bluuebunny/crossref_metadata_2025 huggingface dataset:
Thy‐1 is a differentiation antigen that characterizes immature murine erythroid and myeloid hematopoietic progenitors
Authors: Miller Barbara A. , Lipton Jeffrey M. , Linch David C. , Burakoff Steven J. , Nathan David G.
Published: February 2005

To determine the role of Thy‐1 antigen in murine hematopoietic differentiation, bone marrow was treated with anti‐Thy‐1.2 antibody and complement or complement alone. Growth of immature hematopoietic progenitors, erythroid burst‐forming units (BFU‐E), and granulocyte/macrophage colony‐forming units (CFU‐GM) was greatly reduced following antibody and complement treatment and was not restored by mitogen‐stimulated spleen cell supernatants. In contrast, more mature erythroid and myeloid progenitors, the erythroid colony‐forming unit (CFU‐E) and the macrophage progenitor stimulated by L‐cell‐conditioned media (LCM), were spared by anti‐Thy‐1.2 antibody and complement treatment. Here, to separate the effects of anti‐Thy‐1.2 antibody treatment on accessory cells from those on progenitors, splenic T cells and thymocytes were added to treated marrow at ratios of up to 200%. Growth of BFU‐E and CFU‐GM was not restored. To more precisely replace required accessory cells, male complement‐treated marrow was cocultured with female anti‐Thy‐1.2 antibody and complement‐treated marrow. Even marrow cells failed to restore female BFU‐E and CFU‐GM growth. Fluorescent‐activated cell sorting (FACS) and immune sheep red cell rosetting with anti‐Thy‐1.2‐labeled marrow were then performed to determine if immature hematopoietic progenitors bear Thy‐1.2‐positive fraction, demonstrating the presence of Thy‐1.2 on early murine hematopoietic progenitors. CFU‐E and CFU‐M were present in the Thy‐1.2‐negative fraction following FACS separation. These data demonstrate that Thy‐1.2 is a differentiation antigen, present on at least some murine BFU‐E and CFU‐GM and lost as they mature to CFU‐E and CFU‐M.
Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40
Authors: Steinberg Mark L. , Defendi Vittorio
Published: February 2005

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage‐specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10–15 serial passages (60–90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2–3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S‐methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48–52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two‐dimensional acrylamide gels using either isoelectric focusing (IEF) or non‐equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells came more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.
Cationic amino acid transport by two renal epithelial cell lines: LLC‐PK1 and MDCK cells
Authors: Sepúlveda Francisco V. , Pearson Jeremy D.
Published: February 2005

LLC‐PK1 and MDCK cells take up cationic amino acids (lysine and arginine) by a specific sodium independent transport system. Uptake is inhibited by ornithine in LLC‐PK1 and MDCK cells either in the presence or absence of sodium and by glutamine or homoserine in MDCK cells in the presence of sodium. Trans‐stimulation of uptake occurs in the presence of intracellular cationic amino acids. Experiments with valinomycin or with different extracellular potassium concentrations suggest that uptake is dependent on the membrane potential of these cells. These transport features are similar to those previously ascribed to a transport system denominated y+ in other cells. Further experiments suggested that this carrier system is localised to the basolateral membrane in each cell type.
Ultrastructural analysis of the organization and distribution of insulin receptors on the surface of 3T3‐L1 adipocytes: Rapid microaggregation and migration of occupied receptors
Authors: Smith Robert M. , Cobb Melanie H. , Rosen Ora M. , Jarett Leonard
Published: February 2005

Monomeric ferritin‐insulin and high‐resolution electron microscopic analysis were used to study the organization, distribution, and movement of insulin receptors on differentiated 3T3‐L1 adipocytes. Analysis of the binding to prefixed cells showed that insulin initially occupied single and paired receptors preferentially located on microvilli. The majority of receptors (60%) were found as single molecules and 30% were pairs. In 1 min at 37%C, 50% of the receptors on nonfixed cells were found on the intervillous plasma membrane and more than 70% of the total receptors had microaggregated. By 30 min only 7% of the receptors were single or paired molecules on microvilli. The majority were on the intervillous membrane, with 95% of those receptors in groups. The receptor groups on the intervillous plasma membrane could be found in both noncoated invaginations and coated pits. The concentration of occupied receptors in the noncoated invaginations and the coated pits was similar; however, ten times more noncoated invaginations than coated pits contained occupied insulin receptors. The observations in this study contrast with those reported on rat adipocytes using identical techniques (Jarett and Smith, 1977). Insulin receptors on adipocytes were initially grouped and randomly distributed over the entire cell surface and did not microaggregate into larger groups. Insulin receptors on rat adipocytes were found in non‐coated invaginations but were excluded from the coated pits. The differences in the organization and behavior of the insulin receptor between rat and 3T3‐L1 adipocytes suggest that the mechanisms regulating the initial organization of insulin receptors and the aggregation of occupied receptors may be controlled by tissue‐specific processes. Since both of these cell types are equally insulin sensitive, the differences in the initial organization and distribution of the insulin receptors on the cell surface may not be related to the sensitivity or biological responsiveness of these cells to insulin but may affect other processes such as receptor regulation and internalization. On the other hand, the microaggregates of occupied receptors on both cell types may relate to biological responsiveness.
Butyrate stimulates fibronectin synthesis in cultured rabbit cornea
Authors: Tanaka Hitoshi , Nishida Teruo
Published: February 2005

To understand corneal wound healing, in which fibronectin plays an important role, we investigated the regulatory mechanism of fibronectin synthesis in cultured rabbit corneal blocks in situ. The amount of fibronectin was determined by enzyme‐linked immunosorbent assay. We found that butyrate stimulated fibronectin synthesis in a dose‐response fashion, and that butyrate had a greater stimulatory effect than did any of its derivatives examined. This suggests that butyrate stimulates fibronectin synthesis specifically; 8Br‐cAMP also stimulated fibronectin synthesis. Additivity of stimulation by butyrate and by 8Br‐cAMP was observed at the saturated concentration of each; the present result indicated an independent mechanism of action for these two compounds.
Phospholipid asymmetry in human erythrocyte ghosts
Authors: Williamson Patrick , Algarin Lourdes , Bateman Judy , Choe Hye‐Ryun , Schlegel Robert A.
Published: February 2005

Using phospholipase digestion and the fluorescent probe merocyanine 540 the maintenance of phospholipid asymmetry in the plasma membrane of human erythrocyte ghosts was investigated. Digestion with phospholipase A2 indicated that ghosts prepare in the presence of Mg++ as the only divalent cation retained the normal phospholipid asymmetry characteristic of intact erythrocytes. These ghosts, like normal erythrocytes, also failed to stain with merocyanine 540. However, the presence of as little as 5–10 μM Ca++ during ghost preparation resulted in ghosts in which lipid asymmetry had been abolished, as indicated by phospholipase digestion. Moreover, these ghosts stained with merocyanine 540. In contrast to ghosts, intact erythrocytes treated with ionophore required millimolar levels of Ca++ ions to disrupt membrane lipid asymmetry. To discover the reason for this difference in behavior between ghosts and intact cells, ghosts were prepared from preswollen cells using only small volumes of buffer for lysis. These experiments demonstrated that as the cellular contents of erythrocytes are diluted, the asymmetric arrangement of phospholipids becomes more sensitive to disruption by Ca++.
Induction of differentiation in human myeloid leukemic cells by proteolytic enzymes
Authors: Fibach Eitan , Treves Aliza , Kidron Miriam , Mayer Michael
Published: February 2005

Exogenous serine proteases were found to induce differentiation in human myeloid leukemic cells from either in vitro established long‐term cell lines or in primary cultures of cells derived directly from patients with acute myeloid leukemia. Exposure of the human promyelocytic cell line HL‐60 to trypsin, chymotrypsin, or elastase induced the appearance, within 3–6 days, of neutrophilic granulocytes defined by their morphology, their ability to reduce nitroblue tetrazolium, and their efficient phagocytosis of latex particles. Upon further incubation monocytelike cells appeared. While these cells developed into fully mature macrophages other types of cells disappeared and on day 12 the culture consisted of a pure macrophage population. The inducing effect could be observed when the enzyme was presented alone, whereas a synergistic effect was noted when the protease was added in the presence of subthreshold concentrations of chemicals known to induce differentiation in this cell line such as dimethylsulfoxide, retinoic acid, butyric acid, or hexamethylene bisacetamide. Optimal induction of differentiation by trypsin required a 48 hr continuous exposure to the enzyme. When the protease was removed earlier no appreciable differentiation was noticed. The protease‐induced differentiation involved a direct interaction with the cells and was not due to a proteolytic cleavage of a serum component because it could be obtained in serum‐free cultures. The enzymatic activity of the protease was needed for its effect on cell maturation: Addition of protease inhibitors such as soybean‐trypsin inhibitor or trasylol completely blocked differentiation induced by the proteases but had no effect on differentiation induced by the other inducers. It is still to be determined whether a proteolytic process is a general molecular event in cell differentiation or induction by chemicals involves a mechanism different from that initiated by exogenous proteases.
Serum‐free conditions for the growth of avian granulocyte and monocyte clones and primary leukemic cells induced by AMV, and the apparent conversion of granulocytic progenitors into monocytic cells by a factor in chicken serum
Authors: Dodge W. H. , Sharma S.
Published: February 2005

The supportive activity of chicken serum for the soft‐agar growth of chicken granulocyte and monocyte clones could be replaced with defined ingredients [bovine serum albumin (BSA), conalbumin, selenium, linoleic acid and for routine work, a liquid soy lecithin preparation (59% phospholipids, 39% linoleic acid, and <2% of inositol and choline)]. The lecithin preparation could be replaced with L‐α‐phosphatidylcholine. The source of colony‐stimulating factor was medium conditioned by fibroblasts cultured under protein‐free conditions. AMV‐induced leukemic cells could be cloned under identical conditions. In the presence of both chicken serum (10%) and the replacement ingredients, most of the proliferative clones produced were monocytic (84%). In the presence of serum alone, all of them were monocytic. Under serumfree conditions, all the clones produced were granulocytic when a day‐3 conditioned medium (CM) was used; monocytic ones were also present when a day‐6 CM was used. When the serum was serially diluted in the presence of the replacement ingredients, the number of proliferative monocytic clones progressively decreased while the number of proliferative granulocytic clones progressively increased and the kinetics of each were essentially the same, only opposite in direction. Moreover, the total number of proliferative clones did not change more than 33% at any dilution (or in the absence of serum). We postulate the existence in the chick system of a serum macrophage differentiation factor (M‐DF) which converts early granulocytic progenitors (or exclusively diverts a common progenitor) into monocytic cells.
Pattern of steroid interaction with inducers of aryl hydrocarbon hydroxylase activity in several cloned estradiol‐responsive stromal cell strains from mouse endometrium
Authors: Stols L. , Iannaccone P. M.
Published: February 2005

Several steroid responsive stromal cell strains were isolated from normal mouse endometrium. Both estrogen responsive and progesterone responsive strains were obtained. The estrogen responsive cells were also responsive to diethylstilbestrol (DES). The progesterone responsive strains were inhbited by estrone and estriol. One of the estrogen responsive cell strains, E041, was cloned in the presence and absence of 17‐β‐estradiol (E2). Of 14 clones developed in the absence of E2 and screened for responsivity of growth, six were significantly responsive to E2. Six of 26 clones developed in the presence of E2 were responsive to E2. Microsomes isolated from these clones were shown to have aryl hydrocarbon hydroxylase (AHH) activity which was inducible with either benzanthracene (BA) or 2,3,7,8 tetrachlorodibenzo‐p‐dioxin (TCDD). The basal levels of AHH activity were significantly reduced by E2 in E2 responsive clones. The TCDD induced levels of AHH activity in the E2 responsive and E2 non‐responsive clones were significantly increased by E2 in one of the clones. These results show that mouse endometrial stroma consists of a number of populations of cells with different specific proliferative and metabolic responses to steroids.
The effect of biotin on cellular functions in HeLa cells
Authors: Bhullar R. P. , Dakshinamurti K.
Published: February 2005

HeLa cells cultured in a biotin‐deficient medium show reduced rate of protein synthesis, DNA synthesis and growth. Addition of exogenous biotin to the cells cultured in biotin‐deficient medium results in enhanced protein synthesis, DNA synthesis and cell growth. Continuous protein synthesis is required for the increase in DNA synthesis observed upon the addition of exogenous biotin to the cells cultured in biotin‐deficient medium. These results suggest that cells cultured in biotin‐deficient medium are arrested in the G1 stage of cell cycle and this block is removed upon the addition of biotin to the deficient medium.
The effect of transport system A and N amino acids and of nerve and epidermal growth factors on the induction of ornithine decarboxylase activity
Authors: Rinehart Clifford A. , Viceps‐Madore Dace , Fong Wang‐Fun , Ortiz José G. , Canellakis Evangelo S.
Published: February 2005

The induction of ornithine decarboxylase (EC 4.1 1.17) (ODC) by amino acids and by the peptide hormones nerve growth factor (NGF) and epidermal growth factor (EGF) in salts‐glucose media has been studied. Only those neutral amino acids taken into the cell via one of the Na+ dependent transport systems stimulate ODC activity. Asparagine and the nonmetabolizable α‐amino‐isobutyric acid (AIB) were used as representatives of this class of inducing amino acids, and their intracellular concentrations were related to the levels of ODC induced. A threshold intracellular concentration of asparagine or AIB has to be attained before ODC can be induced. Further slight increases in intracellular concentrations of asparagine or AIB produce disproportionately large increases of ODC, resulting in a sigmoidal curve of ODC by valine is associated with a concurrent decrease in the intracellular level of the inducing amino acid, suggest that the intracellular amino acid level is causally related to the induction of ornithine decarboxylase. Glutamic acid, EGF, and NGF do not induce ODC except in the presence of an inducing amino acid. They act synergistically with the inducing amino acid and produce higher ODC levels at the same intracellular concentration of the inducing amino acid.
Effect of cell density on metabolism in isolated rat hepatocytes
Authors: Jurin Richard R. , McCune Sylvia A.
Published: February 2005

Freshly isolated rat hepatocytes show many changes in metabolic activities as a function of cell density in the incubation flask. Fatty acid synthesis, cholesterol synthesis, general protein synthesis, and rates of accumulation of pyruvate, lactate, citrate, acetyl‐CoA, acetoacetate and β‐hydroxybutyrate decrease as the cell density increases between 1 mg/ml and 60 mg/ml. Glucose release only decreases between 1–5 mg/ml and the concentration of ATP does not vary at any density. There is a small increase in the lactate/pyruvate ratio and a dramatic decrease in the β‐hydroxybutyrate/acetoacetate ratio with increasing cell concentration. When cells at 8 or 28 mg/ml were incubated with added lactate and pyruvate, or alanine, a two fold increase in fatty acid synthesis and 50% decrease in cholesterol synthesis were observed as compared to rates with endogenous substrate. With added glucose the synthetic rates were similar to those obtained with endogenous substrate. However, regardless of the type of substrate used, the less dense cells gave rates up to three times greater than that of the more dense cells. When conditioned medium isolated after incubation of cells at high density was added to the less dense cells, a decrease in the rates of fatty acid synthesis and cholesterol synthesis was observed in the less dense cells; however, when medium from the less dense cells after incubation for the same period was added to the more dense cells, there was no significant change in fatty acid or cholesterol synthesis. These results suggest that a factor may be released into the medium of incubating hepatocytes that progressively inhibits certain metabolic processes as the cell density increases.
Single high affinity binding of interferon α2 to receptors on human lymphoblastoid cells: Internalization and inactivation of receptors
Authors: Faltynek Connie R. , McCandless Sarah , Baglioni Corrado
Published: February 2005

The interaction of human recombinant interferon (rIFN) α2 with its receptor on lymphoblastoid cells was studied using competitive displacement binding. The data were analysed with the LIGAND program, which tests their fit to one‐site or multiple binding site models. The binding at 4° and 37°C fits a one‐site model, with a similar KD for both IFN‐sensitive and resistant cells. Binding at 37°C to Daudi cells at high density fits artifactually a two‐site mode only when the receptor concentration is close to that of the KD. The binding of IFN to its receptor, therefore, follows a simple bimolecular interaction. Furthermore, IFN‐sensitive and resistant cells internalize IFN at similar rates. We have examined whether IFN receptors are also internalized and whether they subsequently recycle to the cell surface. By measuring cell surface and total receptors, we have observed that after 2 h treatment with IFN total receptors remain constant whereas cell surface receptors decrease. After prolonged treatment with IFN, however, there is a loss of total receptors. By inactivating cell surface receptors with proteinase K, we have shown that a fraction of cell surface receptors becomes resistant to inactivation and is apparently internalized. Moreover, experiments which measure IFN receptors either during incubation in the presence of IFN or after IFN has been removed from the medium, show that receptors do not recycle to the cell surface after internalization. The addition of monensin, a drug which has been shown to inhibit receptor recycling, has no effect on the loss of IFN receptors.
Modulation of transferrin receptor expression by inhibitors of nucleic acid synthesis
Authors: Hedley David , Rugg Catherine , Musgrove Elizabeth , Taylor Ian
Published: February 2005

We investigated the effects of the iron chelator desferrioxamine on the expression of transferrin receptors (TfR) by CCRF‐CEM human T‐cell leukaemia and B16 mouse melanoma cells growing in tissue culture. Desferrioxamine (DFOA) enhanced TfR expression when added in the dosse range of 10−5−10−4 to CCRF‐CEM cells, but was toxic to these cells, the lower concentrations producing a slowing of cell growth with a build up in S‐phase, while higher concentrations caused cell death with a block at the G1/S‐phase interface. These toxic effects are compatible with its previously reported inhibition of teh non‐haen iron containing (M2) subunit of ribonucleotide reductase. In marked contrast, DFOA caused the growth of B16 melanoma cells to arrest in G1, without loss of cloning efficiency, and resulted in a fall in TfR expression to approximately 50% of control values. These results suggested that the effects of DFOA on TfR expression were linked to DNA synthesis rather than to a more generalised inhibition of iron‐depdendent cellular processes. It was subsequently found that inhibition of the M2 subunit of ribonucloetide reductase in CCRF‐CEM cells with 5 x 10−5 M hydroxyurea, which is not an iron chelator, also enhanced TfR expression, as did thymidine and Cytosine arabinoside, which have different enzyme targets. By measuring cellular DNA and RNA content simultaneously it was shown that all of these agents caused unbalanced growth, i.e., inhibited DNA synthesis more than RNA synthesis. In contrast, 6‐thioguanine was more inhibitory to RNA synthesis, and treatment with this drug caused a fall in TfR expression. Thus, although CCRF‐CEM cells treated with DFOA show enhanced TfR expression, similar effects are also seen with other inhibitors of DNA synthesis, provided thatRNA synthesis is allowed to continue. These results provide further evidence that the regulation of TfR expression by proliferating cells is specifically linked to DNA synthesis rather than to the iron requirements of other cellular processes.
Characterization of human megakaryocytic colony formation in human plasma
Authors: Solberg L. A. , Jamal N. , Messner H. A.
Published: February 2005

We have analysed the contribution to megakaryocyte colony formation in methylcellulose made by human plasma, serum, media conditioned by phytohemagglutinin (PHA) stimulated leukocytes (PHA‐LCM), erythropoietin (EPO) preparations, and platelets. The culture system was used as a bioassay for megakaryocyte colony stimulating activity (Meg‐CSA) in plasma samples of patients with perturbed megakaryocytopoiesis. Preparations of heparinized platelet‐poor plasma yielded the most consistent results. Platelet‐poor plasma of normal subjects will at best facilitate the occasional growth of small megakaryocyte colonies. Colony frequency and size are reproducibly enhanced in the presence of PHA‐LCM as a source of exogenous Meg‐CSA. Commercially available EPO preparations may vary in their content of activities that influence megakaryocyte colony formation. Addition of these preparations to cultures that contain plasma and PHA‐LCM usually does not enhance colony formation. In contrast to platelet‐poor plasma, platelet rich plasma and serum are less supportive of megakaryocyte colony growth. It is suggested that this loss of activity may be related to the release of inhibitors by activated platelets or alternatively caused by absorption of activities by platelets. Plasma samples from patients with megakaryocytopoietic dysfunction may contain components that promote colony formation without addition of PHA‐LCM or EPO. This phenomenon is consistently observed for patients with severe aplastic anemia and bone marrow transplant recipients after completion of their ablative preparative regimen.
Epidermal growth factor promotes the chemotactic migration of cultured rat intestinal epithelial cells
Authors: Blay Jonathan , Brown Kenneth D.
Published: February 2005

The RIE‐1 cell line is an untransformed, epithelial cell line derived from the rat small intestine. We report that epidermal growth factor (EGF), which regulates the proliferation of RIE‐1 cells, also directs their movement. We measured cell migration through gelatin‐coated filters in blind‐well Boyden chambers. The migration of RIE‐1 cells was stimulated up to ˜ 100‐fold by EGF, with a half‐maximal response at 1–2 ng/ml and a maximal effect at 10 ng/ml. Further analysis showed that the RIE‐1 cells responded directionally to a gradient of EGF in solution. Other growth factors tested did not stimulate RIE‐1 cell migration, and EGF did not stimulate the migration of fibroblasts in this assay. We conclude that EGF is a potent and specific chemo‐attractant for RIE‐1 intestinal epithelial cells and suggest that EGF might influence epithelial cell migration in vivo.
Permissive role of interleukin 3 (IL‐3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture
Authors: Suda Toshio , Suda Junko , Ogawa Makio , Ihle James N.
Published: February 2005

We studied the effects of interleukin‐3 (IL‐3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5‐fluorouracil (FU)‐treated mice. Purified IL‐3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed‐mitogen spleen cell conditioned medium (PWM‐SCM), except that IL‐3 supported eosinophil and neutrophil expression better. Delayed addition of IL‐3 to cultures 7 days after cell plating decreased the number of colonies to one‐half the number in cultures with IL‐3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL‐3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL‐3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL‐3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL‐3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.
Induction of DNA polymerase α in senescent cultures of normal and Werner's syndrome cultured skin fibroblasts
Authors: Pendergrass W. R. , Saulewicz A. C. , Salk D. , Norwood T.
Published: February 2005

DNA polymerase α activity was determined following serum stimulation of early and late passages of human diploid fibroblast‐like (HDFL) cultures derived from apparently normal donors (two strains) and from a patient with Werner's syndrome (one strain). Induction of this enzyme was observed in both low passage, actively proliferating cultures and in postmitotic “senescent” cultures from all three strains. The maximal polymerase activity of early and late passage cells of each strain were nearly identical when normalized to the number of cells present. However, the activity of the enzyme was observed to be significantly lower in late passage cultures when normalized to total protein content apparently because of enlargement of the senescent cells. The behavior of Werner derived cells was similar to that of the normal cells. The induction of DNA polymerase α in senescent cultures indicates that they retain the capacity to carry out some complex metabolic responses to mitogen stimulation. In addition, these results suggest the possibility that dilution of DNA polymerase α and/or other DNA replication factors may play a role in the onset or maintenance of the postmitotic state in the enlarged senescent HDFL cells.
The possible cyclic AMP‐dependence of an early prereplicative event that determines mitosis in regenerating rat liver
Authors: Rixon R. H. , Whitfield J. F.
Published: February 2005

The β‐adrenergic blocker dl‐propranolol prevented a large proportion of regenerating rat liver cells from entering the mitotic phase of their first cell divisions cycle without affecting their ability to initiate or complete DNA replication. The drug, at a dose of 20 or 50 mg/kg of body weight, was most effective in reducing mitosis when injected between 1 and 2 hours after the proliferatively activating partial hepatectomy, which was 22 to 23 hours before the peak of DNA‐synthetic activity. Propranolol also inhibited the early prereplicative surge of total liver cyclic AMP, which occurs shortly after partial hepatectomy, but this effect was not correlated to the mitosis‐inhibiting activity. However, cyclic AMP or dibutyryl cyclic AMP completely reversed propranolol's mitosis‐inhibiting action when injected between 1.5 and 2 hours (but not sooner or later) after partial hepatectomy, which was just before the total liver cyclic AMP content began to rise. Thus, there appears to be a transient, propranolol‐inhibitable, probably cyclic AMP‐initiated event in the early prereplicative development of rat hepatocytes that determines entry into mitosis rather than the initiation of DNA replication.
The mechanism of iron uptake by the rat placenta
Authors: McArdle Harry J. , Douglas Andrea J. , Bowen Barbara J. , Morgan Evan H.
Published: February 2005

The mechanism of iron uptake from transferrin by the rat placenta in culture has been studied. Transferrin endocytosis preceded iron accumulation by the cells. Both transferrin internalisation and iron uptake were inhibited by low temperature. Transferrin endocytosis was less susceptible to the effects of metabolic inhibitors such as sodium fluoroacetate, potassium cyanide, 2,4, dinitrophenol or carbonylcyanide M‐chlorophenyl hydrazone (CCCP) than was iron uptake. Iron accumulation was decreased if the cells were incubated in the presence of weak bases such as chloroquine or ammonium chloride. These results suggest that, following internalisation, the vesicles containing the transferrin and iron became acidified, and that this acidification was a necessary prerequisite for the accumulation of iron by the cell. Further, the results indicate that the intravesicular pH was maintained at the expense of metabolic energy, suggesting that a pump may be involved. The importance of the permeability properties of the vesicle membrane in the iron uptake process was investigated by incubating the cells with labelled transferrin and iron in the presence of different cation and anion ionophores. Irrespective of the normal cation that the ionophores carried, all inhibited iron uptake without altering transferrin levels. In contrast, phloridzin, a Cl‐ transport inhibitor, did not affect either the levels of transferrin within the cells or the amount of iron accumulated.
Adenosine receptor‐mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells
Authors: Jonzon Bror , Fredholm Bertil B. , Nilsson Jan
Published: February 2005

The effects of adenosine and two analogs, L‐phenylisopropyladenosine (L‐PIA) and 5′‐N‐ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet‐derived growth factor (PDGF)‐stimulated initiation of DNA synthesis in growth‐arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose‐dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA > adenosine > L‐PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8‐phenyltheophylline (8‐PT). The stimulatory effect of adenosine was enhanced by 3 μM dipyridamole an adenosine‐uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8‐PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L‐PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin‐stimulated increase in cAMP was inhibited by L‐PIA at nanomolr concentrations. L‐PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1‐ and A2‐subtype on SM‐mediating bidirectional changes of cAMP and DNA synthesis.
Partial purification of a low‐molecular‐weight growth factor from chicken brain
Authors: Carlone Robert L. , Rathbone Michel P.
Published: February 2005

The regenerating amphibian limb serves as a useful model for studying factors influencing cell proliferation and differentiation. In particular, peripheral nerves are thought to provide a stimulus for growth of the blastema, presumably via the elaboration of an as yet unidentified neurotrophic factor. In the present study, pressure ultrafiltration coupled with chromatofocusing have proven to be effective methods of partially purifying a neurotrophic factor from adult chicken brains. This chick brain growth factor (CBGF) appears to be a heat‐stable, basic peptide of low molecular weight (<6,000). It is a potent mitogen in vitro, at nanomolar concentrations, for both blastema cells and Swiss mouse 3T3 fibroblasts. CBGF is apparently distinct from other peptide mitogens and/or neuromodulators that have been reported to stimulate blastema growth in vivo and in vitro. These include substance P, FGF from bovine brain and pituitary, EGF, transferrin (sciatin), and spinal cord growth factor (SCGF). The possible relationship of CBGF to other neural regulatory molecules is discussed.
125I‐EGF binding to responsive and nonresponsive cells in culture: Loss of cell‐associated radioactivity relates to growth induction
Authors: Schaudies R. Paul , Harper Robert A. , Savage C. Richard
Published: February 2005

A comparison of the binding and overall processing of epidermal growth factor (EGF) was made in ten mammalian fibroblast‐like and three epithelial‐like cell lines. EGF stimulated the growth of five fibroblastic cell lines (132 to 224%) after 10 days in the constant presence of EGF and were termed “responsive”. Eight of the lines did not respond or were growth inhibited by EGF (−64 to + 21%) and were listed as “nonresponsive”. Both “responsive” and “nonresponsive” cell lines possessed specific saturable membrane receptors for EGF, but no consistent differences were found between the number of apparent receptors per cell or the concentrations of 125I‐EGF required for half maximal binding. However, a consistent difference between the “responsive” and “nonresponsive” cell lines was observed when the amount of cell associated 125I‐labeled EGF was measured as function of incubation time at 37°C in the constant presence of the hormone. In every cell line we classified as responsive, the binding of 125I‐EGF reached a maximum after 30 minutes incubation at 37°C and the cell associated radiolabel subsequently decreased by 42‐68% within 4 hours. In contrast, the “nonresponsive” cell lines required 1‐2 h to reach maximal binding and showed a minimal decrease of 1–10% during the 4‐h period. These data indicate that a variety of different cell lines can possess receptors with similar binding properties but process EGF in a dissimilar manner. This difference in processing may reflect the specific events which are necessary for the induction of cell growth.
Growth factor production during multistage transformation of epithelium in vitro. I. Partial purification and characterisation of the factor(s) from a fully transformed epithelial cell line
Authors: Wigley Caroline B. , Trejdosiewicz Ludwik K. , Southgate Jennifer , Coventry Ruth , Ozanne Brad
Published: February 2005

Transforming growth factor (TGF)‐like activity is characterised from one of a series of salivary epithelial cell lines, CSG 211, chemically transformed in vitro. In this transformation system, we can demonstrate multiple stages in the acquisition of a malignant phenotype by normal diploid ductal epithelial cells from male mouse submandibular gland. The fully transformed, tumorigenic cell TGF‐like activity in serum‐free supernatants resembles no other well‐characterised growth factor and has an apparent molecular weight (Mr) of 14 kd. There is also evidence of a higher Mr activity, which is separable by anion exchange chromatography. We show that the premalignant, nontumorigenic progenitor cells of this line do not produce demonstrable TGF‐like activity and that this property is therefore acquired as CSG 211 cells become carcinoma producing.
Expression of tracheal differentiated functions in serum‐free hormone‐supplemented medium
Authors: Wu Reen , Nolan E. , Turner C.
Published: February 2005

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum‐free hormone‐supplemented medium differentiate to cilia‐beating and mucus‐secreting cell types. The medium is Ham's F‐12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2–5 × 104 cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin‐like glycoprotein in the HTE cultures. The levels of mucin‐like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum‐free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.
Human spreading factor: Synthesis and response by HepG2 hepatoma cells in culture
Authors: Barnes David W. , Reing Janet
Published: February 2005

Human serum spreading factor (SF) is a cell adhesion and spreading‐promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 μg/ml in 48 hr‐conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically‐labeled with leucine, fucose or glucosamine identified a single from of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin‐treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.
Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells
Authors: Morita Akihito , Kitagawa Yasuo , Sugimoto Etsuro
Published: February 2005

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35Smethionine and radioactive proteins in the medium were analyzed by SDS‐polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.
Glucocorticoid receptors and cell cycle progression in human melanoma cell lines
Authors: Osman A.‐M. , Jansen P. W. M. , Smets L. A. , Benckhuijsen C.
Published: February 2005

Proliferation of six established human melanoma cell lines was inhibited after treatment for 1 h with a high dose of glucocorticoid. Four of the lines with the capacity of colony formation were used to quantify final plating efficiency. Specific glucocorticoid binding sites in these cell lines ranged from 51,000 to 170,000 sites per cell as measured with a whole‐cell assay. Growth inhibition was completely reversible in one cell line, irreversible in another, and partially reversible in two lines. Receptor content er cell correlated with the reduction in final plating efficiency of glucocorticoid‐treated cells, suggesting a receptor‐mediated event. A more than 90% growth inhibition and a 40% reduction in cell survival in the most sensitive cell line, M‐5A, was accompanied by a dual blockage in G1 and G2/M phase that lasted till at least 96 h after treatment with 2.5 μM dexamethasone for 1 h. Evidence is presented of a real arrest of M‐5A cells in G1 phase and a markedly retarded progression through G2; the blockage of G1‐S transition was immediate and complete. Accumulation of G1 cells was observed in two other cell lines but was inconsistent in the fourth line studied by fiow cytometry; in none of the three cell lines was G2/M accumulation observed. Stimulated melanogenesis after glucocorticoid treatment of M‐5A and NKI‐26 cells suggested differentiation of the cells during glucocorticoid‐induced arrest.
Neurite formation by neuroblastoma‐glioma hybrid cells (NG108–15) in defined medium: Stochastic initiation with persistence of differentiated functions
Authors: Krystosek Alphonse
Published: February 2005

Neurite formation and proliferation by NG108‐15 cells were studied in shortterm serum‐free N2 medium. Neuritogenesis by individual cells was observed at widely differing times, suggesting a stochastic component to initiation of differentiation. Cells with and without neurites could also enter one or more rounds of proliferation at varying times. The initial choice between these divergent behaviors influenced subsequent growth. Cells initially extending neurites had a high probability of continuing neuritic elongation. Cells initially dividing had a high probability of yielding further progeny. Addition of dibutyryl cyclic AMP to cells cultured in N2 medium rapidly increased the probability of differentiating and decreased the probability of proliferating. To test whether or not cells with highly differentiated morphologies had irreversibly lost the capacity for proliferation, induced cultures were washed and challenged by the addition of serum‐containing medium. The length of time required for individual cells to divide increased with increasing time of preincubation in the induction medium. However, few cells appeared to be permanently removed from the proliferative pool. These observations suggest that differentiating cells exhibit persistence, a tendency to continue on the differentiation pathway. Persistence is extinguished following one round of proliferation in serum‐contianing medium.
Identification of the cellular mechanisms responsible for platelet‐derived growth factor induced alterations in cytoplasmic vinculin distribution
Authors: Herman B. , Harrington M. A. , Olashaw N. E. , Pledger W. J.
Published: February 2005

Exposure of quiescent density arrested BALB/c‐3T3 cells (clone A31) to platelet‐derived growth factor (PDGF;6–12 ng/ml) results in a rapid, reversible, time‐and dose‐dependent removal of vinculin from adhesion plaques (Herman and Pledger, 1985). Potential cellular mechanisms involved in PDGF‐induced removal of vinculin from adhesion plaques were examined. Removal of vinculin from adhesion plaques following exposure of cells to PDGF was temperature dependent, occurred in many fibroblast cell lines, and could be mimicked by 12‐tetradecanoyl phorbol‐13‐acetate (TPA; 5‐125 nM) or melittin (0.35 μM). Unlike the effect of PDGF, TPA‐or melittin‐induced vinculin disruption was not reversible. The removal of vinculin from adhesion plaques was inhibited by trifluoroperazine (TFP; 2.5 μM). 8‐(N,N‐diethylamino) octyl‐3,4,5‐trimethoxy benzoate (TMB‐8; 1.0 μM), mepacrine (220 μM), n‐α‐p‐tosyl‐L‐lysine chloromethylketone (TLCK; 100 μM), phenylmethoxysulphonylfluoride (PMSF; 500 μM), and e‐aminocaproic acid (e‐ACA; 100 μM); however, amiloride (100 μM), A23187 (20 μM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine > TMB‐8 > TFP > leupeptin > PMSF, whereas A23187 and amiloride had no effect. The return of vinculin to adhesion plaques following PDGF treatment required de novo mRNA transcription and protein synthesis and was associated with PDGF‐stimulated synthesis of vinculin.

The observation that both PDGF‐ and melittin‐induced removal of vinculin from adhesion plaques is inhibited by mepacrine suggests that phospholipase activation may be an early and important step in PDGF‐induced disruption of vinculin from adhesion plaques. In addition, TFP, TMB‐8 and protease inhibitor inhibition of both the PDGF and melittin effects on vinculin distribution, coupled with the finding that TPA can mimic the PDGF or melittin response, suggests that Ca2+, calmodulin, protein kinase C, and /or proteolysis may play an important role(s) in the removal of vinculin from adhesion plaques following PDGF addition. The lack of effect of A23187 addition on vinculin distribution suggests that alterations in cellular Ca2+ is necessary but not sufficient for vinculin removal from adhesion plaques.