bluuebunny/crossref_metadata_2025 huggingface dataset:
Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes
Authors: Gladhaug Ivar P. , Christoffersen Thoralf
Published: February 2005

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature‐ and time‐dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Glad‐haug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride‐treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride‐treated hepatocytes the phorbol ester TPA strongly inhibited high‐affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand‐independent EGF receptor cycling pathway in hepatocytes.
Transcriptional regulation of DF3 gene expression in human MCF‐7 breast carcinoma cells
Authors: Abe Miyako , Kufe Donald
Published: February 2005

The DF3 gene codes for a high molecular weight human breast tumor‐associated glycoprotein. The detection of this antigen in human milk has also suggested that its expression represents a differentiated function of mammary epithelium. The present studies have examined the regulation of DF3 gene expression in human MCF‐7 breast carcinoma cells. These cells express two DF3 transcripts of 4.5 and 7.0 kb. Treatment of MCF‐7 cells with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) was associated with increases in levels of both DF3 mRNAs. When nuclear run‐on assays were used, DF3 gene transcription was at low to undetectable levels in untreated MCF‐7 cells and was increased after TPA exposure. TPA‐induced increases in DF3 mRNA levels were also inhibited by actinomycin D (ACT). MCF‐7 cells exposed to ACT further demonstrated that the half‐lives of the 4.5 and 7.0 kb transcripts are 26 and 11 h, respectively. The results also demonstrate that the protein synthesis inhibitor, cycloheximide (CHX), increases DF3 mRNA levels in MCF‐7 cells. These effects of CHX were sensitive to actinomycin D and not associated with stabilization of the DF3 transcripts. Taken together, these findings indicate that DF3 gene expression is controlled at a transcriptional level in TPA‐ and CHX‐treated MCF‐7 cells.
Effect of cyclic AMP on the cell cycle regulation of ribonucleotide reductase M2 subunit messenger RNA concentrations in wild‐type and mutant S49 T lymphoma cells
Authors: Albert Daniel A. , Nodzenski Edwardine , Yim Gloria , Kowalski Janice
Published: February 2005

Ribonucleotide reductase activity in S49 T lymphoma cells is cell cycle regulated by de novo protein synthesis of the M2 subunit. There is maximal enzyme activity in S and G2/M phase with low activity and low concentrations of the M2 subunit in G1 phase. Pharmacologic concentrations of cyclic AMP arrest S49 cells in the G1 phase of the cell cycle. We investigated the effect of cyclic AMP on M2 messenger RNA concentrations using RNA from exponentially growing and elu‐triated, cell cycle‐enriched populations. To discern whether cyclic AMP‐induced G1 arrest was associated with low concentrations of M2‐specific messenger RNA, we probed blots with a full‐length cDNA for M2. Cell cycle variation in M2 messenger RNA concentrations was similar in wild‐type, hydroxyurea‐resistant cells with amplified M2 activity, and cyclic AMP‐dependent protein kinase‐deficient cell lines. All lines had low amounts of M2‐specific mRNA in early G1, an increase at the late G1/early S phase interface, a decrease in mid S phase, and another increase in late S phase that continued through G2/M. These concentrations did not directly correlate with enzyme activity, suggesting other regulatory effects might participate in determining ribonucleotide rectase activity. Cyclic AMP exposure appeared to induce cell cycle arrest in early G1 with low M2‐specific messenger RNA concentration. This effect reversed upon washout of the cyclic AMP and was dependent on functional cyclic AMP‐dependent protein kinase (PKA). These results suggest that cyclic AMP arrests S49 mouse T lymphoma cells in early G1 prior to transcriptional activation of the M2 gene.
Clonal segregation of multiple and overlapping matrix adhesive responses in dorsal root neuronal derivative cells
Authors: Barletta Emanuela , Culp Lloyd A.
Published: February 2005

Clones of F11 hybrid (neuroblastoma X dorsal root neuron) cells have been tested for adherence and neurite outgrowth on three different substrata on which the parental cells display some competence–;plasma fibronectin (pFN) with its multiple receptors, cholera toxin subunit B(CTB) as a model ganglioside GM1‐binding substratum, and platelet factor‐4 (PF4) as a model proteoglycan‐binding substratum. This paradigm tests for independently segregating and overlapping mechanisms of neuritogenesis via transmembrane processes in pluripotent hybrid cells based on random loss of chromosomes contributed by the parent neural cells. For the nine clones tested, attachment was significantly lower on CTB but much higher on PF4 for all clones when compared to their attachment on pFN. Supplementation of cells with GM1 stimulated attachment of only two clones (on all three substrata). Neurite outgrowth was observed in a substratum‐specific pattern and varied from 0 to >60% on pFN; on CTB and PF4 substrata, the patterns were similar to each other but differed markedly from the pattern on pFN. In contrast, PF4‐ and CTB‐directed neurites differed morphologically from each other while sharing some characteristics with neurites on pFN. Supplementation with GM1 or GT1b, but not GD1a, was inhibitory for neurite outgrowth in certain clones. Cycloheximide pretreatment distinguished several classes of clones based on inhibition of neuritogenesis. While most clones on pFN were unaffected, all clones on CTB and PF4 displayed significant and comparable degrees of inhibitior, suggesting the sharing of some protein intermediate(s) on these substrata. Exposure to cycloheximide only during the active period of neuritogenesis generated higher percentages and longer neurites for all clones, indicating a widelyused negative regulation mechanism. Based on substratum type and cycloheximide protocols, these data have resolved six or more different transmembrane signalling processes for generating different classes of neurites. Some mechanisms have been segregated into individual clones while others overlap in other clones, providing cell systems for biochemical and molecular biological dissection of these processes.
K562 erythroleukemia cells express cytokeratins 8, 18, and 19 and epithelial membrane antigen that disappear after induced differentiation
Authors: Järvinen Maarit , Andersson Leif C. , Virtanen Ismo
Published: February 2005

The effects of differentiation‐modulating drugs were studied on the expression of intermediate filaments (IFs) in the human K562 erythroleukemic cell line. The untreated cells contained typical cytoplasmic coiling bundles, positive for both vimentin and cytokeratin as judged by indirect immunofluorescence microscopy with monoclonal antibodies (Mabs). Some of the cells also showed bright immunoreactivity for epithelial membrane antigen (EMA), as revealed with a Mab and polyclonal antiserum. When exposed to hemin or to sodium butyrate, most of the cells became cytokeratin negative within 3 days and showed dispersion of vimentin fibrils. Upon exposure to the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), the amount of both vimentin and cytokeratin appeared to be greatly increased within 3 days and was found both in dispersed cytoplasmic fibrils, in large spherical, eccentric aggregates, as well as in cytoplasmic fibrils in cells spreading on fibronectin. TPA induced a complete loss of proliferation, as judged by immunostaining with the Mab Ki‐67. The effects of TPA were found to be irreversible and could be induced by only a short exposure to the drug. Western blotting analysis and monoclonal antibodies to individual cytokeratins revealed that untreated K562 cells expressed Mr 52,000 (No. 8), 46,000 (No. 18), and 40,000 (No. 19) cytokeratin polypeptides, which disappeared when the cells were exposed to hemin or to sodium butyrate to induce erythroid differentiation but were greatly enhanced when exposed to TPA. The monoclonal anti EMA antibody reacted in K562 cells with a single Mr 320,000 polypeptide that was also revealed in MCF‐7 breast carcinoma cells. Human bone marrow cells or other leukemic cell lines with erythroid differentiation capacity (HEL and KG‐1) did not contain cytokeratin‐ or EMA‐immunoreactive cells, suggesting that in K562 cells these properties may rather represent abnormal cytodifferentiation or retrodifferentiation toward early embryonic mesenchymal cells, than a more general expression of epithelial features in human leukemic cells.
Use of chlortetracycline fluorescence for the detection of Ca storing intracellular vesicles in normal human erythrocytes
Authors: Engelmann Bernd , Schumacher Udo , Duhm Jochen
Published: February 2005

The uptake of chlortetracycline (CTC) and the nature of the fluorescence of CTC was studied in intact human erythrocytes from apparently healthy donors. The uptake of CTC at 22°C proceeded with a t½ of about 3 min, and after 15 min a stable equilibrium was achieved with an intracellular accumulation by a factor of 5–6 relative to the medium concentration. The accumulation did not change in the range of CTC concentrations tested (20–500 μM). The Ca specificity of the CTC fluorescence spectrum was confirmed by Ca depletion of red cells using A23187 in the presence of EGTA and 0.2 mM Mg. This procedure decreased the total intracellular calcium content by about 70% and reduced the fluorescence intensity to one‐fourth. Fluorescence microscopy of red cells incubated with 100 μM CTC at 22°C showed that the fluorescence originated mainly from the red cell membrane. In addition, in about 15% of erythrocytes one or more fluorescent dots (diameter >0.2 <1μm) were detected. The fluorescence of the dots and membranes was related to calcium, as evidenced by the reduction of their intensity in Ca depleted cells. The number of erythrocytes with fluorescent dots and the frequency of the dots per cell was largely unaffected by lowering the incubation temperature to 0°C, indicating that the dots most probably do not represent endocytotic artifacts induced by CTC. The number of dots was increased in erythrocytes preincubated with primaquine, demonstrating that CTC fluorescence can be applied to monitor the appearance of intracellular Ca storing vesicles. It is concluded that in (at least) 15% of erythrocytes obtained from apparently healthy donors intracellular vesicles containing Ca can be detected by CTC fluorescence microscopy.
Modulation of fibronectin synthesis and fibronectin binding during transformation and differentiation of mouse AKR fibroblasts
Authors: Varani James , Chakrabarty Subhas
Published: February 2005

In previous studies it was shown that transformation of AKR fibroblasts with 3‐methylcholanthrene was associated with a loss of surface fibronectin and that induction of differentiation of the transformed cells with N,N‐dimethylformamide (DMF) was associated with reacquisition of surface fibronectin (Chakrabarty et al., J. Cell. Physiol. 133:415, 1987). It is shown in the present study that changes in surface fibronectin reflect altered fibronectin synthesis and altered fibronectin binding. Both the nontransformed cells (AKR‐2B) and their transformed counterparts (AKR‐MCA) bound 125I‐fibronectin in a receptor‐like fashion, but the AKR‐MCA cells had only 20% of the receptors found on the AKR‐2B cells. Whole cell extracts prepared from the AKR‐2B cells and separated by sodium dodecyl sufate‐polyacrylamide gel electrophoresis under reducing conditions were examined for 125I‐fibronectin binding. Under these conditions, the majority of binding occurred to moieties with molecular weights of 180 kD, 150 kD, and 97 kD. Binding to similar moieties on the AKR‐MCA cells was virtually absent but occurred rapidly after treatment with DMF. The appearance of these moieties paralleled the acquisition of 125I‐fibronectin binding activity by whole cells. Antibodies to the fibronectin receptor isolated from human placenta reacted with the DMF‐sensitive moieties in immunoblot assays. Both the appearance of the fibronectin binding moieties and the acquisition of 125I‐fibronectin binding activity by whole cells occurred within 6 hr of DMF treatment and increased over the suosequent 4 day period. The time course of these events paralleled closely the time course for induction of fibronectin biosynthesis by DMF. These changes in fibronectin binding and fibronectin production were associated with alterations in cell‐substrate adhesion. The AKR‐2B cells rapidly attached and spread on bovine serum albumin‐coated dishes and on fibronectin‐coated dishes, whereas the AKR‐MCA cells were less adhesive on both substrates. Capacity to attach and spread was regained concomitantly with the induction of fibronectin binding and fibronectin production. Adhesion on both substrates was partially inhibited by antibodies to the fibronectin receptor and by RGDS. These studies suggest that fibronectin production and fibronectin binding are coregulated in AKR fibroblasts and that they function together to bring about changes in cell‐substrate adhesion.
Effect of epidermal growth factor on rat pleural mesothelial cell growth
Authors: Van Der Meeren A. , Levy F. , Renier A. , Jaurand M. C. , Katz A.
Published: February 2005

We recently reported that the growth of normal rat pleural mesothelial cells (RPMCs) is inhibited by conditioned media from either in vivo or in vitro transformed RPMCs. In this study we report that the growth of normal RPMCs is inhibited by epidermal growth factor (EGF). This was demonstrated by using three methods of investigation. Two types of studies were carried out with growing cells. First, cell counts indicated that the number of cells was reduced in EGF‐treated cultures when compared with untreated cultures. Second, the percentage of S cells detected by flow cytometry following treatment with EGF was lower than without EGF. In other experiments, incorporation of tritiated thymidine in confluent cells was decreased by EGF treatment, either in the presence or absence of fetal calf serum; these effects were dose dependent and were observed from 2 ng/ml EGF. Lower EGF concentrations did not significantly modify thymidine incorporation when compared with untreated cells. Analysis of 125I EGF binding experiments by the Scatchard method indicated that RPMCs possess EGF receptors (about 105 per cell) with low ligand binding affinity (Kd = 1.7 ± 0.4 nM). These results indicate that EGF might modulate the growth of RPMCs.
Protein disulphide isomerase from human peripheral blood neutrophils
Authors: Bassuk James A. , Capodici Constance , Berg Richard A.
Published: February 2005

Protein disulphide isomerase (PDI) is a 56 kDa resident polypeptide of the endoplasmic reticulum of many cell types. We evaluated the ability of human peripheral blood polymorphonuclear neutrophils (PMN) to synthesize both mRNA and proteins. Using in vitro [35S]‐methionine labeling of purified PMN, followed by immunoprecipitation of cell lysates with immobilized polyclonal and monoclonal antibodies and analysis by gel electrophoresis, PMN were shown to synthesize many proteins, including actin. In contrast, incorporation of [35S]‐methionine into PDI was not detected. Purification of total RNA from PMN and analysis by Northern blots demonstrated the presence in PMN of PDI‐RNA. Western immunoblot evaluations of total PMN protein display an immunoreactive‐PDI of 56 kDa. Indirect immunofluorescence studies suggest an abundance of immunoreactive‐PDI throughout PMN. We therefore conclude that PDI is synthesized in precursor cells of the bone marrow. Phorbol 12‐myristate 13‐aceiate, a reagent known to affect the degranulation of specific granules, causes the release of immunoreactive‐PDI into a post‐centrifugation supernatant. PDI, a ubiquitous endoplasmic reticulum resident protein, is shown here to be associated with specific granules in a cell type which has lost its intracellular membrane network during terminal differentiation.
Influence of guanine nucleotides on vincristine binding in tumor cytosols and purified tubulin: Evidence for an inhibitor of vincristine binding
Authors: Bowman Laura C. , Houghton Janet A. , Houghton Peter J.
Published: February 2005

In cytosols from human rhabdomyosarcoma xenografts, the formation of vincris‐tine (VCR)‐tubulin complex and its stability were increased by GTP (Bowman et al.: Biochem. Biophys. Res. Commun., 135:695–700, 1986). We have further examined this modulation to determine whether (a) GTP was protecting the VCR binding site from denaturation, (b) the enhancement of complex formation was guanosine specific, and (c) whether this influence was a direct interaction between GTP, VCR, and tubulin, or was mediated through another factor. In GTP‐depleted cytosols from tumor xenografts HxRH18 and HxRh12, VCR binding activity was stable for at least 2 hours at 37°C, indicating that the enhancement of complex formation and stability was not due to protection of tubulin integrity as measured by VCR binding; 10 nM GTP increased complex formation slightly, with complex formation increasing as GTP concentrations were increased to 5 μM, where maximum effect was observed. GTP and GDP (0.1 mM) both increased complex formation three‐fold, while GMP, GMP‐PNP, and ITP increased formation 1.5‐fold. IMP, CTP, and ATP had no significant effect. Therefore, the modulation of VCR binding was relatively specific for the guanine nucleotides GDP and GTP. Microtubule protein, purified from Rh18 and Rh12 tumors by cycles of polymerization‐depolymerization, bound VCR rapidly and binding was not influenced by GTP. This suggested that GTP modulation of VCR binding in cytosols was through a soluble factor lost in tubulin purification. In experiments with cytosol fractionated by molecular weight, there was inhibition of VCR binding activity by fractions with an mw range 20–50 kD. This inhibition was decreased by 25% by the addition of GTP. These data suggest that in tumor cytosols there may be competition between VCR and a natural ligand that is modulated by GTP. Two potential models for VCR binding are proposed.
Collagen‐Binding proteins of mammary epithelial cells are related to Ca2+‐and phospholipid‐binding annexins
Authors: Wirl Gerhard , Schwartz‐Albiez Reinhard
Published: February 2005

Three major proteins of 34, 36, and 38 kDa were isolated from mebrane preparations of chemically induced mammary tumors of the rat by collagen type I affinity chromatography and therefore were termed collagen‐binding proteins (CBP). Three proteins in the same molecular weight range isolated from cell extracts by precipitation with calcium, solubilization of the precipitate with EGTA, and chromatography on hydroxylapatite were demonstrated to be immunologically related to CBP. As shown by immunoblot analysis, an antiserum directed against the cluster of the 34–38 kDa proteins reacted strongly with porcine intestinal protein I, weakly with porcine lipocortin I, and very weakly with porcine intestinal protein II. Antiserum against the 34 kDa protein reacted weakly with protein I but strongly with protein II. All three CEP reacted with protein I/calpactin l‐specific antiserum of immunoblots and immunoprecipitation in experiments. However, antisera directed against CBP failed to show cross‐reaction with collagen‐binding protein anchorin II from chicken chondrocytes. Conversely, antisera against anchorin II did not react with CBP. Antiserum AS/87 immunoprecipitated CBP of 38 kDA that was labeled in a lactoperoxydase‐catalyzed iodination, suggesting that this polypeptide is associated with the cell surface. Further, all three CBP were found to be phosphorylated by incubating mammary cells with 32P‐orthophosphate. CBP bound to epithelial cell membranes in a Ca2+ dependent manner (= Triton × 100 insoluble form). Fractionated extraction and immunofluorescence microscopy also show that another form of CBP (= Triton × 100 soluble form) exists in these cells and is associated with a granular fraction. We therefore conclude that mammary collagen‐binding proteins represent members of a family of Ca2+‐binding membrane proteins. The 38 kDa CBP seems closely related to the pp60src kinase substrate protein I/calpactin I monomer, the 34 kDa CBP seems to be related or equivalent to protein II, while the relationship of the 36 kDa CBP to other defined proteins is still unclear.
Human Interleukin‐3 inhibits the binding of granulocyte‐macrophage colony‐stimulating factor and interleukin‐5 to basophils and strongly enhances their functional activity
Authors: Lopez Angel F. , Eglinton Jeffrey M. , Lyons A. Bruce , Tapley Peter M. , To Luen B. , Park Linda S. , Clark Steven C. , Vadas Mathew A.
Published: February 2005

The human T cell‐derived cytokines interleukin (IL)‐3, granulocyte‐macrophage colony‐stimulating factor (CM‐CSF), and IL‐5 were examined for their ability to bind specifically to human basophils and to regulate their function. Scatchard analysis of equilibrium binding studies showed that IL‐3 and GM‐CSF, bound to basophils with apparent dissociation constants (KD) = 8 × 10−11M and 3.9 × 10−11M, respectively. Specificity studies under conditions that prevent receptor internalization showed that the binding of IL‐3, GM‐CSF, and IL‐5 was not inhibited by tumor necrosis factor (TNF)‐α, IL‐1β, interferon (IFN)‐γ, or G‐CSF. However, receptors for IL‐3, GM‐CSF, and IL‐5 interacted with each other on the basophil membrane, showing a unique spectrum of cross‐reactivity, with IL‐3 competing for GM‐CSF and IL‐5 binding, whereas GM‐CSF and IL‐5 showed little or no competition for IL‐3 binding. In order to relate the binding properties of these cytokines to function, they were tested for their ability to influence basophil histamine release in an IgE/anti‐IgE‐dependent system. We found a hierarchy in the stimulation of basophil with the order of potency being IL‐3 >GM‐CSF >IL‐5. In addition, IL‐3 stimulated larger amounts of histamine release than GM‐CSF or IL‐5. The observation that IL‐3 interacts with receptors for GM‐CSF and IL‐5 may have a bearing on its stronger functional effects and suggests a major role for IL‐3 in the pathogenesis of hypersensitivity syndromes.
Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro
Authors: Hunt Richard C. , Davis Alberta A.
Published: February 2005

Actively proliferating human retinal pigment epithelial (RPE) cells grown in tissue culture possess keratin‐containing intermediate filaments that react with a combination of AE1 and AE3 anti‐keratin monoclonal antibodies. Antibody reactivity is lost, however, from RPE cells as the cell population ceases to proliferate when it approaches confluence and attains morphological characteristics more similar to those in vivo. In contrast, clone 8.13 anti‐keratin antibody stains all cells in the culture at all stages of the growth cycle and cell densities. These findings were reflected in vivo using retinal pigment epithelium taken directly from the eye. Normal non‐proliferating RPE cells bound 8.13 antibody to cytoskeletal structures, as judged by indirect immunofluorescence, but did not bind AE1/AE3 antibodies. However, proliferating dedifferentiated RPE cells from the vitreous humor of patients with proliferative vitreoretinopathy possess filaments that bind both AE1/AE3 and 8.13 antibodies. Thus it appears that structures detected by AE1/AE3 antibodies only occur in actively growing RPE cells in vitro and in vivo. Keratins produced by RPE cells were identified using Western blotting. Species with molecular masses of 54 (keratin 7), 52 (keratin 8), 42 (keratin 18), and 40 (keratin 19) kiloDaltons were the most abundant in proliferating cultured cells, but cells isolated directly from the eye were found to lack keratin 7 and 19. Keratin 19 was, however, observed in proliferating RPE cells from some patients with proliferative vitreoretinopathy. The latter findings explain the differential staining observed with AE1/AE3 antibodies in cells in culture and isolated directly from the eye since these antibodies interact primarily with keratin 19 which is absent from non‐proliferating RPE cells. In contrast to the presence of keratin‐containing intermediate filaments in human RPE cells in vivo, there are apparently no detectable vimentin‐containing cytoskeletal structures. However, all RPE cells cultured in vitro develop filaments composed of vimentin which persist in cells that have reached confluence.
Stimulation of plasma membrane and matrix vesicle enzyme activity by transforming growth factor‐β in osteosarcoma cell cultures
Authors: Bonewald L. F. , Schwartz Z. , Swain L. D. , Ramirez V. , Poser J. , Boyan B. D.
Published: February 2005

Transforming growth factor‐β (TGFβ) serves an important role in extracellular matrix formation by stimulating the production of numerous extracellular matrix proteins by connective tissue cells and by osteoblasts or bone‐forming cells. TGFβ has been shown to stimulate alkaline phosphatase (ALPase) activity in the rat osteoblast‐like osteosarcoma cell line ROS 17/2.8. Previous studies have shown that this enzyme is elevated during calcification of bone and that it is enriched in matrix vesicles, an extracellular organelle associated with initial hydroxyapatite formation. To test the hypothesis that TGFβ plays a role in regulating mineral deposition in the matrix, the effects of TGFβ on ALPase and phospholipase A2, two enzymes associated with mineralization, were examined. ROS 17/2.8 cells were cultured at high and low density with recombinant human TGFβ (0.1–10 ng/ml) to examine the influence of cell maturation on response to TGFβ. Maximal stimulation of ALPase activity in the low density cultures was seen at 5 ng/ml; in high‐density cultures, there was further stimulation at 10 ng/ml. There was a dose‐dependent increase in ALPase activity seen in the matrix vesicles and plasma membranes in both types of cultures. Matrix vesicle ALPase exhibited a greater response to factor than did the plasma membrane enzyme. However, in low density cultures, the two membrane fractions exhibited a parallel response with greatest activity consistently in the matrix vesicles. There was a dose‐dependent increase in phospholipase A2‐specific activity in the plasma membranes and matrix vesicles of both high‐ and low‐density cultures. In agreement with previous studies, TGFβ inhibited cellular proliferation 50%. The results show that addition of TGFβ stimulates the activity of enzymes associated with calcification. The effect of TGFβ is dependent on the stage of maturation of the cell. This study indicates that TGFβ may play an important role in induced bone formation, calcification, and fracture repair in addition to its role in promoting chondrogenesis.
Inhibition of gap‐junctional intercellular communication and enhanced binding of fibronectin‐coated latex beads by stimulation of DNA synthesis in quiescent 3T3‐L1 cells
Authors: Shiba Yoshiki , Sasaki Yasuto , Kanno Yoshinobu
Published: February 2005

To clarify the modulation of intercellular communication via gap junctions, associated with the growth induction of quiescent 3T3‐L1 cells, we investigated ‐the gap‐junctional intercellular communication in growth‐stimulated cells that were able to bind fibronectin‐coated beads. When quiescent 3T3‐L1 cells were incubated with fibronectin‐coated beads for the first 2 h after the addition of calf serum, 24.0% of the cells bound and phagocytosed beads. Among the cells with bound beads, the percentage of the cells labeled concurrently with bromodeox‐yuridine was 63.7% when examined 13 h after the addition of calf serum. Transient reduction of dye‐coupling, measured with Lucifer Yellow CH, was observed only in the cells with bound beads 2 h after addition of calf serum, but it was not observed in the cells without bound beads. When the quiescent cells were incubated with fibronectin‐coated beads for 2 h from 4–6 h after the addition of calf serum, the percentage of cells with bound beads increased to 53.1 %, but the decrease in dye‐coupling among the cells with bound beads was slight. These results suggest that the induction of cell growth causes a transient reduction in gap‐junctional intercellular communication in 3T3‐L1 cells with bound fibronec‐tin‐coated beads.
Glutamine regulates glutamine synthetase expression in skeletal muscle cells in culture
Authors: Feng Bo , Shiber Susan K. , Max Stephen R.
Published: February 2005

Metabolite control of glutamine synthetase expression (by glutamine) was studied in L6 skeletal muscle cells. Depletion of glutamine from the culture medium for 24 hours resulted in a 3–4‐fold increase in glutamine synthetase activity. This effect was blocked by cycloheximide but not by actinomycin D. Addition of glutamine to L6 cells maintained in glutamine‐free medium caused a rapid return of glutamine synthetase activity to the control level. As reported, dexamethasone caused a striking increase in the glutamine synthetase mRNA level. In contrast, neither depletion nor addition of glutamine caused a change in the glutamine synthetase mRNA level. Therefore, regulation of glutamine synthetase by glutamine is exerted at a post‐transcriptional level.
Characterization of flat revertants isolated from cells transformed by Abelson murine leukemia virus (Ab‐MuLV)
Authors: Oka Takashi , Yutsudo Masuo , Inoue Hirokazu , Higuchi Fujito , Hakura Akira
Published: February 2005

Transformed Fisher rat fibroblast cell lines by Abelson murine leukemia virus frequently revert to the normal phenotype in usual culture conditions. Molecular Biological analysis of thre revertant clones isolated from the transformants showed that their morphological reversions were due to inactivation of the v‐abl oncogene at multiple steps including transcription, translation or v‐abl protein kinase activity itself without any change in structural gene expression of helper virus. These findings suggest the existence of a specific mechanism(s) for elimination of the v‐abl oncogene by segregation, mutation, or gene rearrangement in these cells.
Prostaglandins do not mediate the actions of cholera toxin on pancreatic acini or gastric chief cells from the guinea pig
Authors: Raufman Jean‐Pierre , Singh Latika , Zakai M. David
Published: February 2005

Recent reports suggest that prostaglandins, rather than cAMP, play a major role in mediating cholera toxin‐induced water and electrolyte secretion from rabbit intestinal loops. We examined the role of prostaglandins in mediating toxin‐induced pancreatic and gastric exocrine secretion. In these tissues, indomethacin, a potent inhibitor of prostaglandin synthesis, did not alter the stimulatory effects of cholera toxin on increases in cellular cAMP or enzyme secretion. Moreover, the addition of cholera toxin did not alter prostaglandin E2 release from either tissue. In contrast to their effects in rabbit intestinal loops, prostaglandins do not regulate cholera toxin‐induced enzyme secretion from the guinea pig pancreas or stomach.
Chondrocytes inhibit endothelial sprout formation in vitro: Evidence for involvement of a transforming growth factor‐beta
Authors: Pepper M. S. , Montesano R. , Vassalli J.‐D. , Orci L.
Published: February 2005

Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti‐TGF‐β antibodies to the cocultures significantly reduces the inhibitory effect of chondrocytes on sprout formation. Chondrocyte‐conditioned medium or exogenously added TGF‐β1 have a similar albeit transient inhibitory effect. Depletion of TGF‐β from chondrocyte conditioned medium with anti‐TGF‐β antibodies and solid‐phase protein‐A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte‐derived TGF‐β‐ like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF‐β.
Epidermal growth factor‐induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts
Authors: Fisher Gary J. , Henderson Patricia A. , Voorhees John J. , Baldassare Joseph J.
Published: February 2005

The enzymatic pathways for formation of 1,2‐diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2‐Diradylglyceride mass was elevated 2‐fold within one minute of addition of EGF. Maximal accumulation (4‐fold) occurred at 5 minutes. Since both diacyl and ether‐linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether‐linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2‐diradylglyceride was diacyl, the remaining 20% being ether‐linked. Addition of EGF caused accumulation of 1,2‐diacylglyceride with out alteration in the level of ether‐linked diglyceride. Thus, the observed induction of 1,2‐diradylglyceride by EGF was due exclusively to increased formation of 1,2‐diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2‐fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF‐induced phospholipase D activity, cells were labelled with exogenous [3H]1‐0‐hexadecyl, 2‐acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3‐fold) at 30 seconds. Consistent with the presence of EGF‐induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D‐catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2‐diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D‐catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2‐diglyceride. Thus, the induction of 1,2‐diacylglycerol by EGF may occur primarily via phospholipase C‐catalyzed hydrolysis of phosphatidylcholine.
Neuraminidase‐induced thrombocytopenia in mice: Effects on thrombopoiesis
Authors: Stenberg Paula E. , Levin Jack , Baker Georgiann , Mok Yuen , Corash Laurence
Published: February 2005

Previous studies to examine the effects of thrombocytopenia on thrombopoiesis have generally utilized immune‐mediated platelet depletion. We have developed a nonimmune model to exclude the possibility that adverse immune‐mediated effects have been misinterpreted as the physiological response to stimulation of thrombopoiesis. Thrombopoiesis was examined in mice after induction of thrombocytopenia with a single injection of the nonimmunologic agent neuraminidase (Ndase). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 8, 12, 24, 48, 72, 96, and 120 hr after administration of Ndase. Eight to 48 hr after induction of acute, severe throm‐bocytopenia (mean platelet count < 50,000/μl), the medians of the platelet sectional area distributions, as measured morphometrically, were significantly greater than the median platelet sectional area of pooled controls. The maximum median value for platelet sectional area was observed at 24 hr. The largest platelets in these samples contained more profiles of endoplasmic reticulum and Golgi cisternae, and a lower concentration of surface‐connected canalicular system, as compared with normal platelets. By 72 hr post‐injection of Ndase, virtually all platelets exhibited normal size and organelle complement. Mean platelet volumes, determined by electrical impedance analysis, paralleled the serial changes in platelet sectional areas. MK frequency and ploidy, measured by two‐color fluorescence activated flow cytometry, were unchanged 12 and 24 hr following Ndase. At 48 hr, total MK frequency increased significantly (P >0.01) from 0.11% to 0.17%, and MK ploidy distribution shifted with a reduction in 16N MK (P >0.005) and an increase in 32N MK (P >0.01). MK ploidy was maximally altered from normal at 72 hr with increased 32N MK frequency (32.0%, P <0.001) and increased 64N MK frequency (2.4%, P >0.005). Morphologic and morphometric examination of MK at all time points did not reveal detectable changes from normal in cytoplasmic appearance or size, respectively. Therefore, we have demonstrated marked alterations of morphology and size of platelets, and of MK ploidy, using this nonimmunologic model. These studies further support our previous observations that megakaryocyte ploidy and platelet volume are independently regulated in response to depletion of the circulating platelet mass, and they show that these changes are not dependent upon the mechanism of thrombocytopenia.
Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses
Authors: Festoff B. W. , Rao J. S. , Hantaï D.
Published: February 2005

Recent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade‐type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin‐binding and cross‐reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43–50 kDa glycoprotein of the serpin superfamily (arg‐serpin class). Purified anti‐protease nexin I antibody (anti‐47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co‐localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia‐derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration Protease nexin I inhibits both tumor cell and myoblast plasminogen activator‐mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post‐translational regulation by specific serpins, in the remodeling that occurs in synapse formation and elimination.
Expression of bone matrix proteins associated with mineralized tissue formation by adult rat bone marrow cells in vitro: Inductive effects of dexamethasone on the osteoblastic phenotype
Authors: Kasugai Shohei , Todescan Reynaldo , Nagata Toshihiko , Yao Kam‐Ling , Butler William T. , Sodek Jaro
Published: February 2005

The nature and tissue distribution of non‐collagenous bone proteins synthesized by adult rat bone marrow cells, induced to differentiate in the presence of dexamethasone (DEX) and β‐glycerophosphate (β‐GP), was studied in vitro to determine the potential role of these proteins in bone formation. Northern hybridization analysis revealed a strong induction of bone sialoprotein (BSP) and osteocalcin in DEX‐treated cultures, whereas the constitutive expression of secreted phosphoprotein I (SPP‐1), type I collagen, SPARC, and alkaline phos‐phatase was stirnulated 6‐, 5‐, 3‐, and 2.5‐ fold, respectively. Metabolic labeling of proteins showed that the sialoproteins (SPP‐1 and BSP) were mostly secreted into the culture medium in the non‐mineralizing (‐β‐GP) cultures, but were the predominant non‐collagenous proteins associated with the hydroxyapatite of the bone nodules in mineralizing cultures (+β‐GP). Extraction of the tissue matrix with 4 M GuHCI and digestion of the demineralized tissue matrix with bacterial collagenase revealed that some BSP was also associated non‐covalently and covalently with the collagenous matrix. SPP‐1 was present in two distinct, 44 kDa and 55 kDa, forms in the conditioned medium of all cultures and was preferentially associated with the hydroxyapatite in the mineralizing cultures. In comparison, SPARC was abundant in culture media but could not be detected in de‐mineralizing extracts of the mineralized tissue. Radiolabeling with [35SO4] demonstrated that both SPP‐1 and BSP synthesized by bone cells are sulfated, and that a 35 kDa protein and some proteoglycan were covalently associated with the collagenous matrix in +DEX cultures. Labeling with [32PO4] was essentially confined to the sialoproteins; the 44 kDa SPP‐1 incorporating significantly more [32PO4] than the 55 kDa SPP‐1 and the BSP. These studies demonstrate that BSP and osteocalcin are only expressed in differentiated osteoblasts and that most of the major non‐collagenous bone proteins associate with the bone mineral. However, some novel proteins together with some of the BSP are associated with the collagenous matrix where they can influence hydroxyapatite formation.
The half‐lives of platelet‐derived growth factor A‐ and B‐chain mRNAS are similar in endothelial cells and unaffected by heparin‐binding growth factor‐1 or cycloheximide
Authors: Gay Cyril G. , Winkles Jeffrey A.
Published: February 2005

Platelet‐derived growth factor (PDGF) is mitogenic and chemotactic for vascular smooth muscle cells cultured in vitro, and, thus, may play a role in the smooth muscle cell proliferation and migration that occurs during atherosclerotic lesion development. Two related PDCF polypeptides, designated as the A and B chains, form functionally active PDGF‐AA, AB, or BB dinners. The PDGF A‐ and B‐chain genes are both transcribed in human umbilical vein endothelial (HUVE) cells and their expression is regulated by cytokines, growth factors, endotoxin, and phorbol ester. We reported previously that the angiogenic polypeptide heparin‐binding growth factor (HBGF)‐I induces PDGF A‐chain gene expression, but does not affect PDGF B‐chain gene expression. In this study, we determined whether mRNA stabilization contributed to this induction by measuring the half‐life of PDGF A‐chain mRNA in quiescent, HBGF‐1‐stimulated, and proliferating HUVE cells. PDGF A‐chain mRNA levels increase when quiescent HUVE cells are treated with the protein synthesis inhibitor cycloheximide; therefore, the effect of cycloheximide on PDGF A‐chain mRNA decay was also investigated. The half‐life of PDGF A‐chain transcripts in quiescent cells was ∼2.4 h and neither HBGF‐1 nor cycloheximide significantly altered this decay rate. We also estimated the half‐life of PDGF B‐chain mRNA under the three different growth conditions and in the absence or presence of cycloheximide. The half‐life in quiescent cells was ∼1.8 h and was unaffected by HBGF‐1 or protein synthesis inhibition. Therefore, the PDGF mRNAs have similar decay rates in HUVE cells, even though the 3′ untranslated region of B‐chain transcripts, but not A‐chain transcripts, contains AU‐rich sequence motifs postulated to confer rapid turnover in vivo.
B16 mouse melanoma cells selected for resistance to cyclic AMP‐mediated growth inhibition are cross‐resistant to retinoic acid‐induced growth inhibition
Authors: Niles Richard M. , Loewy Barbara
Published: February 2005

B16 mouse melanoma cells are grown inhibited by cyclic AMP or by retinoic acid (RA). However, the combination of these two agents results in less growth inhibition than either agent alone. In order to investigate this interaction, cells were selected for resistance to 8‐bromo‐cyclic AMP‐induced growth inhibition. Two clones (3 and 7) which demonstrated significant resistance were isolated. When these two clones were treated with retinoic acid (RA) it was observed that they also exhibited different degrees of resistance to this growth inhibitor. This cross‐resistance did not appear to be due to a lack of uptake or retention of the respective inhibitors, since the mutants took up and retained more 3H‐cAMP and 3H‐RA than wild type cells, suggesting that the dual resistance was not due to an amplification of P‐glycoprotein. The mutation confering cAMP‐resistance did not appear to involve cyclic AMP‐dependent protein kinase, since both catalytic activity and the amount of cAMP protein binding was similar in wild type and mutants. Thus, the mutation must be beyond the interaction of cAMP with cAMP‐dependent protein kinase. We have previously reported that RA induces protein kinase C in B16 melanoma cells (Miles and Loewy: Cancer Res. 49:4483–4487, 1989). Therefore, we measured the ability of RA to induce protein kinase C in the cyclic AMP‐resistant mutants. We found an inverse correlation between RA‐induced protein kinase C activity and growth inhibition in these mutants. The data reported here suggest that cyclic AMP regulates some step in the RA signal transduction pathway.