bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of K+ channel‐blockers on sugar uptake by isolated chicken enterocytes
Authors: Montero M. C. , Calonge M. L. , Bolufer J. , Ilundáin A.
Published: February 2005

The effects of Ba2+, quinine, verapamil, and Ca2+‐free saline solutions on sugar active transport have been investigated in isolated chicken enterocytes. Ba2+, quinine, and verapamil, which have been shown to inhibit Ca2+‐activated K+ channels, decreased basal and theophylline‐dependent 3‐O‐methylglucose (3‐O‐MG) accumulation. Ca2+‐free conditions reduced 3‐O‐MG uptake in the‐ophylline‐treated enterocytes, but it had no effect in control cells. On the other hand, the uptake of a non‐actively transported sugar, 2‐deoxyglucose (2‐DOG), by control or theophylline‐treated cells was not modified by the presence of verapamil or by Ca2+‐removal. 3‐O‐MG increased ouabain‐sensitive Na+‐efflux, but had no effect on either K+ efflux or K+ uptake. However, in the presence of Ba2+, K+ uptake was stimulated by 3‐O‐MG, and this increase was prevented by ouabain. All these findings are discussed in terms of the role that K+ permeability may play in cellular homeostasis during sugar active transport.
Internalization of the fibronectin receptor is a constitutive process
Authors: Sczekan Melanie M. , Juliano R. L.
Published: February 2005

Using a monoclonal antibody specific for the hamster fibronectin receptor (FnR), we have demonstrated that a portion of the CHO cell FnR population is constitutively endocytosed. Three independent techniques were used to study the internalization: (1) after saturation binding of an anti‐FnR antibody (PB1) to cells at 4°C, internalization was initiated by warming to 37°C, and then acid/salt elution of membrane‐bound ligand was used to quantitate the internalized 125I‐PB1; (2) cell vesicular traffic was pharmacologically disrupted with monensin or chloroquine, and the subsequent reduction of the cell surface pool of FnR was monitored; and (3) selective immunoprecipitation was used to separate surface and internalized 125I‐labeled FnR. These experiments indicate that about 30% of the cell surface FnR is endocytosed with a t1/2 of 7 min and that this internalization occurs regardless of the ligation state of the receptor. Other observations indicate that the larger fraction of the cell surface FnR pool (70—75%) is apparently shed from the cell upon ligation with antibody at 37°C. This process occurs much more slowly than receptor internalization and leads to an overall reduction in the amount of cell surface FnR. Our results suggest physically or chemically distinct populations of FnR, one of which is unavailable for internalization and recycling.
Tumor necrosis factor‐alpha induces vitamin D‐1‐hydroxylase activity in normal human alveolar macrophages
Authors: Pryke A. M. , Duggan C. , White C. P. , Posen S. , Mason R. S.
Published: February 2005

The induction of 1‐hydroxylase in alveolar macrophages by tumor necrosis factor‐alpha (TNF) was examined in view of recent evidence suggesting that local production of 1,25‐(OH)2D3 may play a role in the regulation of immune functions. Incubation of pulmonary alveolar macrophages from normal human subjects with recombinant TNF caused a 2‐ to 10‐fold increase in 25‐hydroxyvitamin D3‐1‐hydroxylase activity. The dose‐response curve was linear over the range 0.05—5.0 IU/ml, and no further increase was seen at higher concentrations. The increase in 1‐hydroxylase activity was present after 12 h and reached a maximum after 3 days. The effect of TNF was inhibited in a dose‐dependent manner by the presence of 1,25(OH)2D3 (10−10−10−8 M) in the incubation media for 5 days but was unaffected by 10−9 M 1,25(OH)2D3 after 12 h. The enhancement of macrophage 1‐hydroxylase activity by TNF was comparable to that induced by gamma interferon (IFN) but the effects of maximal doses of both agents were not additive. The presence of antibody to TNF resulted in a 76% inhibition of TNF‐induced 1‐hydroxylase but had no significant effect on IFN‐induced 1‐hydroxylase activity.
Metabolic basis for differential glutamine requirements of human leukemia cell lines
Authors: Kitoh Toshiyuki , Kubota Masaru , Takimoto Tetsuya , Hashimoto Hisako , Shimizu Tsunehiro , Sano Hirohiko , Akiyama Yuichi , Mikawa Haruki
Published: February 2005

We compared the ability of human leukemia cell lines of various origins to grow in glutamine‐deficient media. The growth of B lymphoblastoid cell lines, including promyelocytic HL‐60, is highly dependent on glutamine, whereas T‐cell lines are able to proliferate in glutamine‐free media. Such glutamine dependency has a good inverse correlation with the activity of glutamine synthetase. Moreover, glutamine synthetase can be induced in glutamine‐deficient media, especially in glutamine‐independent cells. In HL‐60 cells, glutamine deprivation results in the decrease of both ATP and dATP levels. The addition of adenine to the culture medium abolishes these changes without restoring cell growth, indicating that the effects of glutamine deprivation on cell growth cannot be fully explained by the perturbation of adenine nucleotide pools.
Distinct genomic copy number in mitochondria of different mammalian organs
Authors: Veltri Karen L. , Espiritu Myrna , Singh Gurmit
Published: February 2005

This study shows that mitochondria in liver, kidney, heart, and brain of the mouse have a distinct mitochondrial density. It also demonstrates that the mtDNA copy number per mitochondrion is organ‐specific. A reliable method of determining mitochondrial density per organ is by stereological analysis of tissue sections while mtDNA quantitation is by the use of radiolabelled mtDNA probe. This is the first study in which a comprehensive examination of mitochondrial density and quantitation of mitochondrial genomes in mouse organs have been done. In summary the variability is not only in mitochondrial density but also in genomic copy number in mitochondria of various tissues.
Signal perception of fibroblasts for directional migration to platelet‐derived growth factor in Boyden‐type chambers
Authors: Adelmann‐Grill Bernhard C. , Cully Zuzana
Published: February 2005

Fibroblast chemotaxis has usually been determined in Boyden‐type chambers with polycarbonate filters, assuming that a stable concentration gradient of the attractant develops that causes directional migration of the cells. This view has been repeatedly challenged, and development of such gradients in vivo is unlikely. The present experiments were designed to test if a stable concentration gradient was required for normal dermal fibroblasts to migrate toward platelet‐derived growth factor. It was found that a brief pulse of the attractant was required and sufficient to induce chemotaxis. The pulse had to contain a specific concentration of attractant and was ineffective when not unilateral. The observed effects could not be attributed to induction of random migration or migration on a mediator‐coated surface. It is not clear which machinery is regulating this cellular behaviour, but it is suggested that cells may migrate in vivo by similar mechanisms, because the establishment of stable concentration gradients of attractants in tissues is deemed unlikely.
Cyclic adenosine monophosphate‐mediated induction of F9 teratocarcinoma differentiation in the absence of retinoic acid
Authors: Goldstein Beth , Rogelj Snezna , Siegel Susan , Farmer Stephen R. , Niles Richard M.
Published: February 2005

F9 teratocarcinoma stem cells differentiate into parietal endoderm‐like cells when given retinoic acid (RA) and dibutyryl cyclic adenosine monophosphate (DB‐cAMP). It is generally accepted that the stem cells are resistant to the action of cAMP alone and need to be primed by RA in order to respond to cAMP. In this report, we demonstrate that F9 stem cells differentiate into parietal endoderm‐like cells in the absence of exogenous RA when treated with cholera toxin and 1‐methyl,3‐isobutyl xanthine (CT/MIX) or 8‐bromo‐cAMP/MIX (8B2‐cAMP/MIX). Cells treated with CT/MIX or 8B2‐cAMP/MIX were morphologically similar to parietal endoderm‐like cells, produced high amounts of plasminogen activator, and synthesized both type IV collagen and laminin mRNA. Conversely, markers made in abundance by stem cells such as stage‐specific embryonic antigen (SSEA‐1) and an mRNA species of 6.8 kb (pST6‐135) were markedly reduced in CT/MIX‐treated cells. To prove that cAMP alone could induce differentiation Lipidex‐1000, a hydrophobio gel, was used to remove 80–;90% of the endogenous serum retinoids. F9 cells grown in this retinoid‐depleted serum and treated with 8B2‐cAMP/MIX differentiated to parietal endoderm‐like cells as shown by both dramatic changes in morphology and induction of type IV collagen mRNA. Our results indicate that the differentiation of F9 to parietal endoderm‐like cells can be induced by increased intracellular cAMP and is not strictly dependent on the addition of RA.
Low temperature blocks exit of pro‐opiomelanocortin from the endoplasmic reticulum but not subsequent delivery to the site of prohormone processing
Authors: Wattenberg Binks W.
Published: February 2005

The effect of reduced temperature on the delivery of the prohormone pro‐opiomelanocortin (POMC) to the site of prohormone processing was investigated in the mouse anterior pituitary cell line AtT20. At 20°C processing was substantially inhibited and was almost completely arrested at 18°C. Earlier studies with membrane glycoproteins indicated that at these temperatures protein movement was blocked at the level of exit from the Golgi apparatus. In contrast it was found here that the inhibition of processing at reduced temperature was due to the retention of POMC in the endoplasmic reticulum. When POMC was allowed to progress to the Golgi before temperature was reduced, subsequent processing was only slightly retarded by incubation at 18°C. This indicates either that Golgi exit is not inhibited at this temperature, or that the processing apparatus exists in the Golgi. A surprising incidental result was that when held in the endoplasmic reticulum at low temperature POMC is apparently subject to post‐translational N‐linked glycosylation.
Quantification of protein transcytosis in the human colon carcinoma cell line CaCo‐2
Authors: Heyman Martine , Crain‐Denoyelle Anne‐Marie , Nath Samir Kumar , Desjeux Jehan‐Francois
Published: February 2005

The transepithelial absorption of food‐type proteins has been shown to proceed by endocytosis along two functional pathways: a minor direct pathway allowing transport of intact protein and a major lysosomal degradative pathway. The human colon carcinoma cell line CaCo‐2 grown on Millipore filters was used here further to characterize these pathways by measuring HRP transport across the cell monolayer in Ussing chambers. In the apical‐basal direction, this transport mainly occurred along the degradative pathway and was inhibited at 4°C (7.41 ± 1.26 pmoles/h·cm2 vs. 27.40 ± 8.91 at 37°C). The amount conveyed via the direct pathway was very small (0.89 ± 0.35 pmoles/h·cm2) and did not diminish at 4°C (1.43 ± 0.59 pmoles/h·cm2). In the basal‐apical direction, HRP transport along the degradative pathway at 37°C was similar to the apical‐basal value and was inhibited at 4°C (16.40 ± 4.50 vs. 2.72 ± 2.52 pmoles/h·cm2), but along the direct pathway, it was eight times the apical‐basal value (8.36 ± 3.11 pmoles/h·cm2) and was inhibited at 4°C (2.43 ± 0.78 pmoles/h·cm2). Intact HRP fluxes were not correlated with the eletrical resistance of the filters, indicating transport via a transcellular route. Monensin at 10−5 M did not affect direct or degradative transport in the apical‐to‐basal direction. These results suggest that in CaCo‐2 cells HRP undergoes bidirectional transcytosis by a fluid‐phase mechanism, but the extent of degradation during that transport varies according to the membrane (apical or basal) where it is presented.
Volume regulation in lens epithelial cells and differentiating lens fiber cells
Authors: Beebe David C. , Parmelee Judith T. , Belcher Karla S.
Published: February 2005

Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cggers a regulatory volume decrease (RVD) that is usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H2O water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 mM sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypoor hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.
Impaired carcinoembryonic antigen release during the process of suramin‐induced differentiation of the human colic adenocarcinoma cell clone HT29‐D4
Authors: Fantini Jacques , Rognoni Jean‐Baptiste , Theveniau Magali , Pommier Gilbert , Marvaldi Jacques
Published: February 2005

The establishment of a differentiated state of the human colic adenocarcinoma cell clone HT29‐D4 can be obtained by two ways: 1) the removal of glucose and its replacement by galactose in the culture medium (Fantini et al.:Biology of the Cell65:163–169, 1989); 2) the addition of suramin, a polyanionic compound, in the glucose‐containing medium (Fantini et al.:Journal of Biological Chemistry264:10282–10286, 1989). We investigated the release of CEA in the culture medium of glucose‐deprived HT29‐D4 cells (HT29‐D4‐Gal) and studied its alteration in suramin‐treated HT29‐D4 cells (HT29‐D4‐S). The amount of CEA released in the medium in function of time in culture of undifferentiated HT29‐D4‐Glu cells was very low (5 to 8 ng/106 cells/24 hours) and almost constant throughout the experiment whereas it increased sharply during differentiation of HT29‐D4‐Gal cells (380 ng/106 cells/24 hours after 9 days in culture). Surprisingly the amount of CEA released by differentiated HT29‐D4‐S cells remained very low and comparable with the one of HT29‐D4‐Glu cells. Moreover suramin, when added to CEA‐producing HT29‐D4‐Gal cells, strongly inhibited its release.

Radioiodination of cell surface proteins followed by immunoprecipitation using an anti‐CEA monoclonal antibody showed the presence of a 180 kDa poly‐peptide, i.e., CEA, predominantly labeled in HT29‐D4‐Gal and ‐S cells. The total CEA cellular content was higher in HT29‐D4‐Glu and HT29‐D4‐S cells than in HT29‐D4‐Gal cells. When HT29‐D4‐Gal or ‐S cells were treated with the bacterial phosphatidylinositol phospholipase C (PI‐PLC) a similar level of CEA was released suggesting a similar type of CEA anchorage. The present data demonstrate that a decrease in CEA release (i.e., in HT29‐D4‐Glu and ‐S cells) corresponds to an increase in its overall cellular expression. These results are in favour of a regulatory mechanism, impaired by suramin, which determines the balance between the soluble and the membrane bound forms of CEA.
Frequent expression of receptors for granulocyte‐macrophage colony‐stimulating factor on human nonhematopoietic tumor cell lines
Authors: Miyagawa Kiyoshi , Chiba Shigeru , Shibuya Kyoichi , Piao Yun‐Feng , Matsuki Shigeru , Yokota Jun , Terada Masaaki , Miyazono Kohei , Takaku Fumimaro
Published: February 2005

Receptors for granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were identified on 9 of 35 (26%) human nonhematopoietic tumor cell lines including non‐small cell lung cancer, stomach cancer, colon cancer, and osteosarcoma cells. GM‐CSF receptors distributed on these human tumor cells were low affinity types with an equilibrium dissociation constant of 1.5–10.0 nM. Cross‐linking studies revealed that the molecular weights of the low affinity GM‐CSF receptors were 65–85 kilodaltons. The high affinity receptors identified on hematopoietic cells were not detected on human nonhematopoietic tumor cells which we studied, and we could detect no effects of GM‐CSF on cell growth of these tumor cells.
Insulin‐like growth factor‐1 (IGF‐1), insulin, and epidermal growth factor (EGF) are survival factors for density‐inhibited, quiescent Balb/c‐3T3 murine fibroblasts
Authors: Tamm Igor , Kikuchi Toyoko
Published: February 2005

The great majority of murine Balb/c‐3T3 fibroblasts in density‐inhibited, quiescent cultures disintegrate and die rapidly when cells are deprived of serum in the medium. Platelet‐derived growth factor (PDGF, 5 ng/ml) used alone and insulin‐like growth factor (IGF‐1, 40 ng/ml) + epidermal growth factor (EGF, 10 ng/ml) prevent most of this cell death and all three factors used together protect close to all cells in the confluent monolayer as determined by counting trypsinized cell suspensions in a Coulter counter. IGF‐1 used alone affords a high level of protection during the first 5 hours of incubation in serum‐free medium but the protective effect declines subsequently unless EGF is also present. EGF alone has little protective activity. The survival‐promoting activity of PDGF used alone or of PDGF + EGF + IGF‐1 is not significantly decreased by selective inhibition of messenger precursor RNA transcription with 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB,20 or 40 μM), which prevents G1 traverse of the cells mediated by the combination of the three growth factors. DRB also does not interfere with the early protective effect of IGF‐1 + EGF, but decreases the late protective effect of this growth factor combination. DRB by itself decreases cell viability in the absence of growth factors or serum. In these experiments viability was assayed by neutral red uptake by using an automated microplate reader. Inhibition of protein synthesis with cycloheximide (CHX, 1 or 5 μg/ml) over a 20‐hour period was associated with decreased survival of cells protected by IGF‐1 + EGF or PDGF + EGF + IGF, but also with decreased survival of cells incubated in the absence of growth factors or serum. The decrease in survival was somewhat more marked when IGF + EGF was present than when PDGF + EGF + IGF‐1 was present. Insulin (1,500 ng/ml) mimics the action of IGF‐1 (40 ng/ml). The cell survival‐enhancing activities of growth factors are concentration dependent. The evidence presented indicates that PDGF, EGF, and IGF‐1 (or insulin) act through distinctive mechanisms in affording protection of cells against death. The short‐term protective effects of the growth factors are independent of gene expression and may be mediated via metabolic events. Long‐term protection may be dependent on gene expression, especially in the case of IGF‐1 + EGF.
Lipoxin A4 and lipoxin B4 stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: Dissociation between lipid remodeling and adhesion
Authors: Nigam Santosh , Fiore Stefano , Luscinskas Francis W. , Serhan Charles N.
Published: February 2005

The profiles of actions of lipoxin A4 (LXA4) and lipoxin B4 (LXB4), two lipoxygenase‐derived eicosanoids, were examined with human neutrophils. At nanomolar concentrations, LXA4 and LXB4 each stimulated the release of [1‐14C]arachidonic acid from esterified sources in neutrophils. Lipoxin‐induced release of [1‐14C]arachidonic acid was both dose‐ and time‐dependent and was comparable to that induced by the chemotactic peptide f‐met‐leu‐phe. Time‐course studies revealed that lipoxin A4 and lipoxin B4 each induced a biphasic release of [1‐14C]arachidonic acid, which was evident within seconds (5–15 sec) in its initial phase and minutes (>30 sec) in the second phase. In contrast, the all‐trans isomers of LXA4 and LXB4 did not provoke [1‐14C]AA release. Lipoxin‐induced release of arachidonic acid was inhibited by prior treatment of the cells with pertussis toxin but not by its β‐oligomers, suggesting the involvement of guaninine nucleotide‐binding regulatory proteins in this event. Dual radiolabeling of neutrophil phospholipid classes with [1‐14C]arachidonic acid and [3H]palmitic acid showed that phosphatidylcholine was a major source of lipoxin‐induced release of [1‐14C]arachidonic acid. They also demonstrated that lipoxins rapidly stimulate both formation of phosphatidic acid as well as phospholipid remodeling. Although both LXA4 and LXB4 (10−8‐10−6M) stimulated the release of [1‐14C]arachidonic acid, neither compound evoked its oxygenation by either the 5‐or 15‐lipoxygenase pathways (including the formation of LTB4, 20‐COOH‐LTB4, 5‐HETE, or 15‐HETE). LXA4 and LXB4 (10−7M) each stimulated the elevation of cytosolic Ca2+ as monitored with Fura 2‐loaded cells, albeit to a lesser extent than equimolar concentrations of FMLP. Neither lipoxin altered the binding of [3H]LTB4 to its receptor on neutrophils. In addition, they did not stimulate aggregation or induce adhesion of neutrophils to human endothelial cells. Results indicate that both LXA4 and LXB4 stimulate the rapid remodeling of neutrophil phospholipids to release arachidonic acid without provoking either aggregation or the formation of lipoxygenase‐derived products within a similar temporal and dose range. Together they indicate that LXA4 and LXB4 display selective actions with human neutrophils and suggest that these eicosanoids possess unique profiles of action which may regulate neutrophil function during inflammation.
Studies on the cell‐cycle dependency of the actions of insulin‐like growth factor‐I in Balb/c 3T3 cells
Authors: Kojima Itaru , Kitaoka Masafumi , Ogata Etsuro
Published: February 2005

The present study was conducted to determine the cell‐cycle dependency of various actions of IGF‐I in Balb/c 3T3 cells. When autophosphorylation of the IGF‐I receptor was determined in [32P]‐labelled cells, IGF‐I increased radioactivity in a 100 K‐Da phosphoprotein, presumably β‐subunit of the IGF‐I receptor, both in quiesent and in primed competent cells. Likewise, IGF‐I stimulated uptake of [3H]deoxyglucose independent of the cell cycle. In contrast, IGF‐I increased calcium entry, radioactivity in [3H]diacylglycerol, and [3H]thymidine incorporation in primed competent cells while these reactions were not induced by IGF‐I in quiescent cells. The latter three reactions were attenuated when cells were pretreated with pertussis toxin. These results indicate that some, but not all, of the actions of IGF‐I are dependent on the cell cycle in Balb/c 3T3 cells. They also suggest that a pertussis‐toxin‐sensitive G protein may be involved in the cellcycle‐dependent actions of IGF‐I.
Differential effects of transforming growth factor‐β and epidermal growth factor on the cell cycle of cultured rabbit articular chondrocytes
Authors: Vivien Denis , Galéra Philippe , Lebrun Emmanuel , Loyau GéRard , Pujol Jean‐Pierre
Published: February 2005

We examined the effect of transforming growth factor (TGF‐β) on the proliferative rate and cell cycle of cultured rabbit articular chondrocytes using cell counting, cytofluorometry, and [3H]‐thymidine incorporation. In the presence of 2% or 10% FCS (fetal calf serum), TGF‐β at 0.01, 0.1, 1, and 10 ng/ml had an inhibitory effect on cell proliferation after 24 h exposure with a dose dependence only for 2% FCS. Flow cytometric analysis of cell DNA content at that time showed that a high proportion of cells were arrested in late S‐phase (SQ or G2Q) in either 2% or 10% FCS‐containing medium. In both cases, a disappearance of the cell blockage occurred between 24 and 48 h after TGF‐β addition. However, whereas a stimulation of cell proliferation rate was observed at that time in cultures containing 10% FCS, a dose‐dependence inhibition of cell growth was detected, in contrast, for 2% FCS‐treated cells. Presence of TGF‐β during the last 24 h was not necessary to release the arrested cells. Furthermore, platelet‐poor plasma at 10% produced the same effects as FCS, suggesting that platelet‐derived factors, such as platelet‐derived growth factor (PDGF), could not be responsible for the release of blocked cells in this case. We compared the effect of TGF‐β to that of epidermal growth factor (EGF), used at an optimal concentration (10 ng/ml). In both slowly growing (2% FCS) and proliferating chondrocytes (10% FCS), EGF caused a significant increase of cell proliferation as early as 24 h. No arrest in late S‐phase but an augmentation of the percentage of cells in S‐ and G2M‐phases were observed. When combined, TGF‐β and EGF did not induce synergistic effect on the chondrocyte proliferation, as estimated by cell counting. [3H]‐thymidine labeling showed that the factors induced identical maxima of incorporation but the peak occurred earlier for TGF‐β than for EGF (approximately 6 h versus 12 h, respectively). Although both factors induce similar cell‐number increases at 48 h in 10% FCS‐containing medium, these proliferative effects were due to different actions on the cell cycle. The present study indicates that TGF‐β induces first a recruitment of chondrocytes in noncycling SQ‐ or G2Q‐blocked cells. Then, the release of these cells may produce either apparent stimulation of cell proliferation if sufficient levels of an unknown serum factor are present (10% FCS) or an inhibition of growth rate when only reduced amounts of this factor are available (2% FCS). This mechanism is quite different from that of EGF, which make more cells enter S‐phase, whatever the serum concentration in medium, and might be related to the transforming capacity of TGF‐β.
Ionic regulation of the plasma membrane potential of rainbow trout (Salmo gairdneri) spermatozoa: Role in the initiation of sperm motility
Authors: Gatti Jean‐Luc , Billard Roland , Christen Richard
Published: February 2005

The ionic dependence of the trout sperm plasma membrane potential was analysed by measuring the accumulation of the lipophilic ions 3H‐tetraphenylphosphonium (TPP) and 14C‐thiocyanate (SCN) following dilution in artificial media isotonic to the seminal fluid. Our data showed that the trout sperm plasma membrane has a mixed conductance: the plasma membrane potential is sensitive upon the transmembrane gradients of K+, Na+, and H+. This potential is negative (< −40mV) in a 125 mM choline chloride media (ChM) at pH 8.5. Replacement of choline by sodium has a small depolarizing effect. The membrane potential is about −15 mV in a 125 mM potassium chloride and falls near zero mV only if valinomycin is added. In ChM changing the external pH (pHe) greatly affects the membrane potential: its value rises from less than −40 mV at pHe 9.0 to −17 mV at pHe 5.0. This pH effect is observed also in presence of sodium or potassium. A decrease in the transmembrane proton gradient produced by increasing internal pH without changing pHe induces also a depolarisation of the plasma membrane. In the different media in which trout sperm remain immotile after dilution (media with [K+]>20–40 mM or a pH <7.5) the plasma membrane is more depolarized than in media allowing motility, suggesting a relationship between the state of membrane polarization and the intracellular effectors of the axonemal movement.
Spontaneous and cAMP‐dependent induction of a resting phase and neurite formation in cell hybrids between human neuroblastoma cells and thymidine auxotrophs of rat nerve‐like cells
Authors: Ishii Kazuhiro , Adachi Yoshifumi , Hatanaka Masakazu , Sakamoto Hiromi , Furuyama Jun‐Ichi
Published: February 2005

Cell hybrids (BIM) were produced between human neuroblastoma cells (IMR‐32) and thymidine auxotrophs (B3T) of rat nerve‐like cells (B103) in order to obtain cell lines undergoing stable neuronal differentiation. BIM cells exhibited the growth properties of partial transformation: 1) When the cell growth reached a plateau, BIM cells ceased to proliferate and expressed a differentiated phenotype. The shape of the cells changed from flat to round and they extended neurites. 2) When cultured in methylcellulose, BIM cells formed colonies, indicating that BIM cells have the ability of anchorage‐independent growth. By Southern blot analysis, BIM cells had both human and rat types of N‐myc genes. The human N‐myc genes were amplified, but the extent of the amplification was lower in BIM cells than that in the parental cell line IMR‐32. The rat N‐myc gene was detected at a similar level in BIM, B3T, B103, and rat fibroblastic cells 3Y1. Therefore, the decrease in amplification of human N‐myc genes may be involved in the properties of partial reverse‐transformation in BIM cells. When treated with various drugs such as db‐cAMP, forskolin, and cAMP with isobutyl‐methylxanthine, BIM cells expressed a nerve‐like phenotype. These findings indicate that cell hybridization yielded partial normalization of transformed nerve‐like cells.
Rapid endocytosis and recycling of wheat germ agglutinin binding sites on CHO cells: Evidence for two compartments in a nondegradative pathway
Authors: Raub Thomas J. , Koroly Mary Jo , Roberts R. Michael
Published: February 2005

The internalization and recycling of CHO cell plasma membrane components have been quantified by using iodinated wheat germ agglutinin (WGA) as an adsorptive tracer. Most of these binding sites are thought to be composed of a subpopulation of plasma membrane proteins called high‐molecular‐weight acidic glycoproteins (HMWAG). Greater than 90% of the WGA bound on the cell surface can be removed by brief treatment with N‐acetylglucosamine (GlcNAc). At 37°C, endocytosis of WGA that had been allowed to bind to the surface at 4°C is curvilinear with an initial rapid phase occurring with a t1/2 of 6–8 min. Within 20 min, accumulation has slowed gradually to steady‐state with 65% of the cell‐associated WGA located intracellularly or resistant to remova by GlcNAc. These portions are unaffected by increasing the extracellular concentration of WGA from 0.003 μM. By using pulse‐chase experiments, the observed decrease in rate of endocytosis is shown to be due to return of the WGA‐HMWAG complexes to the cell surface. More than 60% of the WGA that had been internalized is recycled within 30 min, with a mean t1/12 of 17 min. Recycling involved at least two intracellular populations where a significant fraction ( < 30%) of the WGA‐HMWAG complexes are lost gradually from the rapidly recycling pool. Most of the WGA‐HMWAG complexes that had internalized are not delivered to the lysosome. These results demonstrate the magnitude of rapid and continuous recycling of WGA binding sites between the cell surface and endosomes in fibroblasts.
Participation of transient‐type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells
Authors: Suzuki Nobuyuki , Kano Masaakira , Tanakadate Akihiro , Kudo Yoshihisa , Takagi Hiroshi , Yoshioka Tohru
Published: February 2005

Participation of two types of Ca2+ channels (T‐ and L‐types) in the sustained increase of cytosolic‐free Ca2+ concentration ([Ca2+]i) was studied in thyrotropin‐releasing hormone (TRH)‐stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole‐cell voltage‐clamp and Fura‐2 fluorometry, respectively. Phenytoin (100 μM) and Ni2+ (100 μM) selectively blocked T‐type Ca2+ channels and suppressed the TRH‐induced sustained [Ca2+]i increase in single cells. Synthetic ω‐conotoxin (ω‐CgTX, 2 μM) preferentially blocked L‐type Ca2+ channels, but it did not suppress the TRH‐induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T‐type Ca2+ channels in GH3 cells.
Antigen‐Dependent transition of IgE to a detergent‐insoluble form is associated with reduced IgE receptor‐dependent secretion from RBL‐2H3 mast cells
Authors: Seagrave Jeanclare , Oliver Janet M.
Published: February 2005

In mast cells, basophils, and the RBL‐2H3 tumor mast cell model, crosslinking cell surface IgE‐receptor complexes by multivalent ligands activates a signal transduction pathway that leads to the secretion of histamine, serotonin, and other inflammatory mediators. Receptor crosslinking in RBL‐2H3 cells also changes cell surface morphology and increases F‐actin assembly. Previously, Robertson et al. (J. Immunol., 135: 4565–4572, 1986) demonstrated that crosslinked IgE‐receptor complexes become associated with the Triton X‐100‐insoluble fraction (the “cytoskeleton”) of RBL‐2H3 cells and raised the possibility that receptor‐cytoskeletal association may be a required step in the stimulation of secretion. The studies reported here confirm by flow cytometry that crosslinking cell surface IgE by antigen induces the association of the crosslinked complexes with the detergent‐insoluble fraction. Dose‐response studies, also reported here, indicate that the detergent insolubility of the complexes does not correlate with secretion. Thus, secretion increases with antigen concentration to a maximum beyond which more antigen causes less, not more, secretion. There is little residual detergent‐insoluble IgE at the concentrations of antigen that promote optimal secretion, whereas the association of IgE with the detergent‐insoluble fraction is maximal at the high antigen concentrations that result in reduced secretion. The addition of monovalent hapten to reduce the amount of crosslinking caused by high concentrations of antigen increases secretion and simultaneously reduces the association of IgE with the detergent‐insoluble fraction. Dihydrocytochalasin B, an inhibitor of antigen‐stimulated actin polymerization, also increases the rate and extent of secretion and simultaneously delays the association of crosslinked IgE‐receptor complexes with the detergent‐insoluble fraction. From these data, we propose that the association of crosslinked IgE receptors with the detergent‐insoluble fraction of RBL‐2H3 cells increases with increased receptor crosslinking, is enhanced by antigen‐induced actin polymerization, and is more likely related to the termination than the stimulation of secretion. The ligand‐induced conversion of receptors to a detergent‐insoluble form is not restricted to mast cells but occurs in a variety of cell types. Its general function may be to limit the generation or transmission of transmembrane signals.
Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts
Authors: Mikkelsen Ross B. , Asher Craig R.
Published: February 2005

The effects of hyperthermia (41–43°C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41°C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37°C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+: H+ exchange. Shifting temperature to 41°C increased ouabain‐sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43°C for 60 min, the membrane potential of log phase cells depolarized (20–35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41°C only in the presence of ouabain. At 43°C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43°C, 0.25 mM amiloride inhibited heat‐stimulated Na+ uptake. Na+ efflux was enhanced at 41°C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41°C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures ≥43°C, the Na+ pump is inhibited.
Insulin‐like growth factor (IGF) binding to human fibroblast and glioblastoma cells: The modulating effect of cell released IGF binding proteins (IGFBPs)
Authors: McCusker R. H. , Camacho‐Hubner C. , Clemmons D. R. , Bayne M. L. , Cascieri M. A.
Published: February 2005

The cell surface of human fibroblasts contains not only type IIGF receptors but at least two forms of IGFBPs. Studies were undertaken to analyze the mechanisms by which these IGFBPs alter IGF‐I‐cell surface interactions. Human fetal fibroblasts (GM10) and a human glioblastoma cell line (1690) were chosen for analysis. During assays to quantify [125l]‐IGF‐l binding, both cell lines were shown to release IGFBPs into the binding assay buffer. Under equilibrium conditions, [125I]‐IGF‐I preferentially associates with IGFBPs in the assay buffer (up to 40% of the [125I]‐IGF‐I added) since they have a higher affinity than type IIGF receptors or IGFBPs associated with the cell surface. Likewise the addition of increasing concentrations of unlabeled IGF‐I results in preferential competition for binding to assay buffer IGFBPs. This results in a repartitioning of the [125I]‐IGF‐I that is bound to assay buffer IGFBPs onto cell surface binding sites. The degree of repartitioning is quantitatively related to the amount of [125I]‐IGF‐I bound to released IGFBPs. When cultures are exposed to cycloheximide before the binding assay, both the amount of IGFBPs that are released into the assay buffer and the amount of [125 I]‐IGF‐l that is repartitioned are decreased. In contrast when [Gln3, Ala4, Tyr15, Leu16]‐IGF‐l (IQAYLj‐IGF‐I, an IGF analog that has unaltered affinity for type I IGF receptors) is iodinated and tested, the competition curve with unlabeled IGF‐I shows no repartitioning effect. This form of IGF can be used to quantify type I receptor number independent of the presence of IGFBPs. IGF‐I and the [QAYL]‐IGF‐I compete equally with the [125I]‐[QAYL]‐IGF‐I for binding to cell surfaces, whereas unlabeled [QAYL]‐IGF‐I is > 25‐fold less potent compared to IGF‐I in competing with [125I]‐IGF‐I for cell surface binding. Specific binding of [125I]‐[QAYL]‐IGF‐I to GM10 and 1690 cell surfaces is < 20% of [125I]‐IGF‐I binding. These findings suggest that IGFBPs that are present on human fibroblast surfaces represent a large portion of the IGF binding sites. We conclude that the amount of IGFBPs released into assay buffer is a major determinant of the repartitioning of [125I]‐IGF‐I to cell surface binding sites and that both cell surface and assay buffer IGFBPs modulate type I IGF receptor binding.
Stimulating effects of insulin and low‐density lipoprotein on cell growth and macromolecular syntheses of HeLa cells cultured in K+‐depleted medium
Authors: Hosokawa Keiko , Yamaguchi Hisao , Ikehara Toshitaka , Takahashi Akira , Miyamoto Hiroshi
Published: February 2005

The influence of the intracellular K+ concentration on the effects of growth factors (insulin, EGF, hydrocortisone, and transferrin) and LDL on growth of HeLa cells was investigated. Upon replacenient of K+ in a chemically defined medium (K+‐CDM) by Rb+ (Rb+‐CDM), about 80% of the intracellular K+ was replaced by Rb+ within 24 h, but showed no further change in the next 24 h, irrespective of addition of dialyzed calf serum (5%) or growth factors to the medium. In Rb+‐CDM, cell growth and DNA synthesis were greatly suppressed, although cell viability was not significantly altered for 72 h. The suppression of cell growth was partially restored by addition of serum, insulin (5 μg/ml), or LDL (2.5 mg/ml) to Rb+ ‐CDM. A combination of serum and insulin or insulin and LDL stimulated cell growth to approximately the level in K+‐CDM without any addition, but a combination of serum and LDL did not have more effect than that of serum alone. Unexpectedly, other factors were ineffective in stimulating growth in Rb+‐CDM. In Rb+‐CDM, the effect of insulin was lost in 24–48 h, whereas that of LDL persisted for at least 96 h. Insulin and LDL also enhanced growth in K+‐CDM. After cessation of cell growth in Rb+‐CDM for 24 h, addition of insulin and/or LDL markedly restored cell growth and DNA synthesis. Therefore, insulin and LDL may stimulate certain mechanisms required for cell growth that can operate in K+‐deficient conditions.
Plasminogen activators and their inhibitors in the neuromuscular system: II. Serpins and serpin: Protease complex receptors increase during in vitro myogenesis
Authors: Festoff Barry W. , Rao Jasti S. , Rayford Alan , Hantaï Daniel
Published: February 2005

In the course of studies on the regulation of plasminogen activator‐mediated extracellular matrix degradation in muscle we found the presence of a factor, a cellular inhibitor of serine proteases having features similar to the serpin protease nexin I (PNI). This factor was present in the medium and at maximum concentration following fusion of skeletal muscle cells in culture. The ability of the PNI homologue in mouse muscle to inhibit ECM degradation by urokinase in myo‐blast medium was compared to that of human PNI purified from human fibro‐blasls. Stable (to SDS) 1:1 molar ratio complex formation between PNI and proteases, the proposed means by which these enzymes are regulated and removed, was also detected. Cell surface receptors for protease:PNI complexes, the specific binding sites for inactive complex internalization, were found on multinucleated myotubes, while little or no receptor activity was detected on myoblasts. These data suggest that developmental regulation of (a) increased PNI proteolytic inhibitory activity expression and (b) the appearance of protease:inhibitor complex receptors on muscle cell surfaces during myogenesis may constitute important regulatory features of muscle suface proteolytic activity. They complement previous studies of proteoglycan metabolism in muscle, which itself contains molecules capable of regulating the activity of myotube surface proteases.
Cellular distribution and biological activity of epidermal growth factor receptors in A431 cells are influenced by cell‐cell contact
Authors: Lichtner Rosemarie B. , Schirrmacher Volker
Published: February 2005

The potential significance of cell‐cell interactions on EGF receptor (EGFR) activity was investigated in cultured adherent A431 cells seeded as single‐cell suspensions with different initial cell densities. In dense cultures, EGFRs were mainly localised at cell boundaries and in microvilli as shown by immunofluorescence analysis with an EGFR‐specific antibody while in sparse cultures the distribution of EGFRs was more diffuse. Scatchard analysis showed that as cell density decreased the number of high‐affinity receptors increased considerably. Upon treatment of adherent intact cells with EGF all cells in sparse cultures contained activated EGFRs as demonstrated by immunofluorescence analysis with a phosphotyrosine‐specific antibody, while in dense cultures mainly cells at the periphery of a cluster and especially at their expanding borders exhibited activated EGFRs. EGF‐induced phosphorylation in intact cells was greatly enhanced in sparse compared with dense cultures as demonstrated by immunoprecipitation with a phosphotyrosine‐specific antibody. In contrast to intact cells, in cytoskeleton preparations, obtained after mild detergent treatment of adherent cells, EGFRs were able to undergo EGF‐independent phosphorylation. Pretreatment of cells with EGF led to enhanced tyrosine phosphorylation of cytoskeletal‐associated proteins. Our observations suggest that cell density has a considerable effect on the subcellular localisation as well as biological activity of the EGFR. Thus, in intact A431 cells growing with extensive cell‐cell interactions some negative control mechanisms preventing EGFR activation may be exerted by adjacent cells.
Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: Differentiation response distinguishes KGF from EGF family
Authors: Marchese C. , Messina A. , Faggioni A. , Torrisi M. R. , Frati L. , Ron D. , Rubin J. , Aaronson S. A.
Published: February 2005

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF‐ or EGF‐stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGFα, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.
Human recombinant interleukin‐1β‐ and tumor necrosis factor α‐mediated suppression of heparin‐like compounds on cultured porcine aortic endothelial cells
Authors: Shimada Kazuyuki , Ozawa Toshio , Kobayashi Masashi
Published: February 2005

Cytokines are known to tip the balance of the coagulant‐anticoagulant molecules on the endothelial cell surface toward intravascular coagulation. Their effects on endothelial cell surface‐associated heparin‐like compounds have not been examined yet. Incorporation of [35S]sulfate into heparan sulfate on cultured porcine aortic endothelial cells was suppressed by human recombinant interleukin‐1β (rIL‐1β) or tumor necrosis factor α (rTNFα) in a dose‐ and time‐dependent manner with little effect on cell number, protein content, and [3H]leucine incorporation of cells. Maximal inhibition was achieved by incubation of cells with 100 ng/ml of rlL‐1β or 5 ng/ml of rTNFα for 12–24 hours, resulting in a reduction of the synthesis of heparan sulfate on the cell surface by approximately 50%. The dose dependency was consistent with that seen in the stimulation of endothelial cell procoagulant activity by each cytokine. The suppression of heparan sulfate synthesis was sustained for at least 48 hours after pretreatment of cells with cytokines and was unchanged after the addition of indomethacin or polymyxin B. The rate of degradation of prelabeled 35S‐heparan sulfate on the cell surface was not altered by cytokine treatments. Neither the size, the net negative charge, nor the proportion of the molecule with high affinity for antithrombin III of endothelial cell heparan sulfate was changed by cytokines. Furthermore, specific binding of 125I‐Labeled antithrombin III to the endothelial cell surface was reduced to 40–60% of control by cytokines. In parallel with reduction in binding, antithrombin III cofactor (heparin‐like) activity was partially diminished in cytokine‐treated endothelial cells. Thus, cytokine‐mediated suppression of heparin‐like substance on endothelial cells appears to be another cytokine‐inducible endothelial effects affecting coagulation.