the cells, an increased incorporation of [3H]‐thymidine into DNA indicates increased cell cycling after exposure to TNF. While the increase in cell cycling is not sustained for the duration of the elevation in TER, it does appear to initiate a sequence of events that lead to the sustained increase in TER. A decrease in the lateral intercellular space, observed between these epithelial cells after long‐term TNF exposure, may be a mechanism for the elevated TER following from the mitogenesis and/or transport changes. This overall long‐term tightening of an epithelium in response to TNF may function, in part, as a compensatory action of the epithelium to reestablish its effectiveness as a physiological barrier, following the acute effect of TNF. © 1993 Wiley‐Liss, Inc.
Organization of calsequestrin‐positive sarcoplasmic reticulum in rat cardiomyocytes in culture
Authors: Ioshii Sérgio O. , Imanaka‐Yoshida Kyoko , Yoshida Toshimichi
Published: February 2005

The sarcoplasmic reticulum (SR) regulates the levels of cytoplasmic free Ca2+ ions in muscle cells. Calsequestrin is a major Ca2+ ‐storing protein and is localized at special sites in the SR. To investigate the development of calsequestrin‐positive SR and its interaction with the cytoskeleton, we examined the distribution of calsequestrin in cultured cardiomyocytes from newborn rats by immunofluorescence with anticalsequestrin and antitubulin antibodies and rhodamine‐phalloidin. In frozen sections of neonatal rat heart, anticalsequestrin immunostaining was apparent as cross‐striations at Z‐lines. When newborn cardiomyocytes were isolated, calsequestrin‐positive SR was disorganized and was apparent as small vesicles beneath the sarcolemma, whereas myofibrils accumulated in the center of the cells. As the cells spread in culture, calsequestrin‐positive vesicles spread to the periphery of the cytoplasm, becoming associated with the developing myofibrils. In mature cells, calsequestrin was closely associated with myofibrils, showing cross‐striations at the Z‐lines. Double‐labeling using anticalsequestrin and antitubulin antibodies demonstrated that the distribution of calsequestrin‐positive structures was similar to that of the microtubular arrays. When the microtubules were depolymerized by nocodazole at an early stage, the extension of the SR to the cell periphery was inhibited. In mature cardiomyocytes, nocodazole appeared not to affect the distribution of the SR. These results indicate that the calsequestrin‐positive SR in cardiomyocytes is organized at the proper sites of myofibrils during myofibrillogenesis and that the microtubules might serve as tracts for the transport of components of the SR. © 1994 Wiley‐Liss, Inc.
TGF‐β1 induces the expression of type I collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors
Authors: Reed May J. , Vernon Robert B. , Abrass Itamar B. , Sage E. Helene
Published: February 2005

Fibroblasts have a major role in the synthesis and reorganization of extracellular matrix that occur during wound repair. An impaired biosynthetic or functional response of these cells to stimulation by growth factors might contribute to the delayed wound healing noted in aging. We, therefore, compared the responses of dermal fibroblasts from young and elderly individuals (26, 29, 65, 89, 90, and 92 years of age) to transforming growth factor‐β1 (TGF‐β1) with respect to: (1) the synthesis of type I collagen and SPARC (two extracellular matrix proteins that are highly expressed by dermal fibroblasts during the remodeling phase of wound repair) and (2) the contraction of collagen gels, an in vitro assay of wound contraction. With the exception of one young donor, all cultures exposed for 44 hours to 10 ng/ml TGF‐β1 exhibited a 1.6‐ to 5.5‐fold increase in the levels of secreted type 1 collagen and SPARC, relative to untreated cultures, and exhibited a 2.0‐ to 6.2‐fold increase in the amounts of the corresponding mRNAs. Moreover, the dose‐response to TGF‐β1 (0.1–10 ng/ml), as determined by synthesis of type I collagen and SPARC mRNA, was as vigorous in cells from aged donors as in cells from a young donor. In assays of collagen gel contraction, fibroblasts from all donors were stimulated to a similar degree by 10 ng/ml TGF‐β1. In conclusion, cells from both young and aged donors exhibited similar biosynthetic and contractile properties with exposure to TGF‐β1. It therefore appears that the impaired wound healing noted in the aged does not result from a failure of their dermal fibroblasts to respond to this cytokine. © 1994 Wiley‐Liss, Inc.
Intracellular dynamics of c‐myc mRNA traffic in single cells in situ
Authors: Pallavicini M. G. , George T. , Deteresa P. S. , Amendola R. , Gray J. W.
Published: February 2005

Processing and intracellular transport of RNA transcripts are essential for gene expression. Translational regulation of gene expression may occur by several mechanisms, including control of transcript movement from sites of synthesis to site(s) of translation. We describe temporal analysis of the intracellular translocation of c‐myc transcripts from the site of synthesis in the nucleus to sites of translation in the cytoplasm. Fluorescence in situ hybridization (FISH) and quantitative fluorescence microscopy were used to measure intracellular traffic of c‐myc transcripts in individual recombinant cells following activation of c‐myc sequences linked to a heat shock promoter. C‐myc nuclear transcripts are visible in the nucleus within minutes of heat exposure. Transcripts remain in the nucleus for at least 4 hr after gene activation. Transport of c‐myc transcripts to the cytoplasm begins ∼ 1 hr after cells are returned to 37°C. These data demonstrate the feasibility of measuring intracellular transcript transport following gene activation and provide a description of the kinetics of intracellular traffic of inducible c‐myc transcripts in heated cells in situ. © 1994 Wiley‐Liss, Inc.
Expression of a growth arrest specific gene (gas‐6) during liver regeneration: Molecular mechanisms and signalling pathways
Authors: Ferrero Marina , Desiderio Maria Alfonsina , Martinotti Alessia , Melani Cecilia , Bernelli‐Zazzera Aldo , Colombo Mario P. , Cairo Gaetano
Published: February 2005

A set of growth arrest‐specific (gas) genes negatively regulated by serum has been identified. To define the role of gas genes in a model of cell proliferation in vivo we analyzed the expression of one of these genes (gas‐6) during liver regeneration after partial hepatectomy (PH). We found that gas‐6 mRNA was down‐regulated 4 hours after PH, within the G0 to G1 transition. Later on, gas‐6 mRNA increased over the level found in normal liver with a peak at 16 hours, before the onset of DNA synthesis. This surge was probably triggered by an inflammatory response caused by the surgical trauma, because an increase of similar extent occurring with the same time course was present in livers of sham‐operated and turpentine‐treated rats. Comparison of mRNA steady state levels with nuclear transcription rates indicated that gas‐6 expression is post‐transcriptionally regulated. As we found that down‐regulation of gas‐6 expression was prevented by treatment with Actinomycin D, a labile protein might be involved in the determination of gas‐6 mRNA stability. To investigate the mitogenic signals controlling gas‐6 expression during liver regeneration we treated hepatectomized rats with a specific alpha‐1‐adrenoceptor blocker (prazosin) as well as with drugs which modify intracellular calcium levels. The decrease of gas‐6 mRNA 4 hours after PH was prevented by prazosin and by neomycin, an inhibitor of calcium release from endogenous stores. These findings suggest that down‐regulation of gas‐6 expression during hepatic regeneration is triggered by catecholamines interaction with alpha‐1‐adrenergic receptors and by subsequent calcium release. In addition we found that the rise of gas‐6 gene expression occurring at 16 hours after PH was not affected by prazosin but was inhibited by trifluoperazine. Therefore, we suggest that up‐regulation of gas‐6 gene expression is mediated by the interaction of calcium with calmodulin, independently of catecholamines. © 1994 Wiley‐Liss, Inc.
Stimulatory effect of a phorbol ester on expression of insulin‐like growth factor (IGF) binding protein‐2 and level of IGF‐I receptors in mouse osteoblastic MC3T3‐E1 cells
Authors: Hakeda Yoshiyuki , Yoshizawa Kenji , Hurley Marja , Kawaguchi Hiroshi , Tezuka Ken‐Ichi , Tanaka Kayo , Satoh Takuya , Kumegawa Masayoshi
Published: February 2005

We examined the relationship between signal transduction and the expression of insulin‐like growth factor I (IGF‐I), IGF‐I receptor level, and IGF binding proteins (IGFBPs) in murine clonal osteoblastic MC3T3‐E1 cells. 12–O‐Tetradecanoylphorbol‐13‐acetate (TPA), an activator of protein kinase C, decreased the secretion of immunoreactive IGF‐I into the medium, whereas dibutyryl cAMP (Bt2cAMP) augmented the secretion In contrast, TPA increased the level of type IIGF receptor on the cells. Furthermore, MC3T3‐E1 cells produced and secreted at least three different IGFBPs with molecular masses of 24, 30, and 34 kDa, and the 24‐kDa IGFBP was predominant under normal conditions. However, TPA specifically increased the secretion of the 34‐kDa IGFBP. The N‐terminal amino acid sequence of the purified 34‐kDa IGFBP was nearly identical with that of rat IGFBP‐2. Furthermore, the 34‐kDa IGFBP was immunoreactive to anti‐IGFBP‐2 antiserum. The level of IGFBP‐2 mRNA in the cells was increased by TPA, indicating that the increase in IGFBP‐2 secretion results from the stimulation of IGFBP‐2 production. In contrast, Bt2cAMP affected neither IGF‐l receptor number nor the IGFBP secretion. These results indicate that the production of IGF‐l and the expression of IGF‐l receptors and IGFBP‐2 are up‐regulated by the activation of adenylate cyclase and protein kinase C, respectively, in osteoblastic MC3T3‐E1 cells. © 1994 Willey‐Liss, Inc.
Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study
Authors: Perdomo Juana J. , Gounon Pierre , Schaeverbeke Madeleine , Schaeverbeke Jean , Groult Vanina , Jacob Marie P. , Robert Ladislas
Published: February 2005

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley‐Liss, Inc.
Effects of cytokines on synthesis and function of the hepatic asialoglycoprotein receptor
Authors: Treichel Ulrich , Paietta Elisabeth , Poralla Thomas , Meyer zum Büschenfelde Karl‐Hermann , Stockert Richard J.
Published: February 2005

In this study we have investigated whether cytokines, critical mediators of the immune response, might have a direct effect on the expression and/or function of the human hepatic asialoglycoprotein receptor (ASGPR). Binding and uptake of asialoglycoproteins by the human hepatoma cell line, HepG2, and by freshly isolated rat hepatocytes were inhibited by 50% after 3–6 hours and completely abolished following a 24 hour exposure to tumor necrosis factor (TNF) α, interferon (INF) α or γ, or interleukin‐2 (IL‐2). The loss of ASGPR binding activity mediated by IL‐2 was reversible up to 4 hours of exposure and accompanied by the selective phosphorylatior, of the cell‐surface receptor. Steady‐state levels of total cellular ASGPR protein remained unchanged over the first 6 hours of IL‐2 incubation but declined in a dose dependent manner thereafter. This down regulation of ASGPR expression was due to reduced synthesis as a result of reduced receptor transcript levels. No loss was detected, however, of cell surface‐associated receptor protein even after 24 hours of IL‐2 incubation, suggesting that cytokine induced phosphorvlation constitutes a mechanism to regulate receptor activity. © 1994 Wiley‐Liss, Inc.
Protection of transforming growth factor β activity by heparin and fucoidan
Authors: McCaffrey Timothy A. , Falcone Domenick J. , Vicente Diane , Du Baoheng , Consigli Seth , Borth Wolfgang
Published: February 2005

The transforming growth factor‐β (TGF‐β) family of proteins exert diverse and potent effects on proliferation, differentiation, and extracellular matrix synthesis. However, relatively little is known about the stability or processing of endogenous TGF‐β activity in vitro or in vivo. Our previous work indicated that (1) TGF‐β1 has strong heparin‐binding properties that were not previously recognized because of neutralization by iodination, and (2) heparin, and certain other polyanions, could block the binding of TGF‐β1 to α2‐macroglobulin (α2‐M). The present studies investigated the influence of heparin‐like molecules on the stability of the TGF‐β1 signal in the pericellular environment. The results indicate that heparin and fucoidan, a naturally occurring sulfated L‐fucose polymer, suppress the formation of an initial non‐covalent interaction between 125I‐TGF‐β1 and activated α2‐M. Electrophoresis of 125I‐TGF‐β1 showed that fucoidan protects TGF‐β1 from proteolytic degradation by plasmin and trypsin. While plasmin caused little, if any, activation of latent TGF‐β derived from vascular smooth muscle cells (SMC), plasmin degraded acid‐activated TGF‐β, and purified TGF‐β1, and this degradation was inhibited by fucoidan. In vitro, heparin and fucoidan tripled the half‐life of 125I‐TGF‐β1 and doubled the amount of cell‐associated 125I‐TGF‐β1. Consistent with this protective effect, heparin‐ and fucoidan‐treated SMC demonstrated elevated levels of active, but not latent, TGF‐β activity. © 1994 wiley‐Liss, Inc.
Modifications in the synthesis of membrane‐associated chondroitin sulfate proteoglycans in hemopoietic progenitor cells are accompanied by alterations in their adhesive properties
Authors: Conget Paulette , Minguell José J.
Published: February 2005

In vitro studies in our laboratory have indicated that murine hemopoietic progenitor cell (HPC) lines, irrespective of their differentiation stage, synthesize and accumulate in the cell membrane a unique species of chondroitin sulfate proteoglycan (CS‐PG). It has been postulated that CS‐PG participates in HPC adhesion to pericellular stromal fibronectin by interacting with its heparin‐promoting binding region. To further support this contention, we first attempted to modify CS‐PG synthesis in HPC by the use of chlorate and p‐nitrophenyl (β‐D‐xyloside), which inhibit sulfation and glycosaminoglycan (GAG) addition in proteoglycans, respectively. We then studied the effect that these modifications may have in the adhesive capacity of HPC to interact with fibronectin and its cell‐ and heparin‐promoting binding chymotryptic fragments. Treatment with chlorate which resulted in a decreased sulfation of membrane‐associated 35 S‐labeled CS‐PG, as judged by ion exchange chromatography, did not affect HPC adhesion to fibronectin or its fragments. However, β‐xyloside treatment which reduces the abundance of membrane‐associated CS‐PG, as evidenced by molecular sieve chromatography, produced a major and specific decrease in HPC adhesion to the heparin‐promoting binding fragment of fibronectin. These results indicate that CS‐PG are involved in HPC interaction with fibronectin, in a mode that seems to be dependent on the differentiation stage of HPC. © 1994 wiley‐Liss, Inc.
Cisplatin sensitivity correlates with its ability to cause cell cycle arrest via a wee1 kinase‐dependent pathway in schizosaccharomyces pombe
Authors: Thiebaut Franz , Enns Robert , Howell Stephen B.
Published: February 2005

Mutants of Schizosaccharomyces pombe were used to define genes involved in the cell cycle arrest produced by cisplatin (DDP), an agent that causes both DNA damage and inhibition of DNA synthesis. Previous work has demonstrated that strains with defective or absent wee 1+ function fail to arrest in G2 when DNA is damaged, but do arrest when DNA synthesis is inhibited (Rowley et al., 1992a, Nature, 356:353–355). Strains defective in wee 1+ function, or in the ability of the wee 1+ kinase to regulate cdc2, failed to arrest following DDP exposure, as did a rad1‐1 mutant. All strains failing to arrest in G2 were hypersensitive to DDP. Thus, DNA damage rather than inhibition of DNA synthesis is causative of DDP‐induced cell cycle arrest. In addition, this work shows that the wee 1+ and rad 1+ gene products are required for successful DDP‐induced arrest, and suggests that the ability of S. pombe to arrest is a major determinant of sensitivity to DDP. © 1994 wiley‐Liss, Inc.
Cell cycle controlled histone H1, H3, and H4 genes share unusual arrangements of recognition motifs for HiNF‐D supporting a coordinate promoter binding mechanism
Authors: van den Ent Fusinita M. I. , Van Wijnen André J. , Lian Jane B. , Stein Janet L. , Stein Gary S.
Published: February 2005

Cell cycle and growth control of the DNA binding and transactivation functions of regulatory factors provides a direct mechanism by which cells may coordinate transcription of a multitude of genes in proliferating cells. The promoters of human DNA replication dependent histone H4, H3, and H1 genes interact with at least seven distinct proteins. One of these proteins is a proliferation‐specific nuclear factor, HiNF‐D, that interacts with a key cis‐regulatory element (H4‐Site II; 41 bp) present in H4 genes. Here we describe binding sites for HiNF‐D in the promoters of H3 and H1 genes using cross‐competition, deletion analysis, and methylation interference assays, and we show that HiNF‐D recognizes intricate arrangements of at least two sequence elements (CA‐ and AG‐motifs). These recognition motifs are irregularly dispersed and distantly positioned in the proximal promoters (200 bp) of both the H3 and H1 genes. In all cases, these motifs either overlap or are in close proximity to other established transcriptional elements, including ATF and CCAAT sequences. Although HiNF‐D can interact with low affinity to a core recognition domain, auxiliary elements in both the distal and proximal portions of each promoter cooperatively enhance HiNF‐D binding. Thus, HiNF‐D appears to bridge remote regulatory regions, which may juxtapose additional trans‐activating proteins interacting within histone gene promoters. Consistent with observations in many cell culture systems, the interactions of HiNF‐D with the H4, H3, and H1 promoters are modulated in parallel during the cessation of proliferation in both osteosarcoma cells and normal diploid osteoblasts, and these events occur in conjunction with concerted changes in histone gene expression. Thus, HiNF‐D represents a candidate participant in coordinating transcriptional control of several histone gene classes. © 1994 wiley‐Liss, Inc.
Characterization of regulatory volume decrease in the THP‐1 and HL‐60 human myelocytic cell linesn
Authors: Gallin Elaine K. , Mason Toni M. , Moran Arie
Published: February 2005

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP‐1 and HL‐60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine‐sensitive cation transport mechanism that is independent of the swelling‐induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4‐aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium‐activated conductances, since it occurred normally in Ca‐free medium, in medium containing cadmium, and in BAPTA‐loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP‐1 and HL‐60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5‐diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP‐1 and HL‐60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA‐sensitive anion pathway and a quinine‐inhibitable K efflux pathway, neither of which requires increases in intra‐cellular calcium to be activated. © 1994 wiley‐Liss, Inc.
Characterization of rat aortic smooth muscle cells resistant to the antiproliferative activity of heparin following long‐term heparin treatment
Authors: Bârzu Tereza , Pascal Marc , Herbert Jean‐Marc , Carayon Pierre , Desmoulière Alexis
Published: February 2005

Vascular smooth muscle cells (SMC) do not represent a homogeneous population (Schwartz et al., 1990, Am. J. Pathol. 136:1417–1428). Cellular clones resistant to the antiproliferative activity of heparin were isolated from rat aortic SMC cultures (Pukac et al., 1990, Cell Regul., 1:435–443; San Antonio et al., 1993, Arterioscler. Thromb., 13:748–757) and from explant of human arterial restenotic lesions (Chan et al., 1993, Lancet, 341:341–342). We have shown in the present study that long‐term treatment (growth medium supplemented with 200 μg/ml heparin, from the second to the tenth passage) of rat aortic SMC, without cell cloning, resulted in a significant loss of sensitivity to the growth inhibition by heparin and its derivatives. The heparin resistance was stable after growing cells for two passages in heparin‐free medium, suggesting the selection of a particular phenotype. We tried to characterize these cells and to determine the causes of the resistance to the growth inhibition by heparin. Heparin‐treated SMC (HT‐SMC) were smaller than their control culture at the same passage, expressed less α‐SM actin, and did not overgrow after reaching confluence. As in the heparin‐resistant clones (San Antonio et al., 1993, Cell Regul., 1:435–443) expression of α‐SM actin could be increased in HT‐SMC by heparin addition before Western blotting. Heparin resistance was associated with a tenfold decrease in [3H]‐heparin binding capacity (Bmax=1.9 × 106 sites per cell) compared to control cultures (Bmax = 1.7 × 107 sites per cell), which was irreversible after growing the cells for two additional passages in heparin‐free medium. We also investigated protein kinase C (PKC) in HT‐SMC in terms of both enzymatic activity and protein expression (evaluated by [3H]‐staurosporine and [3H]‐phorbol‐12,13‐dibutyrate binding). We found that HT‐SMC had only half the PKC activity and expression as control SMC. Therefore, long‐term treatment of rat aortic SMC with heparin allowed the selection of a less differentiated subpopulation of cells, exhibiting low sensitivity to the growth inhibition by heparin, which could be related to the low capacity of binding heparin and to a lower PKC activity and/or expression. © 1994 Wiley‐Liss, Inc.