bluuebunny/crossref_metadata_2025 huggingface dataset:
Concanavalin A‐induced impairment of fibroblast spreading on laminin but not on fibronectin
Authors: Codogno Patrice , Doyennette‐Moyne Marie‐Agnès , Botti Joëlle , Aubery Michèle
Published: February 2005

The effect of concanavalin A (Con A) on the adhesion of 8‐day‐old chick embryo fibroblasts (CEFs) to fibronectin (FN) and laminin (LM) was studied. Con A was shown to inhibit the spreading of CEF on a LM substrate. In contrast, no inhibition of CEF spreading on the FN substrate could be detected when the quantity of FN coated varied from 0.5 to 4 pmoles. The effect induced by Con A was specific, since it was abolished by 100 mM α‐methyl‐mannopyranoside. The inhibition of CEF spreading was only observed when the lectin was added during the 20 min following cell plating. In addition, the effect of Con A on CEF spreading on the LM substrate was shown to be dependent upon its presence at the cell surface, since under conditions which accelerate the uptake of the lectin, the effect on cell spreading is no longer detectable. Furthermore, the number of CEFs attached to LM was not modified by the lectin. The molecular weight of the isolated Con A binding sites revealed glycoproteins ranging from 30,000 to 72,000. On the other hand, these Con A binding sites did not interact with LM‐Sepharose. Only a protein with a molecular weight of 68,000 which did not express affinity for Con A bound tightly to the LM‐Sepharose. These data suggested that cell surface Con A binding sites do not interfere with the initial step of CEF adhesion to LM but play a key role during their spreading on this glycoprotein.
Role of IIb‐IIIa‐like glycoproteins in cell‐substratum adhesion of human melanoma cells
Authors: Knudsen Karen A. , Smith Linda , Smith Sena , Karczewski Jerzy , Tuszynski George P.
Published: February 2005

The platelet fibrinogen receptor, glycoprotein complex IIb‐IIIa, was isolated from human platelets by lectin and monoclonal antibody affinity chromatography and a polyclonal antiserum (anti‐IIb‐IIIa) was generated and used to probe for the presence and function of IIb‐IIIa‐like molecules in two adherent human cell lines. Both C32 melanoma cells and W138 fibroblasts expressed a IIb‐IIIa‐like complex on their surface as indicated by immunoprecipitation of detergent extracts of surface radiolabeled cells. When added to cells plated in medium containing 10% serum, the anti‐IIb‐IIIa antiserum perturbed the adhesion of C32 melanoma cells, but not of W138 fibroblasts. In a serum‐free system, anti‐IIb‐IIIa antibodies inhibited attachment and spreading of C32 cells to fibrinogen, vitronectin, and fibronectin adsorbed to glass. Anti‐IIb‐IIIa had no effect on the attachment and spreading of W138 cells to the extracellular matrix proteins, however. Thus, the IIb‐IIIa‐like complex appears to play a predominant role in cell‐substratum adhesion of C32 cells, but not W138 cells, and may result from the fact that, on a protein basis, the C32 melanoma cells express approximately 3 times more complex on their surface than do W138 fibroblasts. The results suggest that the relative abundance of a particular adhesion receptor on the cell surface may govern its importance to cell‐substratum adhesion.
Differential effect of D‐penicillamine on the cell kinetic parameters of various normal and transformed cellular types
Authors: Friteau L. , Jaffray P. , Ronot X. , Adolphe M.
Published: February 2005

The cell‐growth‐inhibitory and phase‐specific effects of D‐penicillamine on cell‐cycle progression were investigated using cell‐proliferation patterns, quantitative cell‐cycle analysis by flow cytometry, and determination of the mitotic index and binucleate cell fraction of normal (rabbit articular chondrocytes, L 809, rabbit fibroblasts) and transformed (HeLa, L 929) cells. D‐penicillamine treatment resulted in an inhibition of growth within a dose range of 5 × 10−4 M to 7.5 × 10−3 M. Examination of DNA by flow cytometric analysis revealed that rabbit articular chondrocytes were preferentially arrested in the G0/1 phase of the cell cycle, whereas the other cell lines were blocked in the G2 + M phase; the increase in the proportion of cells with G2 + M DNA content was partially due to an enhancement of binucleate cells, resulting in a cytokinesis perturbation for HeLa and L 929 cells. These results showed that D‐penicillamine affects cell proliferation through different events according to cell type.
Multiple mechanisms for the inhibition of rRNA synthesis during HL‐60 leukemia cell differentiation
Authors: Schwartz Edward L. , Nilson Laura
Published: February 2005

Treatment of HL‐60 human promyelocytic leukemia cells with inducers of granulocytic differentiation produces a depletion of cellular rRNA, with the anthracycline antibiotics aclacinomycin A (ACM) and marcellomycin (MCM) causing a more rapid loss than dimethylsulfoxide (DMSO). This action is associated with a large reduction in RNA synthesis, which precedes any decreases in protein or DNA synthesis, and is specific for rRNA relative to total polyadenylated RNA synthesis. A 70% reduction in rRNA synthesis occurs within 20 minutes of ACM treatment and by 30 hours of DMSO exposure. Relative to the amount of 28S and 18S rRNA in the cells, there is a nearly complete depletion of the amount of 45S rRNA and other large rRNA precursors in contrast, DMSO treatment produces a more coordinated decrease in 18S and 28S rRNA and rRNA precursors. The anthracycline antibiotics inhibited the synthesis of 5′ proximal and 3′ distal regions of the pre‐rRNA transcript, while actinomycin D had a relatively sparing effect on the transcription of 3′ transcripts. These studies demonstrate that different inducers of HL‐60 differentiation have varying sites of action on rRNA synthesis and/or processing, with depletion of cellular rRNA as a common consequence.
Stimulation of receptor‐mediated low density lipoprotein endocytosis in neuraminidase‐treated cultured bovine aortic endothelial cells
Authors: Sprague Eugene A. , Moser Marianne , Edwards Ellen H. , Schwartz Colin J.
Published: February 2005

Sialic acids, occupying a terminal position in cell surface glycoconjugates, are major contributors to the net negative charge of the vascular endothelial cell surface. As integral membrane glycoproteins, LDL receptors also bear terminal sialic acid residues. Pretreatment of near‐confluent, cultured bovine aortic endothelial cells (BAEC) with neuraminidase (50 mU/ml, 30 min, 37°C) stimulated a significant increase in receptor‐mediated 125l‐LDL internalization and degradation relative to PBS‐treated control cells. Binding studies at 4°C revealed an increased affinity of LDL receptor sites on neuraminidase‐treated cells compared to control BAEC (6.9 vs. 16.2 nM/106 BAEC) without a change in receptor site number. This enhanced LDL endocytosis in neuraminidase‐treated cells was dependent upon the enzymatic activity of the neuraminidase and the removal of sialic acid from the cell surface. Furthermore, enhanced endocytosis due to enzymatic alteration of the 125l‐LDL molecules was excluded. In contrast to BAEC, neuraminidase pretreatment of LDL receptor‐upregulated cultured normal human fibroblasts resulted in an inhibition of 125l‐LDL binding, internalization, and degradation. Specifically, a significant inhibition in 125l‐LDL internalization was observed at 1 hr after neuraminidase treatment, which was associated with a decrease in the number of cell surface LDL receptor sites. Like BAEC, neuraminidase pretreatment of human umbilical vein endothelial cells resulted in enhanced receptor‐mediated 125l‐LDL endocytosis. These results indicate that sialic acid associated with either adjacent endothelial cell surface molecules or the endothelial LDL receptor itself may modulate LDL receptor‐mediated endocytosis and suggest that this regulatory mechanism may be of particular importance to endothelial cells.
Role of plasma membrane and of cytomatrix in maintenance of intracellular to extracellular ion gradients in chicken erythrocytes
Authors: Cameron I. L. , Hunter K. E. , Smith N. K. R. , Hazlewood C. F. , Ludany A. , Kellermayer M.
Published: February 2005

Ultrastructural observations in combination with electron probe X‐ray microanalysis on detergent (Brij 58) permeabilized (disruption of the plasma membrane) nucleated chicken erythrocytes support the view that a large fraction of cytoplasmic and nuclear K+ is not freely diffusible and that adsorption of K+ on detergent released mobilizable proteins exists within the cell. The data also suggest that the detergent proteins are normally immobilized by a detergent‐resistant cytoskeleton so that they are not immediately free to diffuse from the cell for several minutes after detergent disruption of the plasma membrane.
A novel function of glutamine in cell culture: Utilization of glutamine for the uptake of cystine in human fibroblasts
Authors: Bannai Shiro , Ishii Tetsuro
Published: February 2005

Transport and metabolism of glutamine has been investigated in human diploid fibroblasts, IMR‐90. Glutamine was taken up via System ASC (Na+‐dependent amino acid transport system especially reactive with short or polar side chain amino acids). In the routine culture medium the cells contained a large quantity of glutamate; its major source was shown to be glutamine in the medium. Previously we described a transport system that mediates the entrance of cystine in exchange for the exit of glutamate (Bannai, 1986). Since the cystine taken up is reduced to cysteine and the cysteine readily exits to the medium where it is oxidized to cystine, a cystine‐cysteine cycle across the plasma membrane has been postulated. When the cells were cultured in glutamate/glutamine‐free medium, intracellular glutamate decreased, depending on the amount of cystine in the medium; in the absence of cystine, glutamate decreased very slowly. When the cells were cultured in ordinary medium, glutamine in the medium decreased, and glutamate in the medium increased. Both changes were well correlated with cystine concentration in the medium. These results are consistent with the view that the intracellular glutamate, of which the source is glutamine in the medium, is released from the cells into the medium in order to take up cystine and thereby to rotate the cystine‐cysteine cycle. In the routine culture one‐third to one‐half of the total consumption of glautamine seems to be used for the uptake of cystine.
Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium
Authors: Helbig Horst , Korbmacher Christoph , Stumpff Friederike , Coca‐Prados Miguel , Wiederholt Michael
Published: February 2005

The regulation of intracellular pH (pHi) was monitored in a virus‐transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate‐free media using the pH‐sensitive absorbance of 5‐ (and 6‐) carboxy‐4′,5′‐dimethylfluorescein. Under resting conditions, pHi averaged 6.98 ± 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and the withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ ‐depleted cells led to an alkalinization, which was senstive to amiloride, with an IC50 of about 20 μM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.
Metabolism of U6 RNA species in nonirradiated and UV‐irradiated mammalian cells
Authors: Choudhury Kanakendu , Choudhury Indrani , Jones Robert W. , Thirunavukkarasu Chellaiah , Eliceiri George L.
Published: February 2005

We observed a series of rapidly labeled U6 RNA bands, which were hybrid selected with U6 DNA, in nonirradiated human cells. The electrophoretic mobility of these bands in denaturing gels was lower than that of the known mature U6 RNA species, and was equivalent to transcripts up to ∼7 nucleotides longer. These multiple U6 RNA species lost their label during a chase without a proportional increase in radioactivity in the known mature U6 RNA, which suggests that a substantial fraction is not processed into the major mature U6 RNA. During a label chase, the multiple U6 RNA bands appeared first in the cytoplasmic fraction and later in nuclei. One of the major rapidly labeled U6 RNA bands had the electrophoretic mobility of an RNA species one nucleotide shorter than the known mature U6 RNA. UV light induced a UV dose‐dependent, preferential disappearance of recently synthesized molecules of the U6 RNA species of higher gel electrophoretic mobility, including the known mature U6 RNA. Since this effect was seen in cells pulse‐labeled immediately before or after irradiation, it suggests that UV radiation induces the specific degradation of the electrophoretically faster moving species of U6 RNA, which are apparently shorter chains. The effect of UV light was RNA species‐specific, was not seen in molecules synthesized long (e.g., 22 hr) before irradiation, and occurred in human and mouse cells.
Effect of reduced protein synthesis on the cell cycle in sea urchin embryos
Authors: Dubé François
Published: February 2005

We have reinvestigated the existence of cyclical fluctuations of protein synthesis and have examined the effects of reducing it in early embryos of the purple sea urchin, Stronglocentrotus purpuratus. The results show that protein synthesis increases linearly during the first 45–60 minutes after fertilization, then transiently decreases during mitosis, and rises again at first cleavage. Reducing protein synthesis of embryos to 35% its normal value only slightly affects the rate of progression through the cell cycle. It is also shown that the observed retardations of the cell cycle, under depressed protein synthesis, are attributable (by 80%) to a lengthening of the premitotic phase but also, to a lesser extent (20%), to a lengthening of the mitotic phase itself. These results suggest that mitotic proteins, in sea urchin embryos, are stable and little affected by an imposed decrease of protein synthesis during their accumulation phase. This analysis supports the view that specific mechanisms, other than decreased protein synthesis, need be turned on only at appropriate times during the cell cycle in order to explain the destruction or deactivation of mitotic proteins. Finally, a one‐dimensional SDS‐PAGE analysis of synthesized proteins, labeled with 35S‐methionine, reveals the presence of a 50‐kDa cyclin showing the expected characteristics of mitotic proteins deduced from our results.
Wheat germ agglutinin inhibits thrombin‐induced rises in cytosolic free calcium and prostacyclin synthesis by human umbilical vein endothelial cells
Authors: Grulich‐Henn Jürgen , Weksler Babette B. , Watanabe Kentaro , Jaffe Eric A.
Published: February 2005

To characterize the endothelial cell surface membrane glycoproteins that mediate thrombin stimulation of PGI2 synthesis by human umbilical vein endothelial cells (HUVEC), HUVEC were stimulated with thrombin in the presence or absence of different lectins. Of the lectins tested, only wheat germ agglutinin (WGA) inhibited thrombin‐induced rises in cytosolic free calcium ([Ca2+]i), measured using Quin 2‐loaded HUVEC and PGI2 production measured by radioimmunoassay. However, WGA by itself had no influence on baseline [Ca2+]i or PGI2 production and did not inhibit histamine‐induced rises in [Ca2+]i. The inhibition of thrombin‐induced rises in [Ca2+]i and PGI2 production by WGA was dose dependent, with half‐maximal inhibition occurring at 2 μg/ml WGA also inhibited thrombin‐induced release of 3H‐arachidonic acid. These effects of WGA were reversed by N‐acetyl‐glucosamine (GlcNAc) and N‐acetyl‐neuraminic acid, which bind specifically to WGA, but not by unrelated sugars. Succinylated WGA (succ‐WGA), a chemically modified derivative of WGA that binds to GlcNAc but, unlike native WGA, not to sialoglycoproteins, did not inhibit thrombin‐induced rises in [Ca2+]i and PGI2 production. These results suggest that thrombin induces rises in [Ca2+]i and PGI2 production by interacting with an endothelial surface membrane sialoglycoprotein.
Actin depolymerization and inhibition of capping induced by pentoxifylline in human lymphocytes and neutrophils
Authors: Rao K. Murali Krishna , Crawford Jeffrey , Currie Mark S. , Cohen Harvey J.
Published: February 2005

Pentoxifylline is used clinically for the treatment of intermittent claudication. It is believed to exert its effect by altering the rheologic properties of blood. The cytoskeleton plays an important role in the maintenance of cell structure and function. In particular, alterations in the state of actin seem to play an important role in cell motility. Therefore, we examined the effect of pentoxifylline on the actin state in human polymorphonuclear leukocytes (PMN) and mononuclear cells. Pentoxifylline (10 mM final concentration) decreased F‐actin content in both PMN and mononuclear cells. Pentoxifylline also inhibited concanavalin A‐induced capping in PMN and mononuclear cells. Similarly, surface immunoglobulin capping in B lymphocytes was also inhibited. Pretreatment of cells with pertussis toxin did not inhibit pentoxifylline‐induced decrease in F‐actin, suggesting pentoxifylline does not act through pertussis toxin‐sensitive G‐proteins. Dibutyryl cyclic AMP failed to show any significant effect on the F‐actin content in PMN. Therefore, the effect of pentoxifylline cannot be attributed to changes in cyclic AMP levels. Chemotactic peptide‐induced actin polymerization was unaffected in PMN when expressed as percent change in F‐actin. The observations reported here suggest that the rheological effects of pentoxifylline might be due to its effects on the actin state in the cellular elements of the blood. Further studies on the mechanism of action of pentoxifylline on actin state in leukocytes will prove useful in delineating the physiological mechanisms regulating actin state in leukocytes.
Decreased susceptibility of cultured smooth muscle cells from SHR rats to growth inhibition by heparin
Authors: Resink Thérèse J. , Scott‐Burden Timothy , Baur Ursula , Bürgin Maria , Bühler Fritz R.
Published: February 2005

Smooth muscle cell proliferation is regulated through the coordinated action of growth inhibitors and growth factors/mitogens; a specific heparin‐epidermal growth factor (EGF) complementation has been proposed (Reilly et al., 1987, J. Cell. Physiol., 131:149–157). In culture, vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) proliferate more rapidly than VSMC from control Wistar Kyoto rats (WKY). We observed that, compared with WKY‐derived VSMC, cells from SHR were markedly less susceptible to growth inhibition both by heparin and its homopolysaccharide analogue pentosan polysulphate (PPS). SHR‐derived VSMC exhibited a reduced capacity for binding of [3H]heparin to specific extracellular surface receptors, whereas affinities for heparin were comparable between both VSMC isolates. The early (0–2 hr at 37°C) kinetics of internalization did not differ between SHR‐ and WKY‐derived VSMC, but both internalized equivalent proportions (∼ 10%) of initially surface‐bound heparin. VSMC from SHR exhibited a greater capacity, without a changed affinity, for [I125]EGF binding than VSMC from WKY. Pre‐exposure of VSMC to heparin or PPS decreased, in a time‐dependent manner, the EGF binding capacity for both SHR and WKY (by 40–50% after 72 hr). However, in absolute terms, the EGF‐binding capacity of VSMC from SHR exposed to heparinoids was similar to that of nonexposed VSMC from WKY.
Growth‐related changes of oxygen consumption rates of tumor cells grown in vitro and in vivo
Authors: Kallinowski F. , Tyler G. , Mueller‐Klieser W. , Vaupel P.
Published: February 2005

Growth‐related changes of oxygen consumption rates of tumor cells, grown in vitro or in vivo, were investigated. For in vitro investigations, L929 and DS‐carcinosarcoma cells were cultured in artificial media. For in vivo studies, DS‐carcinosarcoma cells were implanted into the abdominal cavity of Sprague‐Dawley rats (ascites tumor, containing malignant cells, leukocytes, lymphocytes, and macrophages). Oxygen uptake was measured photometrically. Parameters of the extracellular medium judged to possibly influence the respiratory activity of tumor cells were monitored at different growth stages (glucose, lactate, and amino acid levels, oxygen and carbon dioxide partial pressures, and pH values). The results obtained clearly show that the oxygen uptake of tumor cells grown in vitro decreased as quiescence developed. In contrast, the respiratory activity of in vivo DS‐carcinosarcoma ascites cells increased as tumor growth reached plateau phase. The differences observed cannot be attributed solely to changes of the environmental conditions monitored. It is likely that an increased respiration rate of activated host cells might profoundly contribute to the elevation of the respiratory capacity of DS‐carcinosarcoma ascites tumors grown in vivo. These data provide evidence that solid tumors in vivo can increase their O2 uptake at an enhanced O2 availability not only due to an enlarged tumor volume with adequate O2 supply but also due to an elevation of the respiratory activity of different cell populations within a tumor.
Regulation of collagenase and collagenase mRNA production in early‐ and late‐passage human diploid fibroblasts
Authors: Sottile Jane , Mann David M. , Diemer Verena , Millis Albert J. T.
Published: February 2005

The levels of collagenase and collagenase mRNA produced by early‐passage (40% of lifespan completed) and late‐passage (>80% of lifespan completed) cultures of human fibroblasts were analyzed. The constitutive levels of collage‐nase and collagenase mRNA produced by the late‐passage cultures were 10‐30x greater than the levels observed in similarly treated early‐passage cultures. Immunofluorescence analysis established that the percentage of collagenase‐positive cells was also greater (77% vs. 4%) in the late‐passage cultures. To determine whether the difference in collagenase production resulted from cell‐derived regulatory factors, collagenase production was examined in cultures plated onto substrates coated with fibroblast extracellular matrix (ECM). Collagenase and collagenase mRNA production was enhanced in both types of cultures, although amounts produced by ECM‐induced early‐passage cultures was significantly less than that produced by similarly treated late‐passage cultures. Collagen‐coated substrates also induced collagenase synthesis.
Evidence for a major route for zinc uptake in human red blood cells: [ZN(HCO3)2CL]−influx through the [CI−/HCO3−] anion exchanger
Authors: Torrubia José Octavio Alda , Garay Ricardo
Published: February 2005

The initial rate of Zn2+ uptake in human red cells was measured by atomic absorption. A very important fraction of Zn2+ uptake was inhibited by DIDS with IC50 = 0.3 (μ)M (and by furosemide and bumetanide with IC50 of 200 and 500 μ M, respectively). DIDS–sensitive Zn2+ uptake exhibited the following properties: (1) It required the simultaneous presence of both external HCO3− and Cl−. (2) In Cl− containing media, it was strongly stimulated by external HCO3− following a sigmoidal (S–shaped) and saturable function, which was fitted by a Hanes equation, with n = 2 and an apparent dissociation constant (for external HCO3−) of 5.3 ± 0.9 mM (mean ± SD of four experiments). The maximal rate of Zn2+ uptake at saturating HCO3− concentrations was 50.7 ± 4.8 mmol (liter cells x h)−1. (3) In HCO3− containing media, it was strongly stimulated by external Cl− following a Michaelis‐like equation with an apparent dissociation constant (for external Cl− of 88 ± 11 mM (mean ± SD of three experiments). 4) Bicarbonate‐stimulated Zn2+ uptake was inhibited by physiological concentrations of phosphate (sulfate was a much less potent inhibitor than phosphate). A kinetic analysis of the data strongly suggested that zinc was transported by the anion carrier in the form of the monovalent anion complex: [Zn(HCO3)2Cl]−.
Characterization of polyclonal antibodies that distinguish acidic and basic fibroblast growth factors by using western immunoblotting and enzyme‐linked immunosorbent assays
Authors: Riss Terry L. , Sirbasku David A.
Published: February 2005

Rabbit polyclonal antibodies were raised against ovalbumin conjugates of purified bovine brain acidic fibroblast growth factor (aFGF) and a synthetic peptide containing the Nα‐terminal 1–24 amino acid sequence of bovine basic fibroblast growth factor (bFGF). Theses antibodies were used to specifically detect 1‐ng quantities of aFGF and bFGF by using enzyme‐linked immunosorbent assay(ELISA)and Western immunoblot procedures. Antibodies raised against aFGF recognized bovine brain aFGF and bovine recombinant aFGF by very poorly recognized recombinant bFGF or purified porcine or bovine pituitary bFGF with ELISA and Western immunoblot procedures. Antibodies raised against bFGF (1–24) recognized purified bovine, procine, and recombinant human bFGF but only very poorly reconized aFGF with ELISA and Western immunoblot procedures. In vitro addition of anti‐bFGF antibodies was able to partially neutralize bFGF‐stimulated 3H‐thymidine incorporation by COMMA‐D mouse mammary epithelial cells while having no effect on aFGF or epidermal growth factor (EGF) stimulation. In vitro additionof anti‐aFGF antibodies had no effect on bFGF‐or EGF‐stimulated 3H‐thymidine incorporation, but surprisingly, had a potentiating effect on aFGF stimulation. Antibodies against aFGF immobilized on protein A‐Sepharose were able to specifically and completely remove mitogenic activity from solutions containing aFGF but had no effect on removal of mitogenic activity from control solutions containing bFGF or EGF. similarly, immobilized anti‐bFGF antibodies completely removed mitogenic activity from solutions of bFGF, but notaFGF or EGF controls. These antibodies have been useful for the identification and characterization of growth factors from tissue and recombinant sources.
Effect of 5‐amino‐4‐imidazolecarboxamide riboside (AICA‐riboside) on the purine nucleotide synthesis and growth of rat kidney cells in culture: Study with [15N]aspartate
Authors: Nissim Itzhak , Yudkoff Marc , Nissim Ilana , States Beatrice
Published: February 2005

The present investigation evaluates the effect of AICA‐Riboside on the synthesis of purine nucleotide and the growth of normal rat kidney cells in culture. Experiments in the presence and absence of various concentrations of AICA‐Riboside were conducted with Dulbecco's Modified Eagle's Medium supplemented with either 1 mM [15N] aspartate or [14N]aspartate. Addition of 50 μM AICA‐Riboside to the incubation medium significantly stimulated intracellular adenine nucleotide concentrations following incubation for 48 hours. This stimulation was associated with augmented cell growth and DNA concentration. In contrast, with concentrations above 100 μM of AICA‐Riboside in the incubation medium, there was a remarkable inhibition of cell growth and a significant depletion of intracellular pools of adenine nucleotides and DNA. Experiments with [15N] aspartate showed that the initial rate (0‐24 hours) of [6‐15NH2] adenine nucleotide formation from 1 mM [15N] asparate was 38.8 ± 9.6, 67.9 ± 12.5, and 20.1 ± 3.8 pmol h−1/106 cells in the presence of O (control), 50 μM and 500 μM AICA‐Riboside, respectively. These observations indicate that the main effect of AICA‐Riboside is on the formation of AMP from aspartate and IMP via the sequential action of adenylosuccinate synthetase and adenylosuccinate lyase. The current studies suggest that AICA‐Riboside could be used as a factor mediating renal cell mitosis in culture. AICA‐Riboside has a biphasic effect on the growth of renal epithelial cells in culture and on their intracellular purine nucleotides and DNA concentration.
Modulatory function of protein kinase C in the activation of ornithine decarboxylase and in cAMP production in rat osteoblasts
Authors: van Leeuwen J. P. T. M. , Bos M. P. , Herrmann‐Erlee M. P. M.
Published: February 2005

The effect of activation of protein kinase C on stimulation of ornithine decar‐boxylase (ODC) activity and cAMP production was studied in fetal rat osteoblasts. Both phorbol 12‐myristate, 13‐acetate (PMA), an activator of protein kinase C, and 4α‐phorbol, ineffective in activating protein kinase C, failed to stimulate ODC activity and cAMP production. We tested the effect of protein kinase C on stimulation of ODC activity by parathyroid hormone (PTH) and forskolin. In contrast to PTH‐stimulated ODC activity, which was not affected by PMA, forskolin‐stimulated (1 and 10 μM) ODC activity was dose dependently reduced. PMA (400 nM) reduced both 1 and 10 μM forskolin‐stimulated ODC activity to the same level, ∼ 3 nmol CO2/mg protein, which suggests a controlling role of protein kinase C in forskolin‐stimulated ODC activity. The study of the effect of protein kinase C on PTH‐ and forskolin‐stimulated cAMP production also revealed differences between PTH and forskolin. When PMA was added simultaneously with PTH (4 and 20 nM) or forskolin (1 and 10 μM) the PTH‐stimulated cAMP production was dose‐dependently potentiated by PMA, whereas forskolin‐stimulated cAMP production was not affected. However, both PTH‐ and forskolin‐stimulated cAMP production was dose‐dependently augmented when PMA was added 3 min prior to PTH or forskolin. With increasing preincubation periods (up to 24 h) with PMA instead of a potentiation an inhibition was observed. This inhibition is not due to PTH receptor desensitization, although, on basis of the present results desensitization can not completely be excluded. In all cases 4α‐phorbol was without effect. The present results show that protein kinase C modulates stimulation of ODC activity and cAMP production in fetal rat osteoblasts. The modulation of both ODC activity and cAMP production appears to be dependent on the nature of the stimulator. The present data suggest a role for protein kinase C in limiting the cAMP‐mediated stimulation of ODC activity in these cells. Furthermore, it is suggested that protein kinase C can interfere at more than one site in the cAMP‐generating system.
Analysis of cell variants showing differential susceptibilities to radiation‐ or chemical‐induced neoplastic transformation: Differences in their responses to growth factors
Authors: Tatsuka Masaaki , Orita Satoshi , Kakunaga Takeo
Published: February 2005

The induction of DNA synthesis in quiescent, density‐arrested Balb/c 3T3 cells is known to be controlled by the sequential action of at least two functionally distinct sets of growth factors, so‐called “competence factors” and “progression factors.” Here we examined this induction pathway in Balb/c 3T3 A31‐l variants, which showed differential susceptibilities to radiation‐ and chemical‐induced neoplastic transformation despite their similar susceptibilities to radiation‐ or chemical‐induced cell killing and mutagenesis. DNA synthesis was acquired only with the exposure to progression factors in a highly susceptible cell variant (A31‐l‐13) whereas both competence factors and progression factors were required for a less susceptible cell variant (A31‐l‐1). The competent state constitutively produced by an autologous mechanism in the highly transformation‐susceptible A31‐l‐13 cells suggests the existence of an endogenous promoter that acts for the expression of the transformed phenotype in an autocrine fashion when the cells have been initiated by radiation or chemical carcinogens. The growth factor requirements acting as a determining factor for susceptibilities to transformation are discussed.
Direct evidence for nuclear and cytoplasmic colocalization of proteasomes (Multiprotease complexes) in liver
Authors: Tanaka Keiji , Kumatori Atsushi , Ii Kunio , Ichihara Akira
Published: February 2005

Subcellular localization of the large multicatalytic protease complexes called proteasomes, which have been found in soluble fractions of various cells, was examined by biochemical, immunological, and immunohistological methods. Rat liver nuclei, purified by two different procedures, showed high activities for degrading [3H]methylcasein and various fluorogenic oligopeptides with neutral and weakly alkaline pH optima. On gel filtration, all of these peptidase activities were recovered in a single peak with the unusually large molecular weight of about 600,000. Properties of the proteolytic activity in crude extracts of the nucleus and the cytoplasm were very similar. Immunoelectrophoretic blot analysis showed the presence of appreciable concentrations of proteasomes with similar immunoreactivity in isolated nuclear and cytosolic fractions. Moreover, immunohistochemical staining of human liver showed that proteasomes were predominantly localized in the nuclear matrix but also were present diffusely in the cytoplasm of hepatocytes. These findings indicate the nuclear and cytoplasmic colocalization of proteasomes.
Prostaglandin E2 receptor heterogeneity and dysfunction in mammary tumor cells
Authors: Fulton Amy M. , Laterra John J. , Hanchin Cynthia M.
Published: February 2005

We have reported previously that murine mammary tumor cell subpopulations isolated from one spontaneous adenocarcinoma are heterogenous in terms of prostaglandin E2 (PGE2) syntnetic capacity. We have also shown that tumor‐PGE2 contributes to the ability of these cells to grow and metastasize in vivo (Fulton and Heppner: Cancer Research 45:4779–4784, 1985). In the present study, we have asked whether exogenous PGE2 has direct effects on the proliferation of these cells in vitro and if such responses can be attributed to the capacity of these cells to (1) bind PGE2 and (2) activate adenylate cyclase via the PGE2 receptor. We report that PGE2, at concentrations below 1 × 10−5 M, does not affect the proliferation rate of these cells. This unresponsiveness is not due to the absence of receptors for PGE2. However, marked heterogeneity in receptor binding and function was detected in these closely related cell lines. Two metastatic lines (66 and 410.4) have high‐affinity receptors for PGE2 (average Kd = 4.3 × 10−9 M/L and 4.2 × 10−9 M/L, respectively) and similar binding capacities (4.1 × 104 and 2.9 × 104 binding sites, respectively). Two nonmetastatic lines, 410 and 67, have receptors with lower affinity (Kd = 8.3 × 10−9 M/L and 1.6 × 10−7 M/L, respectively) and binding capacities of 2.8 × 105/410 cell or 7.3 × 104/67 cell. A third nonmetastatic line (168) exhibits no specific binding. PGE2 receptor stimulation leads to elevated intracellular cAMP in lines 66, 410, and 67. Line 410.4 cells appear to have a functional lesion in the PGE2 receptor resulting in a failure to elevate cAMP in response to receptor occupancy. Adenylate cyclase can, however, be activated in these cells by cholera toxin, NaF, or forskolin. In comparison to the other cell lines, line 168 cells respond poorly to all cAMP‐stimulating agents. Thus, we have found that PGE2 binding is a heterogenous property for these cells, and, in addition, we have identified an apparent uncoupling of PGE2 receptor to the adenylate cyclase system in one cell line.
Cytochalasin D‐mediated hyperinduction of the substrate‐associated 52‐kilodalton protein p52 in rat kidney fibroblasts
Authors: Hiccins Paul J. , Ryan Michael P. , Chaudhari Panna
Published: February 2005

Regulation of certain differentiated and housekeeping functions in cultured mammalian cells is significantly influenced by cell shape. The shape‐modulating agent cytochalasin D (CD) was used, therefore, to elucidate potential cytoarchitectural influences affecting synthesis of a major 52 kDa secreted/substrateassociated protein (p52) of normal rat kidney (NRK) fibroblasts. Biosynthetic labeling experiments indicated that treatment of NRK cells with CD increased, by 10‐18‐fold, the medium content of an M, 52,000 protein. Two‐dimensional gel electrophoresis and peptide fragment mapping confirmed that the 52 kDa protein produced in abundance as a consequence of CD treatment was identical to p52 constitutively expressed by NRK cells. A lower mw protein (p50; M, 50,000) was also resolved which, based on pl microheterogeneity, protease fragmentation profile, and sensitivity to tunicamycin, could be identified as a less‐glycosylated form of p52. p50 and p52 were both detected in the matrix and medium compartments of NRK and NRK/CD cells. The matrix p52 content of CD‐induced and uninduced cells, however, was significantly greater (by 200‐500‐fold) than the corresponding medium levels. This differential compartmentalization, the time course of p52 accumulation in the matrix of NRK/CD cells compared to its appearance in the medium, and the kinetics of p52 pulse‐chase from the matrix collectively indicated that the matrix is the initial site of p52 deposition. Low levels of CD (1 μM) produced extensive disruptions of cellular microfilaments but did not result in an overall cell shape change nor a hyperinduction of p52. Morphologic rounding (seen in 10–100 μM CD) coincided with augmented p52 production. Transition from a flat to a round phenotype in NRK cells, or at least the generation of sufficient microfilament fragmentation to compromise cellsubstrate adhesivity, appears to be an essential aspect of CD‐mediated p52 hyperinduction.
Epidermal growth factor stimulation of prostacyclin production by cultured aortic smooth muscle cells: Requirement for increased cellular calcium levels
Authors: Blay Jonathan , Hollenberg Morley D.
Published: February 2005

We have examined the ability of epidermal growth factor (EGF) to regulate prostacyclin production by cultured A10 smooth muscle cells. EGF by itself had no effect on prostacyclin production, but it augmented the response to arg8‐vasopressin. An EGF stimulation of prostacyclin production was also observed in the presence of the calcium ionophore A23187; it therefore seemed likely that the key event required for EGF to stimulate prostacyclin production might be an increase in the available cellular Ca2+. Studies with 45Ca2+ showed that vasopressin both mobilised Ca2+ from intracellular stores and increased the influx of extracellular Ca2+ into A10 cells. The increase in prostacyclin production caused by vasopressin and the augmentation by EGF were both abolished by TMB‐8, an antagonist ofCa2+mobilisation, by EGTA, a chelator of Ca2+ ions, or by incubating cultures in the absence of added Ca2+. These results were consistent with a central role for Ca2+ in the responses and showed that both intracellular and extracellular sources of Ca2+ were important for the triggering of prostacyclin production. The increases in prostacyclin production were only marginally affected by nifedipine, and no responses were seen (either in the absence or presence of EGF) when KCL was used to depolarise the cell membrane. These data indicated that uptake of Ca2+ ions via voltage‐dependent channels was unlikely to be a major factor in the stimulation of prostanoid production. We conclude that the ability of EGF to stimulate prostacyclin production in A10 smooth muscle cells depends upon a concurrent stimulus that will increase available intracellular Ca2+ levels.
Effect of scatter factor on motility of epithelial cells and fibroblasts
Authors: Stoker Michael
Published: February 2005

The scatter factor is a protein released by fibroblasts that causes dispersal of epithelial cell colonies and disruption of intercellular junctions, as well as an alteration of morphology with ruffling and rapid extension and movement of pseudopodia. To find out if the scatter factor has a direct effect on cell migration, the Boyden chamber assay was used to determine the effect of partially purified factor on the migration of cells through 8 μm pore size filters. The results showed that the mobility of Madin‐Darby canine kidney (MDCK) cells was stimulated, and usually maximal at 100 ng per ml, which is equivalent to 100 to 200 units of activity in the standard assay based on the morphology and arrangement of cells. The migration was due to chemotaxis and chemokinesis. A keratinocyte cell line was also sensitive as were, to a lesser extent, BSCI monkey kidney cells. The motility of freshly isolated fibroblasts and fibroblast cell lines, however, was not significantly affected. The results confirm the cell specificity and paracrine role of the scatter factor and show that this fibroblast‐derived molecule can directly stimulate the movement of epithelial cells.
EGF and TGF‐alpha are potent chemoattractants for endothelial cells and EGF‐like peptides are present at sites of tissue regeneration
Authors: Grotendorst Gary R. , Soma Yoshinao , Takehara Kazuhiko , Charette Marc
Published: February 2005

We report here that epidermal growth factor (EGF) isolated from male mouse salivary glands is active as a potent chemoattractant for a rat heart vascular endothelial cell line (RHEC). Human EGF and transforming growth factor‐alpha (TGF‐alpha) produced in Escherichia coli are also active as chemoattractants for these cells. The chemotactic response of the cells is induced by concentrations of murine EGF that ranged from 0.01 to 1.0 nM with similar specific activities for both the human EGF and TGF‐alpha. Other growth factors such as fibroblast growth factor (FGF), TGF‐beta, platelet‐derived growth factor (PDGF), insulin, or IGF were not active as chemoattractants for these cells. The chemotactic response exhibited similar kinetics to those reported for connective tissue cells responding to PDGF (2–6 h) and required both RNA and protein synthesis. However, in contrast to NIH/3T3 cells responding to PDGF, the proliferating cultures of RHEC respond as well as growth‐arrested cultures in the chemotaxis assay. In in vivo studies of connective tissue regeneration it appears that EGF‐like factors are present in wound fluid collected from rats. Acid extracts of wound fluid contained a RHEC chemotactic activity that was neutralized with anti‐EGF antisera. These studies indicate that EGF‐like factors may function in vivo to control in part the angiogenic events that occur during tissue repair.
Bone marrow‐derived stromal cell line expressing osteoblastic phenotype in vitro and osteogenic capacity in vivo
Authors: Benayahu D. , Kletter Y. , Zipori D. , Wientroub S.
Published: February 2005

Marrow stroma has been shown to have osteogenic potential. Here we report the characterization of a unique stromal cell line derived from mouse bone marrow (MBA‐15), which expresses osteoblastic phenotype in vitro and forms bone in vivo. More than 70% of cells in culture were histochemically positive for alkaline phosphatase. The enzyme levels were enhanced threefold when cultures were treated with dexamethasone. Gel electrophoresis of [3H]‐proline‐labeled cultures showed that MBA‐15 cells produced only type I collagen. These cells were responsive to PTH, as indicated by a 50‐fold increase in intracellular cAMP. Prostaglandin E2, but not calcitonin, stimulated cAMP up to 70‐fold. When cultures were grown to confluence and fed daily with ascorbic acid and β‐glycerophosphate, the cells formed a Von Kossa positive, thick extracellular matrix, shown to contain hydroxyapatite crystals. MBA‐15 cells produced mineralized bone when implanted in diffusion chambers. These results indicate that the MBA‐15 cell line possesses osteoblastic features in vitro and osteogenic capacity in vivo.
Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form
Authors: Presta M. , Maier J. A. M. , Rusnati M. , Ragnotti G.
Published: February 2005

Basic fibroblast growth factor (bFGF) binds to heparin‐like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM‐bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell‐associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM‐bound bFGF by a neutral 2 M NaCI wash. Soluble heparin and heparan sulfate reduce the amount of ECM‐bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase‐like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM‐bound bFGF.
Regulation of the sodium‐potassium pump in cultured rat skeletal myotubes by intracellular sodium ions
Authors: Brodie Chaya , Sampson S. R.
Published: February 2005

The properties of the Na‐K pump and some of the factors controlling its amount and function were studied in rat myotubes in culture. The number of Na‐K pump sites was quantified by measuring the amount of [3H]ouabain bound to whole‐cell preparations. Activity of the pump was determined by measurement of oua‐bain‐sensitive 86Rb‐uptake and component of membrane potential. Chronic treatment of myotubes with tetrodotoxin (TTX), which lowers [Na]i, decreased the number of Na‐K pumps, the ouabain‐sensitive 86Rb uptake, and the size of the electrogenic pump component of Em. In contrast, chronic treatment with either ouabain or veratridine, which increases [Na+]i, resulted in an elevated level of Na‐K pump sites. This effect was blocked by inhibitors of protein synthesis. Neither rates of degradation nor affinity of pump sites in cells treated with TTX, veratridine, or ouabain differred from those in control cells. The number and activity of Na‐K pump sites were unaffected by chronic elevation in [Ca]i or chronic depolarization. We conclude that alterations in the level in intracellular Na ions play the major role in regulation of Na‐K pump synthesis in cultured mammalian skeletal muscle.
Enhanced messenger RNA stability and differentiation of HL 60 cells treated with 1, 25‐dihydroxyvitamin D3 and cordycepin
Authors: Mathew Poruneloor A. , Ellis Linda K. , Studzinski George P.
Published: February 2005

The effect of inhibitors of RNA synthesis on 1,25(OH)2 vitamin D3‐induced monocytic differentiation was studied in a well‐differentiating clone AB 47 of HL 60 cells. The concentrations of these inhibitors were chosen to permit the maintenance of cell viability for at least 48 hours, and resulted in 40‐60% inhibition of total cellular RNA synthesis. No impairment of 1,25(OH)2 vitamin D3‐induced monocytic differentiation was observed with all inhibitors tested, and the presence of cordycepin actually enhanced differentiation. The phenotypic evidence of monocytic differentiation correlated with the increased levels of mRNA for c‐fos and c‐fms measured by hybridization to appropriate nick‐translated cDNA probes. In contrast, nuclear run‐on experiments showed the expected inhibition of transcription of these genes by the compounds used. The data suggest that interference by external agents with transcription of genes essential for a differentiation program brings into play compensatory mechanisms which permit the program to continue. Thus, differentiation appears to have a high priority among various competing intracellular pathways in 1,25(OH)2 vitamin D3‐treated HL 60 cells. Stabilization of messenger RNA levels evident in this study may therefore represent a general cellular mechanism for the correction of unwanted effects of xenobiotics on the cell.