bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of hexose transport in rat myoblasts during growth and differentiation
Authors: Chen S. R. , Lo T. C. Y. 
Published: February 2005

AbstractWe report here the effects of growth conditions and myogenic differentiation on rat myoblast hexose transport activities. We have previously shown that in undifferentiated myoblasts the preferred substrates for the high (HAHT)‐ and low (LAHT)‐affinity hexose transport systems are 2‐deoxyglucose (2‐DG) and 3‐O‐methyl‐D‐glucose (3‐OMG), respectively. The present study shows that at cell density higher than 4.4 × 104 cells/cm2, the activities of both transport processes decrease with increasing cell densities of the undifferentiated myoblasts. Since the transport affinities are not altered, the observed decrease is compatible with the notion that the number of functional hexose transporters may be decreased in the plasma membrane. Myogenic differentiation is found to alter the 2‐DG, but not the 3‐OMG, transport affinity. The Km values of 2‐DG uptake are elevated upon the onset of fusion and are directly proportional to the extent of fusion. This relationship between myogenesis and hexose transport is further explored by using cultures impaired in myogenesis. Treatment of cells with 5‐bromo‐2′‐deoxyuridine abolishes not only myogenesis but also the myogenesis‐induced change in 2‐DG transport affinity. Similarly, alteration in 2‐DG transport affinity cannot be observed in a myogenesis‐defective mutant, D1. However, under myogenesis‐permissive condition, the myogenesis of this mutant is also accompanied by changes in its 2‐DG transport affinity. The myotube 2‐DG transport system also differs from its myoblast counterpart in its response to sulfhydryl reagents and in its turnover rate. It may be surmised from the above observations that myogenesis results in the alteration of the turnover rate or in the modification of the 2‐DG transport system. Although glucose starvation has no effect on myogenesis, it is found to alter the substrate specificity and transport capacity of HAHT. In conclusion, the present study shows that hexose transport in rat myoblasts is very sensitive to the growth conditions and the stages of differentiation of the cultures. This may explain why different hexose transport properties have been observed with myoblasts grown under different conditions.
Regrowth kinetics of cells from different regions of multicellular spheroids of four cell lines
Authors: Freyer James P. , Schor Patricia L. 
Published: February 2005

AbstractA basic understanding of the recruitment of quiescent tumor cells into the cell would be an important contribution to tumor biology and therapy. As a first step in pursuing this goal, we have investigated the regrowth kinetics of cells from different regions in multicellular spheroids of rodent and human origin. Cells were isolated from four different depths within the spheroids using a selective dissociation techinque. The outer cells were proliferating and resumed growth after replating with a 0–8‐hour lag period, similar to cells from exponentially growing monolayers. with increasing depth of origin, the lag periods prior to regrowth increased to 2–3 times the monolayer doubling time; cells from plateau‐phase monolayers showed a lag period of 1–1.5 times the doubling period. After resuming grwoth, all cells of a given cell line grew with the same doubling time and achieved the same confluency level. The inner spheroid cells and cells from plateau‐phase monolayers had reduced clonogenic efficiencies. The inner cells were initially 1.5–3 times smaller than the outer cells, but began to increase in volume within 4 hours of replating. The fractions of S‐phase cells were greatly decreased with increasing depth of origin in the spheroids; there were long delays prior to s‐phase recovery after plating, to a maximum of 1–1.5 times the normal doubling time. These results suggest that those quiescent cells from spheroids and monolayers which are able ot reenter the cell cycle are predominantly in the G1‐phase. However, quiescent cells from the innemost spheroid region require approximately twice as long ot enter normal cell cycle traverse as cells from plateau‐phase monolayers. The selective dissociation method can isolate very pure populations of proliferating and quiescent cells in a rapid and nonpeturbing manner; this system will be valuable in further characterizing quiescent cells from spheroids.
A mammary‐derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells
Authors: Müller T. , Kurtz A. , Vogel F. , Breter H. , Schneider F. , Angström U. , Mieth M. , Böhmer F.‐D. , GrosseVogel R. 
Published: February 2005

AbstractThe aim of the present study was to investigate the expression of the mammary derived growth inhibitor (MDGI) and the subcellular localization of MDGI related antigens in bovine mammary glands. Cell‐free translation of poly (A+) = RNA, immunoprecipitation with rabbit anti‐MDGI‐antibodies, and estimation of the relative contents of MDGI by a radioimmunoassay in mammary tissue of different functional statess revealed that the 13 kDa MDGI was dramatically increased in terminally differentiated mammary tissue compared with the proliferating tissue from pregnant animals. To address the question of tissue localizationl, polyclonal anti‐MDGI antibodies and antibodies directed aganist a sythetic peptide corresponding ot residues 69 to 78 of MDGI were used. Western blotting of tissue fractions revealed the cytosolic and microsomal localization of MDGI. Additionally, both types of antibodies a 70‐kDa antigeninthe unclear fractionof differentiated mammary glands. Salt extraction and DNase I digestion of isolated unclei, as well as chromatin purification, indicated an association of the 70‐kDa antigen with the chromatin. By means of the immunogold technique, MDGI‐related antigens were localized within euchromatic unclear regions of epithelial cells in the intact differentiated mammary gland. The immunostaining was markedly diminished in the proliferating tissue. This finding raises the possibility that MDGI and the 70‐kDa antigen influence cell proliferation by acting on geneexpression within the unclei of mammary glands.
Heparin interactions with cultured human vascular endothelial and smooth muscle cells: Incidence on vascular smooth muscle cell proliferation
Authors: Herbert Jean‐Marc , Maffrand Jean‐Pierre 
Published: February 2005

AbstractThe binding, internalization, and metabolism of [3H]‐heparin by human umbilical vein endothelial cells (HUVEC) and human umbilical arterial smooth muscle cells (HUASMC) have been characterized using size‐exclusion HPLC. Incubation of HUVEC awith [3H]‐heparin demonstrated selective binding of high‐molecular‐weight (MW) components (MW = 21 kd), which was followed by rapid, temperature‐dependent internalization. Over the next 3 hours, this internalized [3H]‐heparin was degraded to low‐MW fragments (MW = 0.9 kd). Primary cultures of HUASMC selectively bound extremely high‐MW components (MW = 40 kd) and also smaller components whose MW (0.9 kd) corresponded to that of the heparin metabolite(s) formed by HUVEC. Subcultured HUASMC bound only the 40‐kd components. Internalization of heparin by smooth muscle cells (SMC) was significantly slower than that determined for HUVEC, and even after 4 hours there was no evidence of the heparin being metabolized. However, when incubating primary rabbit aortic SMC with purified low‐MW heparin fragment(s) produced by culture by HUVEC, a significantly lower proliferative response of these cells (IC50 = 18.4 μg/ml) was obtained. Virtually no effect was observed with subcultured SMC in the range of the tested concentrations (0–20 μg/ml). These fragments were 10‐ to 15‐fold more effective in inhibiting primary SMC growth than was standard heparin. Furthermore, heparin fractions in the same range of molecular weights, purified either after nitrous acid or heparinase depolymerization of standard heparin, showed no activity on primary SMC growth, thus indicating a high degree of selectivity of the heparin metabolite(s) produced by HUVEC in culture.
Metabolism of small RNAs in cultured human cells
Authors: Choudhury Kanakendu , Choudhury Indrani , Eliceiri George L. 
Published: February 2005

AbstractThere are gaps in what is known about the metabolism of some mammalian small RNA species. Our present observations can be summarized as follows. The level of radiolabeled mature U1 RNA doubled between 2 and 24 hr of label chase, while that of all other small RNA species tested did not change. These results are compatible with the possibility that the nucleotide precursor pool for U1 RNA transcription may be partly segregated, or that there may be a second pathway of U1 RNA formation. Precursors of nucleolar U3 RNA were detected whose electrophoretic mobilities are equivalent to those of transcripts ∼ 14 and ∼ 8 nucleotides longer than the mature species, and which are apparently cytoplasmic. The ladder of U2 RNA precursors showed a gap, suggesting that some of the cleavages during U2 RNA processing are endonucleolytic. We detected an apparent U5 RNA precursor whose electrophoretic mobility is equivalent to that of a species ∼ 1 nucleotide longer than mature U5 RNA. There was a predominant band in the middle of the ladder of U4 RNA precursors (apparently ∼ 3 nucleotides longer than mature U4 RNA) which suggests that U4 RNA maturation may pause briefly at that intermediate. There are apparently two additional species of mature hY3 RNA, which are less abundant and are about 1 and 2 bases longer than the major hY3 RNA species. An apparent hY3 RNA precursor was detected, which may be ∼ 2–3 nucleotides longer than the main mature hY3 RNA species.
Effects of vanadate on tyrosine phosphorylation and the pattern of glycosaminoglycan synthesis in rabbit chondrocytes in culture
Authors: Owada M. Koji , Iwamoto Masahiro , Koike Tatsuya , Kato Yukio 
Published: February 2005

AbstractThe present study examined the effects of high doses of vanadate on glycosami‐noglycan (GAG) synthesis and tyrosine phosphorylation in rabbit chondrocytes in confluent cultures. Although 6 μM vanadate increased the incorporation of [3H] glucosamine into chondroitin sulfate proteoglycans twofold, 40–60 μM vanadate suppressed this incorporation fourfold. Although 6 μM vanadate had little effect on [3H] glucosamine incorporation into hyaluronate, 40–60 μM vanadate increased this incorporation threefold. Chemical analyses confirmed that the increase in ∥3H∥glucosamine incorporation into hyaluronate and the decrease in the incorporation into chondroitin sulfate proteoglycan correlated with increased hyaluronate content and decreased chondroitin sulfate content in the cell layers of vanadate‐transformed cells. Chondrocytes exposed to 40–60 μM vanadaje became typically transformed spindlelike cells. Furthermore, vanadate, at 6 and 60 μM, increased the overall level of phosphotyrosine by 8‐ and 31‐fold, respectively, and 60 μM vanadate enhanced phosphorylation of many phosphotyrosine‐containing proteins. These observations suggest that vanadate induces transformation‐associated changes in the pattern of GAG synthesis when it induces excess phosphorylation on tyrosine in chondrocyte proteins.
Dynamic interactions of c‐fos protein in serum‐stimulated 3T3 cells
Authors: Vosatka Robert J. , Hermanowski‐Vosatk Anne , Metz Richard , Ziff Edward B. 
Published: February 2005

AbstractThe c‐fos gene, the cellular homologue of the transforming gene of the FBJ osteosarcoma virus, v‐fos, is strongly induced in quiescent BALB/c 3T3 cells by growth factors and in other cell types by a wide variety of transmembrane signalling agents. c‐fos is a member of a family of structurally related proteins which includes the fos‐related antigens (fra). We have studied the dynamic state of the c‐fos protein with an antibody prepared by immunizing rabbits with a plasmid‐encoded fos fusion protein. In serum‐stimulated BALB/c 3T3 cells, the antibody recognizes a nuclear antigen which resolves on SDS‐PAGE as a 60–68‐kD group of bands corresponding to c‐fos, a doublet at 44–45‐kD corresponding to the noncovalently associated p39 protein, as well as an approximately 50‐kD band corresponding to a fra. We show that although c‐fos protein synthesis is only transiently induced by serum, the c‐fos protein persists within the cell after its synthesis has ceased, and it decays with a half‐life of 2 hours. Significantly, newly synthesized p39 continues to appear in the immune‐precipitated complex even at times when c‐fos is no longer synthesized. These kinetics indicate that even following shutoff of c‐fos protein synthesis, p39 is newly synthesized and can complex with c‐fos protein or a fos‐related antigen. During this time, c‐fos also undergoes an extensive posttranslational modification. The modification is partially reversed by phosphatase treatment, which implicates protein phosphorylation. Together these results suggest that both interaction with p39 and phosphorylation may progressively modify the properties of c‐fos and/or the fos‐related antigens over a period of 4–8 hours following the shutoff of fos synthesis. We discuss the implications of the dynamic state of c‐fos and fra protein interactions for the function of these proteins.
Potassium depletion and hypertonic medium reduce “non‐coated” and clathrin‐coated pit formation, as well as endocytosis through these two gates
Authors: Carpentier Jean‐Louis , Sawano Fumio , Geiger Dominique , Gorden Phillip , Perrelet Alain , Orci Lelio 
Published: February 2005

AbstractIntracellular potassium depletion inhibits receptor‐mediated endocytotic processes occurring through clathrin‐coated pits. Besides the clathrin‐coated pit route, flask‐shaped invaginations that do not bear a typical clathrin coat have been recently implicated in receptor‐mediated endocytosis of cholera toxin. These invaginations are called “non‐coated” to distinguish them from the typical clathrin‐coated pits. In the present study, we have investigated whether “non‐coated” invaginations are sensitive, as are clathrin‐coated pits, to potassium depletion and whether hypertonic medium, which inhibits receptor‐mediated endocytosis, also affects “non‐coated” invaginations. We found that 1) both potassium depletion and hypertonic medium reduce “non‐coated” invaginations on the cell surface; 2) similar to potassium depletion, hypertonic medium markedly decreases the number of clathrin‐coated pits; 3) these changes are accompanied by an inhibition of the internalization (measured morphologically) of cholera toxin‐gold through “non‐coated” invaginations, as well as of α2‐macroglobulin‐gold taken up by clathrin‐coated pits; and 4) in addition, both the hypertonic medium and potassium depletion inhibit the uptake of horseradish peroxidase, a marker of fluid‐phase endocytosis.
Ionic dependence of glycylsarcosine uptake by isolated chicken enterocytes
Authors: Calonge M. L. , Ilundain A. , Bolufer J. 
Published: February 2005

AbstractDipeptide transport was studied in chicken enterocytes and its properties compared with those of Na+‐dependent sugar transport. Results showed that 1) isolated cells were capable of accumulating glycylsarcosine (Gly‐Sar) against a concentration gradient (2.5‐ to 3.0‐fold accumulation). This uptake was maximal at pH 6.0, and it was inhibited by Na+‐free medium and by ouabaine; 2) uptake of Gly‐Sar was not affected by methionine and was competitively inhibited by carnosine, with a Ki of 12 mM; 3) the protonophore FCCP inhibited both Gly‐Sar and 3‐oxy‐methyl‐D‐glucose (3‐OMG) uptake by the cells; 4) amiloride, a well‐known inhibitor of the Na+/H+ exchanger system stimulated 3‐OMG uptake and inhibited Gly‐Sar uptake, its effects being greater at pH 7.4; 5) and monensine prevents the effects of amiloride on both sugar and dipeptide uptake. In summary, Gly‐Sar uptake depends on extracellular Na+ in an indirect manner via its effect on H+ efflux, and it appears to be dependent on an inward H+ gradient.
Effects of 1,25‐dihydroxyvitamin D3 on cell‐cycle kinetics of T 47D human breast cancer cells
Authors: Eisman John A. , Sutherland Robert L. , McMenemy M. Lynne , Fragonas Jean‐Charles , Musgrove Elizabeth A. , Pang Grace Y. N. 
Published: February 2005

AbstractThe replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25‐(OH)2D3, which inhibit cellular replication, on the cell‐cycle kinetics of a 1,25‐(OH)2D3‐responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25‐(OH)2D3 in the range 10−9 M to 10−6 M caused a time‐ and concentration‐dependent decrease in cell numbers. Treatment of cells growing in charcoal‐treated fetal calf serum with 10−8 M 1,25‐(OH)2D3 for 6 days reduced cell numbers to 49% ± 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% ± 0.5% (n = 11) to 19.6% ± 2.3% (n = 9), significant by paired analysis (P > 0.002). At higher concentrations of 1,25‐(OH)2D3 (10−7 −10−6 M), there was a concentration‐dependent decline in S phase and increases in both Go/G1 and G2 + M phase cells.Detailed analysis of the temporal changes in cell‐cycle phase distribution following treatment with 2.5 × 10−8 and 10−7 M 1,25‐(OH)2D3 showed an initial accumulation of cells in Go/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in Go/G1 phase and an increase in G2 + M. In contrast cells treated with 10−7 M 1,25‐(OH)2D3 had significantly elevated % Go/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from Go/G1 following 4 days of exposure to 10−7 M 1,25‐(OH)2D3. This accumulation of cells in Go/G1 was accompanied by a fall in % S phase cells. After 3 days of treatment the % Go/G1 phase cells began to decline, due presumably to a reduced rate of entry into this phase caused by the transition delay through G2 + M which was present at early times but increased rapidly between days 2 and 4 to reach a maximal effect between 4 and 6 days. These studies confirm the 1,25‐(OH)2D3 effect on progression through G1 phase previously reported in hemopoietic cells. Furthermore they document an additional transition delay in G2 + M which is a major contributor to the known growth inhibitory effects of this hormone in T 47D human breast cancer cells. Further study of these effects may lead to a better understanding of hormonal regulation of cellular replication in human breast cancer.
Stimulation of a Mr 80,000 protein phosphorylation by EGF in EGF receptor‐hyperproducing human tumor cells
Authors: Hirai Masamichi , Shimizu Nobuyoshi 
Published: February 2005

AbstractNA and Ca9‐22 cells derived from squamous cell carcinomas of the tongue possess a large number of epidermal growth factor (EGF) receptors (2.0 × 106 and 1.3 × 106 receptors/cell, respectively). In these cell lines, EGF stimulated receptor autophosphorylation and phosphatidylinositol (PI) turnover. Furthermore, EGF enhanced the phosphorylation of an acidic protein of Mr 80,000. Phosphorylation of this protein was also stimulated by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a phorbol ester tumor promoter, and was mainly at serine residues. Phosphopeptide mapping using protease V8 or trypsin indicated that Mr 80,000 proteins isolated from the EGF‐ and TPA‐treated cells were identical. The Mr 80,000 protein was present mainly in the cytosol, but it became closely associated with the membrane as a phosphorylated form upon EGF or TPA stimulation. These results suggest that the EGF‐stimulated phosphorylation of the Mr 80,000 acidic phosphoprotein in EGF receptor‐hyperproducing tumor cells is mediated through the activation of PI turnover and protein kinase C.
Cortisol induction of metallothionein in primary culture of rainbow trout hepatocytes
Authors: Hyllner Sven Johan , Andersson Tommy , Haux Carl , Olsson Per‐Eric 
Published: February 2005

AbstractIn the present experiment, metabolically active primary cultures of rainbow trout hepatocytes were utilized to study the inducibility of metallothionein after cortisol treatment. Glucocorticoid induction of metallothionein has previously only been demonstrated in mammalian systems. We now demonstrate the inducibility of rainbow trout metallothionein by cortisol treatment of primary hepatocytes. A 90% elevation above control levels was achieved within 8 days of treatment. Zinc treatment was performed to evaluate the system, and 100 μM zinc in the culture medium resulted in a 350% increase of the metallothionein levels. We conclude that primary culture of rainbow trout hepatocytes constitutes an efficient system for studies of MT induction kinetics.
Interleukin 1 stimulates prostaglandin synthesis and cyclic AMP accumulation in Swiss 3T3 fibroblasts: Interactions between two second messenger systems
Authors: Burch Ronald M. , White Marsha F. , Connor Jane R. 
Published: February 2005

AbstractBiochemical events elicited by interleukin 1 (IL–1) were studied in Swiss 3T3 fibroblasts. One hour after its addition, IL–1 stimulated synthesis of prostaglandin E2 (PGE2), which continued to accumulate for 4 days. IL–1 also stimulated cAMP accumulation. Indomethacin blocked cAMP accumulation in response to IL–1, suggesting that PG2 was responsible for the increase. Addition of exogneous PGE2 to indomethacin‐treated cells restored cAMP accumulation. IL–1 enhanced thymidine incorporation, and indomethacin attenuated responses to lower concentrations. Thus, PGE2 appeared to play a role in the ability of low concentrations of IL–1 to stimulate thymidine incorporation. PGE2 augmented thymidine incorporation by increasing cAMP accumulation because in the presence of indomethacin addition of exogenous cAMP enhanced thymidine incorporation in response to low concentrations of IL–1. Elevated cAMP further stimulated PGE2 synthesis. Thus, PGE2 and cAMP interacted to potentiate their mutual accumulation. In summary, IL–1 stimulates PGE2 synthesis. PGE2, in turn, stimulates cAMP accumulation which potentiates IL–1–stimulated PGE2 synthesis and thymidine incorporation.
HeLa cell mutants resistant to epidermal growth factor ricin A‐chain conjugate
Authors: Banker Deborah , Herschman Harvey R. 
Published: February 2005

AbstractEpidermal growth factor (EGF) was linked to the toxic A chain of ricin toxin (RTA) to produce an EGF‐receptor‐specific cytotoxic agent, EGF‐RTA. Three EGF‐RTA‐resistant mutants of the human HeLa cell line were selected. These mutant cell lines are 10‐fold to more than 100‐fold more resistant to EGF‐RTA when compared to HeLa cells. The EGF‐RTA‐resistant mutants have at least as many EGF receptors as parent cells; the basis for the EGF‐RTA‐resistant phenotype must be distal to EGF binding. The EGF‐RTA‐resistant cells are not cross‐resistant to ricin or to diphtheria toxin; their mutant phenotype appears to be EGF specific. The EGF‐RTA‐resistant mutants are able to internalize and degrade EGF. However, the mutants have altered EGF receptor down‐regulation and phorbol 12‐tetradecanoate 13‐acetate modulation properties. EGF‐RTA/ammonium chloride and EGF‐RTA/adenovirus co‐treatment data suggest that the mutant defect(s) which confers EGF‐RTA resistance is either in the endosome or at a step(s) in the intracellular EGF processing pathway between the endosome and the lysosome.