bluuebunny/crossref_metadata_2025_split huggingface dataset:
Action of phorbol esters in cell culture: Mimicry of transformation, altered differentiation, and effects on cell membranes
Authors: Weinstein I. Bernard , Lee Lih‐Syng , Fisher Paul B. , Mufson Alan , Yamasaki Hiroshi 
Published: May 2005

AbstractThe carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast t o initiating carcinogens, the action of the tumor‐promoting phorbol esters does not appear t o involve covalent binding t o cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose‐glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor‐promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane‐related effects include enhanced uptake of 2‐deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor t o cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of “two stage” carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.
Introduction of metastatic heterogeneity by short‐term in vivo passage of a cloned transformed cell line
Authors: Talmadge James E. , Starkey Jean R. , Davis William C. , Cohen Arthur L. 
Published: May 2005

AbstractAn experimental system for the study of metastasis has been developed using an epithelioid cell line of hepatic origin which had previously been chemically transformed in vitro. These metastatic cells were studied in the syngeneic rat strain. The cloned parent cell line metastasizes only to the lungs following in travenous, subcutaneous, or intraperitoneal injection. The metastatic phenotype is stable during in vitro passage, and subclones from the parent clone have a metastatic capacity statistically similar to that of the parent clone. Following ascites passage of the parent cell line, the cell population obtained exhibits the same metastatic ability as the parent clone. However, subclones obtained from the ascites‐passaged population exhibit metastatic heterogneity. This heterogeneity is introduced by the host passage and not by in vitro culture or subcloning. In the case of the two metastatic variants examined, the difference in the metastatic phenotype is found not to be due to differences in arrest or trapping of the cells but appears to be related to long‐term survival and proliferation of the tumor cells following their arrest in the lungs. Morphologically the variants are very similar, and growth of the metastatic foci provokes a vigorous inflammatory response by the host.
An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts
Authors: Furcht L. T. , Wendelschafer‐Crabb G. , Mosher D. F. , Foidart J. M. 
Published: May 2005

AbstractFibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti‐peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non‐periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.
Light‐driven primary sodium ion transport in halobacterium halobium membranes
Authors: Lanyi Janos K. 
Published: May 2005

AbstractLight‐induced sodium extrusion from H halobium cell envelope vesicles proceeds largely through an uncoupler‐sensitive pathway involving bacteriorhodopsin and a proton/sodium antiporter. Vesicles from bacteriorhodopsin‐negative strains also extrude sodium ions during illumination, but this transport is not sensitive to uncouplers and has been proposed to involve a light‐energized primary sodium pump. Proton uptake in such vesicles is passive, and under steady‐state illumination the large electrical potential (negative inside) is just balanced by a pH difference (acid inside), so that the protonmotive force is near zero. Action spectra indicated that this effect of illumination is attributable to a pigment absorbing near 585 nm (of 568 for bacteriorhodopsin). Bleaching of the vesicles by prolonged illumination with hydroxylamine results in inactivation of the transport; retinal addition causes partial return of the activity. Retinal addition also causes the appearance of an absorption peak at 588 nm, while the absorption of free retinal decreases. The 588 nm pigment is present in very small quantities (0.13 nmole/mg protein), and behaves differently from bacteriorhodopsin in a number of respects. Vesicles can be prepared from bacteriorhodopsin‐containing H halobium strains in which primary transport for both protons and sodium can be observed. Both pumps appear to cause the outward transport of the cations. The observations indicate the existence of a second retinal protein, in addition to bacteriorhodopsin, in H halobium, which is associated with primary sodium translocation. The initial proton uptake normally observed during illumination of whole H halobium cells may therefore be a passive flux in response to the primary sodium extrusion.
Visualization of the turkey erythrocyte β‐adrenergic receptor
Authors: Durieu‐Trautmann O. , Delavier‐Klutchko C. , Vauquelin G. , Strosberg A. D. 
Published: May 2005

AbstractWe have recently described the affinity chromatography purification of the turkey erythrocyte β‐adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10‐fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine‐sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l‐epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4–6 pmoles of 3H‐dihydroalprenolol binding sites per mg of membrane protein.A single step of affinity chromatography on alprenolol‐sepharose of the soluble digitonin extract resulted in an additional 1,000‐fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations.Electrophoresis in SDS‐polacrylamide of iodinated purified β‐receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with β‐mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l‐propranolol. A fourth 52,000‐dalton component (60,000 daltons after reduction with β‐mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l‐propranolol.Our results suggest that the β‐adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.
Induction and long‐term maintenance of Thy‐1 positive T lymphocytes: Derivation from continuous bone marrow cultures
Authors: Tertian Gérard , Yung Yee Pang , Moore Malcolm A. S. 
Published: May 2005

AbstractIn the present study we investigated the presence of T‐lymphocyte progenitors in the long‐term murine bone marrow culture system described by Dexter: mature Thy‐1 antigen‐bearing T lymphocytes are lost in these cultures after a few days. By culturing nonadherent cells from such cultures in the presence of a supernatant of concanavalin A‐stimulated spleen cells, a source of T‐cell growth factor, we found that Thy‐1 positive blast cells proliferated together with a second population of Thy‐1 negative cells. These two populations of cells have been maintained in long‐term in vitro cultures by passaging the cells in fresh conditioned medium at regular intervals. Moreover, we have been able to establish pure cultures of the Thy‐1‐bearing blast cells after separating them from the non‐T cells using their adherence property to plastic surfaces. Long‐term cultures of T lymphocytes can thus be established from long‐term marrow cultures as well as from the spleen, thymus or fresh bone marrow.
Total and exchangeable calcium in lymphocytes: Effects of PHA and A23187
Authors: Lichtman Andrew H. , Segel George B. , Lichtman Marshall A. 
Published: May 2005

AbstractCalcium has been suggested as an internal second messenger when lymphocytes are stimulated by mitogens to enter the cell cycle. We have assessed the effect of 2 lymphocyte stimulants, the plant lectin phytohemagglutinin (PHA) and the calcium ionophore A23187, on human lymphocyte nucleic acid synthesis, total cell calcium content, and 4 5Ca labeling. We have used an ultrasensitive method for the measurement of total cell calcium in the same samples used for radiolabeling. Mitogenic concentrations of A23187 (∼ .25 μ mole/liter) caused an increase in both total cell calcium and 4 5Ca labeling. These increases were almost completely blocked by inhibitors of mitochondrial respiration, suggesting that the calcium increment after ionophore treatment was located in the mitochondria. In contrast, total cell calcium was not altered at optimal mitogenic PHA concentrations (0.1 μg/ml and above). However, at the minimum PHA concentrations that caused stimulation (0.025 to 0.1 μg/ml), the dose response of 4 5Ca uptake was very similar to that of DNA sysnthesis. Importantly, we could not stimulate DNA synthesis with PHA without increasing lymphocyte 4 5Ca labeling. Thus, an increase in total cell calcium is not essential for mitogenesis; however, an increase in 4 5Ca exchange is closely associated with the mitogenic effects of A23187 and PHA.
H‐2 I alloantigens and recall of memory cytotoxic responses
Authors: Orosz Charles G. , Macphail Stuart , Bach Fritz H. 
Published: May 2005

AbstractAQR mice were immunized with H‐2K and H‐2 I encoded alloantigens presented by (Ax6R)F1 splenocytes. Spleen cells from these alloimmune mice were subsequently restimulated in vitro with B10.A lymphocytes and/or B10.T(6R) lymphocytes, thus presenting them with the immunizing H‐2K and H‐2 I alloantigens independently. When stimulated with B10.A lymphocytes, alloimmune lymphocytes develop significant cytotoxicity against the immunizing H‐2K target antigens. When stimulated with a similar number of B10.T(6R) spleen cells, alloimmune lymphocytes undergo a prominant proliferative response, but develop little, if any, cytotoxicity against the immunizing H‐2 K target antigens. The most efficient restimulation of cytotoxicity occurs when the alloimmune spleen cells are simultaneously restimulated by B10.A and B10.T(6R) lymphocytes. Stimulation with the immunizing H‐2 I alloantigens alone is not sufficient for regeneration of detectable cytotoxic responses from alloimmune spleen populations. Stimulation with the immunizing H‐2K alloantigens alone appears to be both necessary and sufficient to stimulate alloimmune cytotoxic responses. Although the immunizing H‐2 I alloantigens are apparently not required to generate alloimmune cytotoxic responses, they markedly potentiate the cytotoxic responses induced by the immunizing H‐2K alloantigens.
An in vitro assay for T lymphocyte progenitors (CFU‐preT)
Authors: Acuff Bonita R. , Cohen J. John 
Published: May 2005

AbstractThy‐1.2 negative progenitors give rise to Thy‐1.2 positive colony cells when mouse bone marrow is cultured in vitro. The bone marrow cells are immobilized in a viscous medium containing methyl cellulose; discrete colonies are identifiable at 2 days and contain 30–60 cells by day 3 of culture. Colonies are tightly packed spheres (raspberries) and grow suspended in the gel. Growth of the raspberry colonies is absolutely dependent upon the presence of the appropriate serum (horse or human; not fetal calf) and conditioned medium from pokeweed mitogen‐stimulated mouse spleen cells. As little as 0.1% of the conditioned medium is sufficient to promote raspberry colony growth. Under these conditions, nude mouse bone marrow yields as many colonies (1 per 1,000 nucleated cells plated) as normal marrow. Thymus, lymph node; and spleen (normal or nude) do not form colonies. Colony precursors are predominantly in S phase of the cell cycle, as determined by tritiated thymidine suicide of fresh bone marrow. Their numbers fall with age. Because the cells in colonies are Thy‐1 positive, peanut agglutinin‐positive, and active in a pre‐T cell synergy assay, we conclude that their precursors are early committed T cell progenitors, and propose that they be called CFU‐preT.
Catalytic activities associated with the enzymes II of the bacterial phosphotransferase system
Authors: Saier Milton H. 
Published: May 2005

AbstractThe phosphotransferase system (PTS) in Escherichia coli is a multifunctional, multicomponent enzyme system. Its primary functions deal with carbon source acquisition, while its secondary functions are concerned with the regulation of bacterial physiology. The primary functions of the system include (1) extracellular detection, (2) unidirectional and exchange transmembrane transport, and (3) phosphoenolpyruvate‐dependent and sugar phosphate‐dependent phosphorylation of the sugar substrates of the system. The secondary functions include (1) regulation of the activities of adenylate cyclase and various non‐PTS permeases and (2) regulation of the induced synthesis of several PTS enzymes. Both the primary and secondary functions appear to be elicited by the binding of a sugar substrate to an Enzyme II complex. One of these integral transmembrane enzymes, the mannitol Enzyme II (IImtl), has been solubilized with detergent, purified to homogeneity, and reconstituted in an artificial membrane system. The molecular weight of this protein, IImtl, is 60,000 daltons. It possesses an extracellular sugar binding site and distinct intracellular combining sites for sugar phosphate and phospho‐HPr. An essential sulfhydryl group and an antibody combining site are localized to the cytoplasmic surface of the enzyme, while a dextran combining site is localized to the external surface. Preliminary experiments suggest that the different functions of the Enzyme IImtl can be dissected by genetic and biochemical techniques. These studies emphasize the functional complexity of the PTS and its integral membrane protein constituents.
Interaction of polypeptide neurotoxins with a receptor site associated with voltage‐sensitive sodium channels
Authors: Catterall William A. , Beneski Daniel A. 
Published: May 2005

AbstractAnthopleurin A, a polypeptide toxin from the Pacific sea anemone Anthopleura xanthogrammica, enhances persistent activation of voltage‐senstive sodium channels by the alkaloid toxins veratridine and batrachotoxin with K0.5 = 20 nM. This effect is inhibited by depolarization. There is a close correlation between enhancement of sodium channel activation and block of [125I] scorpion toxin binding by unlabeled scorpion toxin, sea anemone toxin II from Anemonia sulcata, and Anthopleurin A, indicating that these three polypetide toxins interact with a common receptor site in modifying sodium channel function. Photoactivable derivatives of scorpion toxin label a single Mr ∼ 250,000 polypeptide chain at the polypeptide toxin receptor site. Labeling is blocked by unlabeled scorpion toxin or depolarization and is not observed in variant neuroblastoma clones, which lack sodium channels. These results identify a protein component of the polypeptide toxin receptor site of voltage‐sensitive sodium channels.
Interaction of serum and cell spreading affects the growth of neoplastic and non‐neoplastic fibroblasts
Authors: Tucker R. W. , Butterfield C. E. , Folkman J. 
Published: May 2005

AbstractBoth growth factor availability and cell‐to‐cell contact have been mechanisms used to explain cell growth regulation at high cell density. Recently Folkman and colleagues have shown that changes in cell shape, rather than cell‐to‐cell contact, can regulate the growth of fibroblasts. However, in those studies the relation between serum and shape regulation of growth was not studied, nor were neoplastic and non‐neoplastic cells compared. In this report we have studied these aspects by varying cell spreading and serum concentration independently for 2 non‐neoplastic and 3 neoplastic cell lines. Cell spreading (projected cell area) was controlled by decreasing the adhesiveness of tissue culture plastic plates with poly (hydroxyethyl methacrylate) [poly (HEMA)]. Cell growth was measured as the increase in cell number/day. We have found that more spreading increased net growth of both neoplastic and non‐neoplastic cells, while less spreading (toward rounded configuration) depressed growth. There were also quantitative differences between neoplastic and non‐neoplastic cells. Neoplastic cells continued to grow under conditions of cell rounding, which completely prevented the growth of their non‐neoplastic counterparts. Some neoplastic cells also tended to show little or no increase in net cell number for serum concentrations above 10% as cells became more spread; in contrast, all non‐neoplastic cells grew more with increasing concentrations of serum as they became well spread. Thus, in normal cells, it appears that the sensitivity of cells to humoral factors is governed by cell spreading. This interaction between serum and cell shape is less prominent in some neoplastic cells.
Cleavage of cell surface proteins by thrombin
Authors: Moss Martin , Cunningham Dennis D. 
Published: May 2005

AbstractThis study was based on our previous findings that the mitogenic action of thrombin on cultured fibroblasts can result from interaction of thrombin with the cell surface in the absence of internalization, and that the proteolytic activity of thrombin is required for stimulation of cell division. This prompted us to look for thrombin‐mediated cleavages using 2‐dimensional gel electrophoresis of labeled cell surface proteins. Surface membrane components were labeled by 3 procedures: (1) proteins were labeled by lactoperoxidase‐catalyzed iodination using 125I−; (2) galactose and galactosamine residues of glycoproteins were oxidized with galactose oxidase and reduced with 3H‐NaBH4; and (3) glycoproteins were metabolically labeled by incubating cells with 3H‐fucose. Labeling with the first 2 procedures was carried out after thrombin treatment; in contrast, cells metabolically labeled with 3H‐fucose were subsequently treated with thrombin to look for proteolytic cleavages. Collectively, these studies indicated that only about 5 cell surface proteins were thrombin‐sensitive, consistent with the high specificity of this protease. Each of the labeling procedures revealed a thrombin‐sensitive cell surface glycoprotein which was identified as fibronectin by immunoprecipitation experiments. In addition, cell surface proteins of about 140K and 55K daltons were thrombin‐sensitive. However, cell surface proteins of about 45K daltons and 130K to 1 50K daltons were increased after thrombin treatment. These experiments were conducted on an established line of Chinese hamster lung cells with the eventual goal of studying thrombin‐mediated cleavages of cell surface proteins in a large number of cloned populations derived from this line that are either responsive or unresponsive to the mitogenic action of thrombin. This approach should permit identification of proteolytic cleavages that are necessary for thrombin‐stimulated cell division.
Cell Cycle activation and ouabain‐sensitive ion movements of 3T3 and C3H‐10T½ fibroblasts
Authors: Wenner Charles E. , Cheney John C. , Tomei L. David 
Published: May 2005

AbstractThe introduction of either PGF2α (10−7 M) or TPA (10−7 M) stimulated, ouabain‐sensitive 86Rb+ influx at 30 min in postconfluent 3T3‐4 mouse fibroblast cultures by 117% and 124%, respectively. Both TPA and PGF2α at these concentrations stimulated the incorporation of 3H‐TdR into DNA. TPA had the greatest stimulatory effect, which was similar to that obtained with 10% fetal calf serum. In accord with the idea that modulation of membrane processes such as Na+/K+ pump activity in fibroblasts may reflect important events related to the initiation of DNA synthesis, it was observed that in both 3T3‐4 and C3H‐1 0T½ cells there were parallel increases in 3H‐TdR incorporation and ouabain‐sensitive 86Rb+ influxes with 10−7 M TPA, whereas PGF2α stimulated a significant increase in 3H‐TdR incorporation in 3T3‐4 but not C3H‐10T½ cells and only marginal increases in ouabain‐sensitive 86Rb+ influx in both. Therefore, although there appears to be a close correlation between Na+/K+ pump activation and subsequent S‐phase entry following TPA stimulation, a similar correlation for PGF2α cannot be confirmed.
Purification and characterization of multiplication‐stimulating activity (MSA) carrier protein
Authors: Knauer Daniel J. , Wagner Fred W. , Smith Gary L. 
Published: May 2005

AbstractThe rat liver cell line, BRL‐3A, is known to produce a family of polypeptides referred to as multiplication‐stimulating‐activity (MSA). Serum‐free conditioned medium from this cell line is a rich source for the purification of these somatomedin‐like molecules. Somatomedins in serum, as well as MSA produced by BRL‐3A cells in culture, exist primarily as a high molecular weight complex bound to specific carrier proteins. This study describes the purification of the MSA carrier protein (MCP) from conditioned medium using affinity chromatographic procedures. The purified carrier protein is shown to specifically bind labeled MSA and generates a complex with an apparent molecular weight of 60,000–70,000 daltons. Characterization of the carrier protein indicates that it consists of two different noncovalently linked protein chains with apparent molecular weights of 30,000 and 31,500 daltons. The availability of a pure carrier protein should provide a unique opportunity to investigate the functional significance of the carrier protein in the biological activity of the somatomedins.