bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents
Authors: D'amore Tony , Lo Theodore C. Y. 
Published: February 2005

AbstractThe importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N‐ethylmaleimide (NEM) and p‐chloromer‐curibenzenesulfonic acid (pCMBS) inhibited 2‐deoxy‐D‐glucose (2‐DOG) transport in a time and concentration‐dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70–80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2‐DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D‐glucose or 20 mM 2‐DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2‐DOG transport, pCMBS and NEM had much less of an effect on 3‐O‐methyl‐D‐glucose (3‐OMG) transport. For example, 1 mM NEM inhibited 2‐DOG transport by 66%, whereas 3‐OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2‐DOG transport system, whereas the low affinity transport system is unaffected. 3‐OMG is preferentially transported by the low affinity system.
Endocytosis of mannose‐6‐phosphate binding sites by mouse T‐lymphoma cells
Authors: Bourguignon Lilly Y. W. , Balazovich K. , Suchard Suzanne J. , Hindsgaul O. , Pierce M. 
Published: February 2005

AbstractThe endocytosis and intracellular transport of mannose‐6‐phosphate conjugated to bovine serum albumin (Man‐6‐P:BSA) by mouse T‐lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man‐6‐P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man‐6‐P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent or 125I‐labeled Man‐6‐P:BSA at 0°C revealed a uniform distribution of the Man‐6‐P binding sites over the cell surface. Competition experiments indicate that the Man‐6‐P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37°C, endocytosis of Man‐6‐P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5–15‐min incubation of cells at 37°C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man‐6‐P:BSA in the Golgi region after 15–30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man‐6‐P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30‐min incubation at 37°C, the internalized Man‐6‐P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co‐stained for acid phosphatase. These results demonstrate a temporal participation of clathrin‐containing coated vesicles during the initial endocytosis of Man‐6‐P binding sites and that one step in the Man‐6‐P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.
Regulation of hexose transporters in chicken embryo fibroblasts: Stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters
Authors: Yamada Kazuo , Tillotson Loyal G. , Isselbacher Kurt J. 
Published: February 2005

AbstractAs has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) develop a 3‐ to 4‐fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two‐fold enhancement of stereospecific D‐glucose transport activity as well as D‐glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis.
Phenotypic heterogeneity in lymphoblastic cell lines
Authors: Smith L. J. , McCulloch E. A. 
Published: February 2005

AbstractFour T‐cell and two B‐cell lines from patients with lymphoblastic leukemia were examined with a panel of monoclonal antibodies for a variety of lineage and differentiation stage‐associated antigens during growth in liquid suspension. In five of the lines, markers normally associated with the granulopoietic lineage were found and the level of expression of these markers varied during culture. The sixth line, MOLT‐3, was examined in more detail. Using clonal selection it was found that phenotypic heterogeneity could readily be obtained and that subclones could be acquired that expressed a wide range of markers, typically in reproducible kinetic patterns, that were not detected on the parent clone. Previous results were confirmed showing that treatment with the drug 5‐azacytidine (5‐aza) prior to selection promoted the expression of the granulopoietic lineage associated antigen 80H.5 on MOLT‐3 subclones; however, treatment with 5‐aza appeared to inhibit substantially the expression of other novel markers by subclones following a second selection compared to untreated controls. It appears that the expression of markers normally associated with other lineages on leukemic lymphoblasts (lineage infidelity) may be characteristic of such lines and that phenotypically variant subclones expressing lineage infidelity can readily be obtained by clonal selection.
Necessity of transferrin for RNA synthesis in chick myotubes
Authors: Shoji Akiko , Ozawa Eijiro 
Published: February 2005

AbstractChick transferrin (Tf) is essential not only for growth and differentiation but also for the maintenance of chick myotubes in culture. Its removal from the culture medium gives rise to degeneration of the myotubes. The analysis of this process revealed that the removal resulted in decrease in total and messenger RNA content in the myotubes; this was mainly due to a decrease in RNA synthesis. Activity of in vitro RNA synthesis in isolated nuclei from myotubes cultured without Tf was lower than the activity in nuclei from myotubes cultured with Tf and increased with the addition of FeCl3. Although RNA degradation in myotubes was also enhanced following Tf removal, the degree was small. The synthesis of most proteins was reduced. In contrast to this, a few new proteins of unknown nature were synthesised in myotubes cultured in Tf‐free medium.The role of Fe ion carried into the cells by Tf in promoting myogenic cell growth and differentiation and in preventing the myotubes from degeneration can be explained, at least in part, on the basis of its effect on RNA synthesis.Since we have found that Fe is required for activation of RNA polymerase purified from embryonic muscles (Shoji and Ozawa, 1985b), these effects may be ascribed to this activating effect.
Endothelial cell injury in vitro is associated with increased secretion of an Mr 43,000 glycoprotein ligand
Authors: Sage Helene , Tupper Joan , Bramson Rachel 
Published: February 2005

AbstractA novel, serum albumin‐binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem. 259:3993–4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70–100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three‐fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed ‘culture shock’), was also accompanied by a significant increase in the secretion of 43K protein.Pulse‐chase studies revealed that the initial product secreted within 1.5 h was of Mr 38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr 43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr 40,000–50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti‐activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids.These studies identify a unique Mr 43,000 glycoprotein that is associated with cellular stress or injury in vitro. As a secreted but nonmatrix macromolecule, this protein may be part of a ‘survival kit’ used by the endothelium to cope with cellular injury.
Internalization of transforming growth factor‐β and its receptor in BALB/c 3T3 fibroblasts
Authors: Massagué Joan , Kelly Brenda 
Published: February 2005

AbstractThe fate of 125I‐labeled transforming growth factor‐β (125I‐TGFβ) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I‐TGFβ to cellular receptors at 4°C is pH‐sensitive, being markedly decreased at pH < 6. Most (∼ 90%) of the 125I‐TGFβ bound to cells at 4°C can be removed by a brief treatment with acidic medium but is converted into an acid‐resistant state rapidly after shifting the cells to 37°C. Cell‐bound 125I‐TGFβ is degraded at 37°C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I‐TGFβ from the cells. Cells allowed to accumulate 125I‐TGFβ intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X‐100; this treatment caused the cross‐linking of internalized 125I‐TGFβ with the 280‐kilodalton TGFβ receptor component. Under conditions in which sustained binding and degradation of saturating 125I‐TGFβ concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGFβ binding the TGFβ:receptor complex becomes rapidly internalized and that TGFβ is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGFβ receptors recycled after internalization or supplied by a large intracellular pool.
Stress induces an increased hexose uptake in cultured cells
Authors: Warren A. P. , James M. H. , Menzies D. E. , Widnell C. C. , Whitaker‐Dowling P. A. , Pasternak C. A. 
Published: February 2005

AbstractTemperature‐sensitive mutants have revealed a region of the herpes simplex virus 1 genome that affects both the uptake of hexose and the synthesis of heat shock proteins. Other inducers of heat‐shock proteins, namely heat shock itself and arsenite, likewise induce an increased uptake of hexose. The increased uptake, like that induced by insulin, is insensitive to the presence of actinomycin D or cycloheximide. It is concluded that an increased hexose uptake, reflecting an activation or relocation of existing hexose transport protein, is general biochemical response of stressed cells.
Ultrastructural studies of the intracellular translocation of endocytosed alpha‐foetoprotein (AFP) by cytochemistry and of the uptake of 3H‐arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells
Authors: Geuskens Maurice , Naval Javier , Uriel José 
Published: February 2005

AbstractA covalent conjugate of alpha‐foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow, at the ultrastructural level, the pathway of AFP uptake and translocation in a rat rhabdomyosarcoma cell line. The cells were incubated for several times at 4°C and/or 37°C, and fixed. AFP‐HRP was found to enter the cells via coated pits and receptosomes and to move to tubular elements of the trans‐reticular portion of the Golgi. Some observations suggest that AFP can be recycled back to the cell surface. On the other hand, the cells were incubated with a noncovalent conjugate of AFP and 3H‐arachidonic acid [3H‐(20:4)], and the uptake of the fatty acid molecules studied by ultrastructural autoradiography. The cytoplasmic labeling, very low after an incubation in the presence of [3H‐(20:4)]‐AFP for 2 hours at 4°C, increased rapidly after transfer of the cells for 5 minutes to 37°C. These observations support the hypothesis that AFP plays a role in the intracellular delivery of polyunsaturated fatty acids.
Functional differences in the interactions of glycosylation‐deficient cell lines with fibronectin, laminin, and type IV collagen
Authors: Hughes R. Colin , Mills Gary 
Published: February 2005

AbstractFibronectin isolated from the conditioned medium of monolayer cultures of baby hamster kindey (BHK) cells and several ricin‐resistant (Ric®) mutants derived from them express differences in N‐glycosylation. The asparaginelinked oligosaccharides of BHK cell‐derived fibronectin consist largely of complex chains, whereas hybrid and/or high‐mannose chains are present in the fibronectins of mutant cell lines. The fibronectins exhibiting different glycosylation patterns are incorporated to similar extents into the cell‐layer of human skin fibroblasts. In contrast, mutant cells retain significantly less endogenously produced fibronectin than BHK cells and also incorporate less human cellular fibronectin into a pericellular matrix. In vitro adhesion assays show that mutant cells attach to and spread relatively poorly on fibronectinor type IV collagen‐coated substrata but interact as well as do BHK cells with a laminin substratum. These results indicate that asparagine‐linked oligosaccharides of fibronectin are not required for the binding and incorporation of the molecule into cell layers, but, as constituents of other cellular glycoproteins, they do modulate the ability of BHK cells to interact with some matrix components.
Heparin protects basic and acidic FGF from inactivation
Authors: Gospodarowicz D. , Cheng J. 
Published: February 2005

AbstractThe ability of heparin or that of hexuronyl hexosaminoglygan sulfate (HHS‐4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the growth of baby hamster kidney (BHK‐21) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at −80°C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65°C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS‐4, basic and acidic FGF retained their potency. The effect of heparin and HHS‐4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS‐4 concentrations. This indicates that heparin and HHS‐4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.
Thrombin‐induced chemotaxis and aggregation of neutrophils
Authors: Bizios Rena , Lai Linda , Fenton John W. , Malik Asrar B. 
Published: February 2005

AbstractThrombin‐induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (108 cells/ml) exhibited maximum chemotactic migration towards 10−8 M human α ‐thrombin, 10−8 M γ‐thrombin (which lacks the fibrinogen site), and 10−12 MD‐Phe‐Pro‐Arg‐CH2‐α‐thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (108 cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10−11 M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, α‐thrombin, and γ‐thrombin. The responses of both human and sheep neutrophils to D‐Phe‐Pro‐Arg‐CH2‐α‐thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin‐induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.
Glucocorticoid agonists as well as antagonists are effective inducers of mouse mammary tumor virus RNA in mouse mammary tumor cells treated with inhibitors of ADP‐ribosylation
Authors: Johnson George S. , Ralhan Ranju 
Published: February 2005

AbstractGlucocorticoids increase expression of specific genes by a mechanism involving binding to and “activation” of a specific receptor protein. Other steroids, such as RU 486, bind to the glucocorticoid receptor but the resultant steroidreceptor complex is unable to activate glucocorticoid sensitive genes. In the present study we have observed that steroid regulation of the glucocorticoidregulated mouse mammary tumor virus (MMTV) genome in cultured mouse mammary tumor cells is altered by treatment of the cells with inhibitors of (ADP‐ribose)n synthetase. The ability of glucocorticoid agonists to increase MMTV is about 2‐fold increased by the inhibitor treatment. Interestingly, RU 486 and other steroids that are normally inactive in control cells are very good inducers of MMTV in the treated cells. This alteration in MMTV expression is associated with a 37% increase in nuclear binding of the glucocorticoid, triamcinolone acetonide, and also RU 486 in the inhibitor‐treated cells. Steroids that do not bind to the glucocorticoid receptor are not inducers in control or in treated cells. The results point to a role for ADP‐ribosylation of proteins as a negative regulator of MMTV expression and suggest a mechanism for activation of steroid‐sensitive genomes.
Both normal and tumor cells produce basic fibroblast growth factor
Authors: Moscatelli David , Presta Marco , Joseph‐Silverstein Jacquelyn , Rifkin Daniel B. 
Published: February 2005

AbstractWe have previously purified from human placenta a basic fibroblast growth factor (FGF)‐like molecule which stimulates the production of plasminogen activator (PA) and collagenase, induces DNA synthesis, produces an increase in motility in cultured bovine capillary endothelial (BCE) cells, and induces angiogenesis in vivo. The ability of basic FGF to stimulate PA production in BCE cells was used as an assay for the presence of basic FGF‐like molecules in extracts of both normal and tumor‐derived cultured cells. The identity of the PA‐stimulatory activity with basic FGF was confirmed by its high affinity for heparin and by its cross‐reactivity with antibodies to human placental basic FGF. Basic FGF‐like molecules were identified in eight of ten cell lines tested, and the amount of FGF‐like activity present in these cells bore no relation to their origin from normal or tumor tissue. The test cells, BCE cells, had one of the highest levels of FGF‐like activity, suggesting that it may have an autocrine role in these cells.
Isolation and characterization of mink lung epithelial cell mutants resistant to transforming growth factor β
Authors: Chinkers Michael 
Published: February 2005

AbstractMink lung epithelial cells resistant to growth inhibition by transforming growth factor β (TGF‐β) have been isolated by chemical mutagenesis and growth in the presence of platelet extracts enriched in TGF‐β. Several resistant clones were isolated, at least one of which stably retained its resistance to TGF‐β when grown in the absence of the factor. The cells of this clone were similar to the parent cells in morphology and growth properties. However, unlike the parent cells, the resistant cells did not show any of the following responses to TGF‐β: (1) inhibition of DNA synthesis and proliferation; (2) morphological changes involving increased cell spreading; or (3) stimulation of synthesis of a 48‐kilodalton secreted protein. The resistant cells do, however, retain a functional TGF‐β receptor. The TGF‐β resistant cell lines may be useful in genetic studies designed to identify the biochemical events required for inhibition of epithelial cell growth by this factor.