bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of cyclic adenosine 3′:5′‐monophosphate phosphodiesterases: Altered pattern in transformed myoblasts
Authors: Seth Prem K. , Rogers Jackilynn , Narindrasorasak Suree , Sanwal Bishnu D.
Published: February 2005

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10–16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus‐transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an “acquisition” of some new property by transformed cells.
Regulation of self‐renewal of human T lymphocyte colony‐forming units (TL‐CFUs)
Authors: Wu A. M.
Published: February 2005

Formation of human T lymphocyte colonies in semisolid medium from T lymphocyte colony‐forming units (TL‐CFUs) under stimulation of phytohemagglutinin (PHA) has been reported by several authors. These TL‐CFUs were present in unsensitized lymphocyte populations. We report here that such TL‐CFUs are capable of renewing themselves. This was observed when colony cells from primary T cell colonies that developed in the presence of PHA were replated in methylcellulose medium containing irradiated autologous leukocytes and PHA. We have also been able to demonstrate serial transfer of TL‐CFU for up to six passages. At each passage, colony‐forming frequency was determined from the proportional relationship between the number of new colonies obtained and the number of colony cells plated. Examination of the number of new colonies derived from each individual T cell colony (“burst size of TL‐CFU”) showed that most colonies contained very few new TL‐CFU and only a very small number of colonies contained many new TL‐CFU. The distribution of burst sizes could be well fitted to a gamma distribution, in agreement with prediction from a stochastic model. We have identified an activity that enhanced the mean TL‐CFU burst size three to ten times. This work provides the first evidence in vitro that self‐renewal of human T lymphocyte progenitor cells can be stimulated by specific regulatory proteins.
Separate intracellular pathways for insulin receptor recycling and insulin degradation in isolated rat adipocytes
Authors: Marshall Stephen , Olefsky Jerrold M.
Published: February 2005

To gain insight into the sequence of events that follow endocytotic uptake of insulin receptor complexes, we have examined the interrelationship between the degradative pathway of the insulin ligand and the recycling pathway of the insulin receptor. Tris(hydroxymethyl)aminomethane and other nonamphoteric amines were found to selectively impair insulin receptor recycling while leaving the insulin‐degradative pathway intact. In contrast, low concentrations of the lysosomotropic agent chloroquine markedly inhibited intracellular insulin degradation but had little or no affect on the recycling of internalized receptors. Thus, we conclude: (1) that insulin dissociates from its receptor after endocytotic uptake and both receptor and ligand follow a separate intracellular pathway; and (2) that receptor recycling and insulin degradation can be selectively inhibited by Tris and chloroquine, respectively, highlighting the potential usefulness of these agents as intracellular probes in the study of receptor‐ligand metabolism.
Occurrence of a monocyte/macrophage colony‐stimulating factor in the continuous ambulatory peritoneal dialysis fluids and its chromatographic behaviors and antigenicity compared with human urinary colony‐stimulating factor
Authors: Sakai N. , Tsuneoka K. , Shikita M.
Published: February 2005

Continuous ambulatory peritoneal dialysis (CAPD) fluid from three patients with chronic renal failure exhibited the activity of colony‐stimulating factor (CSF) in amounts varying from 5 to 40 units per ml. Like the CSF obtained from normal human urine, the peritoneal CSF predominantly produced monocyte/macrophage colonies in soft‐agar culture of mouse bone marrow cells. Semipurified peritoneal CSF showed its isoelectric point at pH 3.6 and 4.9 before and after the treatment with neuraminidase. Under the same conditions, the urinary CSF was focused at pH 3.1 and 4.6. The position of elution of the peritoneal and urinary CSF in ordinary gel‐filtration chromatography corresponded to a molecular weight of 62,000 and 117,000, whereas both CSFs exhibited a molecular weight of 28,000 upon gel‐filtration in the presence of 6 M guanidine HCI. Furthermore, the two CSFs from the human sources were neutralized by antimouse L cell CSF serum in the same manner. We conclude that the peritoneal CSF is a sialoglycoprotein which is nearly identical with the urinary CSF despite processing of the latter through kidneys.
Delayed and reduced cell replication and diminishing levels of DNA polymerase‐α in regenerating liver of aging mice
Authors: Fry Michael , Silber John , Loeb Lawrence A. , Martin George M.
Published: February 2005

Kinetics of cell replication was compared in regenerating livers of Mus musculus at ages ranging between 6 and 32 months. Incorporation of [3H]‐thymidine into DNA and autoradiographic analysis showed that the maximal extent of DNA replication following partial hepatectomy became delayed with age. Furthermore, the total fraction of parenchymal and nonparenchymal cells in S phase at different intervals during regeneration diminished as mice aged. The specific activity of DNA polymerase‐α, the putative replicative enzyme, declined progressively during aging. The specific activity of DNA polymerase‐β, the purported repair enzyme, declined to an appreciably lesser extent during the lifespan of the mouse. No evidence was found for the appearance of a specific inhibitor of polymerase‐α in senescent mouse liver. Also, the bulk of the activities of both hepatic DNA polymerase‐α and ‐β remained localized in the cell nucleus throughout the lifetime of the animal and were mainly associated with chromatin.
Inhibition of vascular smooth muscle cell migration by heparin‐like glycosaminoglycans
Authors: Majack Richard A. , Clowes Alexander W.
Published: February 2005

Previous studies have suggested that heparin‐like glycosaminoglycans may be endogenous inhibitors of smooth muscle proliferation in the vessel wall. The purpose of this study was to determine the effects of exogenous glycosaminoglycans on rat vascular (aortic) smooth muscle cell migration following wounding in vitro. Our data indicate that heparin and related molecules (iota carrageenan, dextran sulfate), but not other glycosaminoglycans (hyaluronate, chondroitin, and dermatan sulfates), inhibit smooth muscle cell motility in a cell‐specific, dose‐dependent, and reversible fashion. The effect of heparin was maximal (60% inhibition) at 10 μg/ml; a half‐maximal effect was observed at 1 μg/ml; Heparin did not significantly affect the migration of bovine aortic endothelium or Swiss 3T3 cells. These observations support the concept that heparin‐like glycosaminoglycans may be important regulators of vascular smooth muscle cell function.
Isolation of plasma membrane, Golgi apparatus, and endoplasmic reticulum fractions from single homogenates of mouse liver
Authors: Croze Edward M. , Morré D. James
Published: February 2005

Procedures to isolate plasma membrane, Golgi apparatus, and endoplasmic reticulum from a single homogenate of mouse liver are described. Fractions contain low levels of contaminating membranes as determined from morphometry and analyses of marker enzymes. The method requires only 2–3 gm of liver as starting material and yields approximately 0.7, 0.7, and 0.5 mg protein/gm liver, respectively, for endoplasmic reticulum, Golgi apparatus, and plasma membrane. Golgi apparatus fractions show high levels of galactosyltransferase activity and consist of cisternal stacks and associated secretory vesicles and tubules. Endoplasmic reticulum fractions are enriched in both glucose‐6‐phosphatase and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)‐cytochrome c reductase and contain membrane vesicles with attached ribosomes. K+‐stimulated p‐nitrophenyl phosphatase and (Na+ K+) adenosine triphosphatase activity are enriched in the plasma membrane fraction. This fraction consists of membrane sheets, many with junctional complexes, and bile canaliculi that are representative of the total hepatocyte plasma membrane. The fractionation procedure is designed to utilize small amounts of tissue (e.g., with liver slices), to reduce the total time required for fractionation, and to permit comparisons of constituents of plasma membrane, Golgi apparatus, and endoplasmic reticulum prepared from the same starting homogenates.
Density‐dependent inhibition of growth: Inhibitorydiffusible factors from 3T3‐ and rous sarcoma virus (RSV)‐transformed 3T3 cells
Authors: Harel Louise , Chatelain Gilles , Golde Alice
Published: February 2005

We recently fractionated, from the culture medium of 3T3 cells, a thermolabile inhibitory diffusible factor (IDFN) with a molecular weight of about 40,000 daltons, which decreased nucleic acids synthesis of stimulated target 3T3 cells. In the present publication the inhibitory activities of IDFN (produced by 3T3 cells) and IDFT (produced by RSV‐transformed 3T3 [3T3 SRA/H] cells) on 3T3 and 3T3 SRA/H cells have been compared. The inhibitory activity of IDFN decreased (by a mean of 57%) when it was tested on transformed instead of 3T3 cells. On the other hand IDFT was able to decrease 14C‐inosine incorporation in target 3T3 cells. However, the inhibitory activity of IDFT decreased (by mean 50%) when tested on 3T3 SRA/H instead of 3T3 cells. Therefore, transformed cells produced an inhibitory factor but were less sensitive than 3T3 cells to its inhibitory activity. The inhibitory activity of IDFT on 3T3 SRA/H cells was only 20% of the inhibitory activity of IDFN on 3T3 cells. This appreciable difference is of particular interest, since it could explain the release of density‐dependent inhibition of growth (DDI) in transformed 3T3 SRA/H cells. Furthermore, it provides more evidence for the hypothesis that, in 3T3 cells, DDI of growth is due to the release of an inhibitory molecule into the medium, and that IDFN is in fact, the inhibitory molecule involved in this phenomenon.
The effects of serine protease inhibitors on morphological differentiation of murine neuroblastoma cells (NB 15)
Authors: Gibson William H. , Burack Shari L. , Picciano Anne
Published: February 2005

Morphological differentiation of neuroblastoma cells (NB15) was induced by cAMP effectors in the presence and absence of serine protease inhibitors. In all conditions tested, the percent differentiation was inhibited by protease inhibitors antipain, diisopropylfluorophosphate (DFP), leupeptin, and soybean trypsin inhibitor (SBTI). The level of morphological differentiation obtained in medium containing fetal calf serum was significantly less than the percent differentiation obtained with serum‐free medium alone, so serum‐free medium was the principal method of induction and comparisons were made to control uninduced cultures or cultures induced with the phospho‐diesterase inhibitor R020–1724. Secreted or cell surface caseinolytic protease activity was higher in differentiating cells than in control cultures and was inhibited by the serine protease inhibitors. The effects of the protease inhibitors on growth and differentiation are discussed.
Protein degradation in 3T3 cells and tumorigenic transformed 3T3 cells
Authors: Gronostajski Richard M. , Pardee Arthur B.
Published: February 2005

To study the relation of overall rates of protein degradation in the control of cell growth, we determined if transformation of fibroblasts to tumorigenicity affected their rates of degradation of short‐ and long‐lived proteins. Rates of protein degradation were measured in nontumorigenic mouse Balb/c 3T3 fibroblasts, and in tumorigenic 3T3 cells transformed by different agents. Growing 3T3 cells, and cells transformed with Moloney sarcoma virus (MA‐3T3) or Rous sarcoma virus (RS‐3T3), degraded short‐ and long‐lived proteins at similar rates. Simian virus 40 (SV‐3T3)‐ and benzo(a)pyrene (BP‐3T3)‐transformed cells had slightly lower rates of degradation of both short‐ and long‐lived proteins. Reducing the serum concentration in the culture medium from 10% to 0.5%, immediately caused about a twofold increase in the rate of degradation of long‐lived proteins in 3T3 cells. Transformed lines increased their rates of degradation of long‐lived proteins only by different amounts upon serum deprivation, but none of them to the same extent as did 3T3. Greater differences in the degradation rates of proteins were seen among the transformed cells than between 3T3 cells and some transformed cells. Thus, there was no consistent change in any rate of protein degradation in 3T3 cells due to transformation to tumorigenicity.
Effects of valinomycin on hexose transport and cellular ATP pools in mouse fibroblasts
Authors: Yamanishi Kiyofumi
Published: February 2005

The K+ ionophore valinomycin at concentrations of 1 × 10−8 M and over, stimulated 2‐deoxy‐D‐glucose (2DG) and 3‐O‐methylglucose (3OMG) uptake in Swiss 3T3 fibroblasts. The rate‐limiting step of 2DG uptake was transport rather than phosphorylation, in the control or valinomycin‐treated cells. Kinetic analysis showed that valinomycin increased the Vmax for 2DG uptake without change of the Km. The valinomycin‐stimulated 2DG uptake was insensitive to 10 μg/ml cycloheximide, and extracellular K+ concentrations between 0.1 and 50 mM. On the other hand, valinomycin at the concentration of 1 × 10−8 M and over, induced a rapid decrease in cellular ATP content, followed by stimulation of 2DG uptake and recovery of the ATP content. A similar relationship between the reduction of cellular ATP content and the subsequent stimulation of 2DG uptake was observed when the cells were treated not only with 2,4‐dinitrophenol and iodoacetic acid, but also with other monovalent cation ionophores or inhibitors of oxidative phosphorylation. These results suggest that valinomycin may posttranslationally stimulate hexose transport by increasing the number of functional carriers of hexose or changing their mobility, and the rapid decrease in cellular ATP pools by valinomycin may be a trigger of the stimulation of the hexose transport in Swiss 3T3 fibroblasts.
The purification of an acid‐ and heat‐labile transforming growth factor from an avian sarcoma virus‐ transformed rat cell line
Authors: Yamaoka Kazuko , Hirai Reiko , Tsugita Akira , Mitsui Hiromi
Published: February 2005

A new class of transforming growth factor (TGF), with chemical characteristics differing from previously reported TGFs, was isolated and purified from an avian sarcoma virus‐transformed rat cell line, 77N1. Purification steps were simple and consisted of ion‐exchange chromatography on diethylaminoethyl (DEAE)‐Sephacel, ammonium sulfate precipitation, Chromatofocusing, and DEAE‐Sephadex A‐25 chromatography. The purified TGF is a heat‐ and acidlabile protein with a molecular mass of 12,000 daltons and isoelectric point of 5.2–5.4. Because of the acid lability of this TGF, purification was carried out at neutral pH. The TGF induced DNA synthesis in growth‐arrested BALB 3T3 cells and promoted anchorage‐independent growth of nontransformed BALB 3T3 cells in soft agar; the latter activity is specific for the peptide growth factors, called TGFs, but it did not compete with epidermal growth factor (EGF) for binding to the EGF membrane receptors. The TGF activity was not potentiated by EGF. The purified preparation of the TGF stimulated BALB 3T3 cells to grow progressively in soft agar at a dose of 20 ng/ml.
Cyclic AMP‐dependent and ‐independent protein kinases and protein phosphorylation in human promyelocytic leukemia (HL60) cells induced to differentiate by retinoic acid
Authors: Fontana Joseph A. , Emler Carol , Ku Katherine , McClung J. Keith , Butcher Fred R. , Durham John P.
Published: February 2005

The human leukemia cell line HL60 which resembles promyelocytes can be induced to differentiate to cells displaying features of the mature myeloid phenotype by a variety of agents including retinoic acid (RA) and agents that elevate intracellular adenosine 3:5 cyclic monophosphate (cyclic AMP) levels, e.g., 8‐bromo‐cyclic adenosine 3:5 monophosphate (8‐Br‐cyclic AMP), cholera toxin. Since most, if not all the effects of cyclic AMP, are mediated by adenosine 3:5 cyclic monophosphate‐dependent protein kinase (cyclic AMP‐dPK), we investigated the role of cyclic AMP‐dPK and adenosine 3:5 cyclic monophosphate‐independent protein kinase (cyclic AMP‐iPK) in the induced differentiation of HL60 cells. Marked stimulation of cyclic AMP‐dPK and cyclic AMP‐iPK appears to be intimately involved with and specific for HL60 myeloid differentiation as evidenced by: (1) Stimulation of cyclic AMP‐dPK and cyclic AMP‐iPK early during HL60 myeloid differentiation and prior to phenotypic changes. (2) RA and dimethylformamide (DMF), agents that induce differentiation along the myeloid pathway, cause a marked increase in the type I cytosolic cyclic AMP‐dPK and cyclic AMP‐iPK (protamine kinase) while no such increases are noted in cells treated with 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) which induces differentiation along the monocyte/macrophage pathway. (3) Both native polyacrylamide gel electrophoresis as well as photoaffinity labeling with 8‐azido‐cyclic AMP demonstrate marked increases in type I cyclic AMP‐dPK in the cytosols of cells exposed to agents that induce myeloid differentiation but no increase in TPA‐differentiated cells. (4) The appearance and disappearance of specific cyclic AMP‐dependent and ‐independent protein phosphorylations are associated with the induced myeloid differentiated state.
Hydrocortisone stimulates fibronectin synthesis in cultured fibroblasts
Authors: Lien Yeong‐Hau , Wong Mitchell J. , Golbus Mitchell S. , Stern Robert
Published: February 2005

The effect of hydrocortisone on fibronectin synthesis was investigated in cultured skin fibroblasis. Confluent cells were treated with hydrocortisone (10−7 M to 10−5 M) for 2 days and labeled with [3H]proline for 24 h. Fibronectin levels in both the culture medium and the cell layer were studied by gelatin‐Sepharose affinity chromatography and SDS‐polyacrylamide gel electrophoresis. In control cultures of human fetal skin fibroblasts, fibronectin constituted 8% of the total labeled proteins in the medium. The proportion of fibronectin increased to 13.1% at 10−7 M hydrocortisone, 15.5% at 10−6 M and to 19.4% at 10−5 M. The proportion of fibronectin associated with the cell layer remained at 2‐3% of total [3H]prolne‐labeled proteins and did not increase with hydrocortisone exposure. The stimulating effect of hydrocortisone on medium fibronectin was also demonstrated in cultured human newborn foreskin fibroblasts and in rabbit skin fibroblasts.
Adenosine inhibits cell division and promotes neurite extension in PC12 cells
Authors: Huffaker Tom , Corcoran Teresa , Wagner John A.
Published: February 2005

Low concentrations (10–50 μM) of adenosine (EC50 = 17 μM) or chloroadenosine (EC50 = 23 μM) prevent the division of PC12 cells. This inhibition is not mimicked by guanosine, inosine, 3′,5′ dideoxyadenosine, phenylisopropylad‐enosine, or adenylylimidodiphosphate. The growth inhibition is not relieved by addition of uridine or deoxycytidine, nor is it potentiated by homocysteine thiolactone. Inhibition of adenosine uptake does not inhibit adenosine‐de‐pendent growth arrest. PC12 variants that are deficient in adenosine kinase are as sensitive as wild‐type cells to the growth‐inhibitory effects of adenosine. These experiments suggest that adenosine prevents cell division at an adenosine receptor rather than acting after being metabolically altered. The adenosine receptor that inhibits cell division does not appear to be the adenosine receptor that stimulates adenylate cyclase for these reasons: (1) phenylisopropyladenosine, which is a potent agonist of this receptor, does not inhibit cell division; (2) 3′,5′ dideoxyadenosine does not antagonize the effect of adenosine on cell division; and (3) theophylline does not affect growth inhibition by adenosine. Thus, these experiments suggest the existence of a second adenosine receptor that can inhibit cell division.

Adenosine also promotes the morphological differentiation of PC12 cells. In the presence of the adenosine deaminase inhibitor, erythro‐9‐(2‐hydroxy‐3‐nonyl)adenosine (EHNA), adencsine causes the formation of short neurites (one‐half to one and one‐half cell diameters in length). Adenosine also increases the rate of neurite formation of both long and short neurites in response to NGF.
Intravesicular pH and iron uptake by immature erythroid cells
Authors: Paterson S. , Armstrong N. J. , Iacopetta B. J. , McArdle H. J. , Morgan E. H.
Published: February 2005

The intravesicular pH of intact rabbit reticulocytes was measured by two methods; one based on the intracellular:extracellular distribution of DMO (5, 5, dimethyl + oxazolidin‐2,4‐dione), methylamine, and chloroquine and the other by quantitative fluorescence microscopy of cell‐bound transferrin. The latter method was also applied to nucleated erythroid cells from the fetal rat liver. A pH value of approximately 5.4 was obtained with both methods and in both types of cells. Treatment of the cells with lysosomotrophic agents, metabolic inhibitors, and ionophores elevated the intravesicular pH and inhibited iron uptake from transferrin. When varying concentrations of NH4Cl were used, a close correlation was observed between the inhibition of iron uptake and elevation of the intravesicular pH. At pH 5.4 iron release from rabbit iron‐bicarbonate transferrin in vitro was much more rapid than from iron‐oxalate transferrin. The bicarbonate complex donates its iron to rabbit reticulocytes approximately twice as quickly as the oxalate complex. It is concluded that the acidic conditions within the vesicles provide the mechanism for iron release from the transferrin molecule after its endocytosis and that the low vesicular pH is dependent on cellular metabolism.
The relationship between purines, pyrimidines, nucleosides, and glutamine for fibroblast cell proliferation
Authors: Engström Wilhelm , Zetterberg Anders
Published: February 2005

Previous studies indicate that glutamine is a critical requirement for cell proliferation in vitro. We recently showed that depletion of glutamine from the cuture medium supporting growing cells significantly reduced the proportion of cells undergoing DNA synthesis. Similarly glutamine depletion significantly reduced the stimulatory response of quiescent cells to 10% serum. This study shows that the inhibitory effects of depletion of glutamine—in either of these two situations—can be overcome by the addition of adenine or adenosine. Adenine was the only nitrogen base and adenosine was the only nucleoside for which this effect was observed. Such effects could, however, also be achieved by addition of the purine metabolites hypoxantine and inosine. Furthermore, it was found that glutamine (or adenine/adenosine) is only required during a limited interval coinciding with the late part of the G1‐phase and the beginning of S‐phase. These data suggest the possibility that glutamine exerts its main regulatory effects on cell proliferation by acting as a precursor for adenine and adenosine.
Initiation of DNA synthesis by human thrombin: Relationships between receptor binding, enzymic activity, and stimulation of 86Rb + influx
Authors: Stiernberg Janet , Carney Darrell H. , Fenton John W. , Lablle Edward F.
Published: February 2005

Stimulation of amiloride‐sensitive sodium (Na+) influx and the subsequent activation of NA+, K+‐ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin‐initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human α‐thrombin was converted to γ‐thrombin, nitro‐α‐thrombin, and diisopropylphospho (DIP)‐α‐thrombin. These derivatives retain either the capacity to bind cell surface α‐thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of α‐thrombin or the various thrombin derivatives, only α‐thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP‐α‐thrombin that saturate the α‐thrombin recptors (up to 2 μg/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP‐α‐thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either γ‐thrombin or nitro‐α‐thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.
Structural determinants of the capacity of heparin to inhibit the proliferation of vascular smooth muscle cells
Authors: Castellot John J. , Karnovsky Morris J. , Beeler David L. , Rosenberg Robert D.
Published: February 2005

Previous work from our laboratory has demonstrated that both anticoagulant and nonanticoagulant heparin species can inhibit the proliferation of vascular smooth muscle cells in vivo and in vitro. In this communication, we report studies on the structure‐function relationships of heparin to its antiproliferative effect on vascular smooth muscle cells. These structure‐function studies were carried out by preparing discrete sizes of heparin fragments and by chemically modifying heparin. The compounds were tested for their ability to inhibit rat and calf aortic smooth muscle cell growth. The minimum fragment size which retains some growth inhibitory activity is a hexasaccharide; maximal antiproliferative activity was obtained with dodecasaccharide and larger fragments. Both O‐sulfation and N‐substitution were found to be important for the growth inhibitory effect. Comparison of the antiproliferative and anticoagulant activities of the different heparin species has allowed us to identify several heparin molecules which have lost their anticoagulant properties, but retain antiproliferative activity.
An antibody that inhibits fibronectin‐independent adhesion of fibroblasts to extracellular matrix material
Authors: Kelleher P. J. , Juliano R. L.
Published: February 2005

Chinese hamster ovary (CHO) fibroblasts adhere to the extracellular matrix by both fibronectin‐dependent and ‐independent mechanisms (Harper and Juliano, 1981a,b). Previous studies have suggested that a trypsin‐sensitive, 265,000‐dalton membrane glycoprotein (gp265) is involved in the fibronectin‐independent adhesion process. Using a polyclonal antibody against soluble products obtained from trypsin‐treated CHO cells, we have been able to further analyze this involvement. This antibody immunoprecipitates a trypsin‐sensitive 265,000‐dalton protein from detergent‐solubilized cells. Incubation of AdvF11, a variant cell line that does not utilize fibronectin for adhesion, with this antibody blocks their adhesion to extracellular matrix material (ECM). The immunoglobulin fraction will also partially block adhesion of the parental cell line to ECM particularly when the ECM is first treated with an antifibronectin antibody. Taken together these results add support for the involvement of gp265 in fibronectin‐independent adhesion and provide a methodology for further characterization.
Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts
Authors: Tramacere Mariarosaria , Petronini Pier Giorgio , Borghetti Angelo F.
Published: February 2005

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L‐phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity‐treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L‐Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.
Temperature‐dependent transmembrane potential changes in cells infected with a temperature‐sensitive moloney sarcoma virus
Authors: Lai C. N. , Gallick G. E. , Arlinghaus R. B. , Becker F. F.
Published: February 2005

Normal rat kidney cells (NRK) infected with the temperature‐sensitive (ts) transformation mutant of Moloney murine sarcoma virus yielded a clone of cells, 6m2, that exhibited a transformed morphology at 33°C and a normal morphology at 39°C. Transmembrane potential (Em) was measured fluorometrically using a cyanine dye diS‐C3‐(5). Fluorescence was inversely correlated with Em. Cells at 33°C had lower Em. Em changes were recorded within 15 minutes of temperature shift from 33°C to 39°C in both directions, increasing in the 33°C to 39°C direction and decreasing in the 39°C to 33°C direction. Uninfected NRK cells when shifted under the same condition exhibited small fluorescence changes in the 33°C to 39°C direction. Shifting from 39°C to 33°C resulted in Em changes similar to those in 6m2 cells. Also studied was a cell line infected with a spontaneous revertant of the ts mutant, designated 54‐5A4; it was transformed at both temperatures. Shifting from 33°C to 39°C in both directions yielded small changes. Transmembrane potential changes in 6m2 cells precede other transformation‐specific changes that occur after a temperature shift.
The role of increased proteolysis in the atrophy and arrest of proliferation in serum‐deprived fibroblasts
Authors: Gronostajski Richard M. , Goldberg Alfred L. , Pardee Arthur B.
Published: February 2005

When cultured fibroblasts are deprived of serum, the degradation of long‐lived proteins and RNA increases, the cells stop proliferating, and they decrease in size. To determine the role of the increased protein catabolism in these responses, we studied the effects of inhibitors of intralysosomal proteolysis in Balb/c 3T3 cells. When these cells were placed in serum‐deficient medium (0.5% serum), the rate of degradation of long‐lived proteins increased about twofold within 30 min. This increase was reduced by 50‐70% with inhibitors of lysosomal thiol proteases (Ep475 and leupeptin) or agents that raise intralysosomal pH (chloroquine and NH4Cl). By contrast, these compounds had little or no effect on protein degradation in cells growing in 10% serum. Thus, in accord with prior studies, lysosomes appear to be the site of the increased proteolysis after serum deprivation. When 3T3 cells were deprived of serum for 24‐48 hours, the rate of protein synthesis and the content of protein and RNA and cell volume decreased two‐ to fourfold. The protease inhibitor, Ep475, reduced this decrease in the rate of protein synthesis and the loss of cell protein and RNA. Cells deprived of serum and treated with Ep475 for 24‐48 hours had about twice the rate of protein synthesis and two‐to fourfold higher levels of protein and RNA than control cells deprived of serum. The Ep475‐treated cells were also about 30% larger than the untreated cells. Thus, the protease‐inhibitor prevented much of the atrophy induced by serum deprivation. The serum‐deprived fibroblasts also stopped proliferating and accumulated in the G1 phase of the cell cycle. The cells treated with Ep475 accumulated in G1 in a manner identical to untreated serum‐deprived cells. Other agents which inhibited protein breakdown in serum‐deprived cells also did not prevent the arrest of cell proliferation. Thus the enhancement of proteolysis during serum deprivation appears necessary for the decrease in size and protein synthesis, but probably not for the cessation of cell proliferation. When cells deprived of serum in the presence or absence of Ep475 were stimulated to proliferate by the readdition of serum, the larger Ep475‐treated cells began DNA synthesis 1–2 hours later than the smaller untreated cells. Thus, after treatment with Ep475, the rate of cell cycle transit following serum stimulation was not proportional to the cell's size, protein, or RNA content, or rate of protein synthesis.
Apparent heterogeneity in the response of quiescent swiss 3T3 cells to serum growth factors: Implications for the transition probability model and parallels with “cellular senescence” and “competence”
Authors: Brooks Robert F. , Richmond Frances N. , Riddle Peter N. , Richmond K. M. Veronica
Published: February 2005

When subconfluent, Swiss 3T3 cells made quiescent by serum deprivation are stimulated with low concentrations of serum (ca. 1%), only a proportion of them (roughly 50%) enter S phase despite daily replacement with fresh, low‐serum medium. The cells that fail to enter S phase are not incapable of doing so, since most of them initiate DNA synthesis after transfer to 10% serum. It would appear that individual cells vary in their growth factor requirements. Using time‐lapse cinemicroscopy a few of the cells that respond to low serum were seen to give rise to several generations of progeny, while the majority of cells failed to divide at all, or divided once at most. Despite this, differences between cells in growth factor requirements do not seem to be heritable in the long term, since attempts to enrich for responding cells by prolonged culture in 1% serum have been unsuccessful. Rather, it would appear that the capacity to respond to low serum is an unstable property lost after a few generations in low serum. The loss of responsiveness shows parallels with “cellular senescence” and could conceivably result from decay of the platelet‐derived growth factor‐induced state of “competence.” But regardless of why some cells respond to low serum while others do not, it is clear that the kinetics of entry into S phase after serum stimulation of quiescent 3T3 cells are not strictly first‐order, since the labelling index plateaus after roughly 3 days at values substantially below 100%. As such, the kinetics, though not contradicting the transition probability model, cannot be taken to support it as was previously thought.
Cell cycle regulation by environmental pH
Authors: Taylor Ian W. , Hodson Pamela J.
Published: February 2005

Purified populations of quiescent human tumour cells were isolated from plateau phase cultures of PMC‐22 cells by centrifugal elutriation. Dilution into fresh medium resulted in these quiescent cells entering S phase exponentially with a t1/2 of 12 hr, after a 18‐20‐hr lag period during which cellular RNA content increased. Subsequent studies showed that recruitment of quiescent cells into the cell cycle could be regulated by extracellular pH. When exponentially growing PMC‐22 cells were exposed to acidic extracellular pH levels, three growth patterns were observed: (1) Normal growth between pH 7.2 to pH 6.8; (2) A reduction in growth rate associated with accumulation of cells with a G1 DNA content between pH 6.7 and 6.4 (this was also shown to occur in a number of other tumour cell lines); (3) Non‐cell‐cycle‐phase‐specific arrest of growth at pH levels less than 6.3. Further studies with purified quiescent cell populations showed the possible existence of a pH‐dependent restriction point in the G1 phase of these tumour cells. The implications of these observations to tumour biology are discussed.
Phorbol esters inhibit the binding of low‐density lipoproteins (LDL) to U‐937 monocytelike cells
Authors: Rouis M. , Goldstein S. , Thomopoulos P. , Berthelier M. , Hervy C. , Testa U.
Published: February 2005

The present study demonstrates that U‐937 monocytelike human cells possess specific LDL receptors. 125I‐LDL binds at 4°C on the cell surface. The bound molecules are releasable by heparin. The reaction requires Ca2+ and the binding sites are sensitive to proteolysis. Unlabeled LDL compete with 125I‐LDL, whereas HDL are ineffective. At 37°C, LDL are internalized and degraded by a chloroquine‐sensitive pathway. Tumor‐promoting phorbol esters inhibit the binding of 125I‐LDL to its receptor on U‐937 cells. This inhibition exhibits temperature, time, and concentration dependence. At 37°C, inhibition is 50% at 5 × 10−9 M of TPA. After removal of phorbol esters, treated cells recover their 125I‐LDL‐binding activity in 60 min. The inhibitory activities of various phorbol esters are proportional to their tumor‐promoting activities. Inhibition appears to be due to a reduction in the number of available LDL receptors rather than a decrease in receptor affinity.
Correlation between trypsin binding to a specific receptor and prostacyclin production in cultured vascular endothelial cells
Authors: Savion Naphtali , Naveh‐Floman Nava
Published: February 2005

The correlation between the binding and processing of trypsin and its effect on prostacyclin (PGI2) production in cultured adult bovine aortic endothelial (ABAE) cells was studied. ABAE cells demonstrated an ability to produce PGI2 in a dose‐response manner to trypsin at the range of 0.1–2.0 μg/ml with a saturation at a concentration of 1 μg/ml; Likewise, 125I‐trypsin binding to the cultured cells increased in a dose‐response way and reached saturation at a concentration of about 1 μg/ml; 125I‐trypsin was bound to a specific high‐affinity cell‐surface receptor with a dissociation constant (Kd) of 1.5 × 10−8 M and each of the confluent ABAE cells has about 1.2 × 105 such receptors sites. The cell‐surface receptor for trypsin displayed specific characteristics and an excess amount of unlabeled trypsin successfully abolished 125I‐trypsin binding while thrombin in excess failed to compete for 125I‐trypsin binding. Only a small fraction of the cell‐surface‐bound 125I‐trypsin was internalized and subsequently degraded by ABAE cells as compared to the process of 125I‐trypsin internalization by human skin fibroblasts (HSF). This study demonstrated that the stimulatory effect of trypsin on prostacyclin production and release by ABAE cells might be mediated by a specific cell‐surface receptor for trypsin on these cells distinct from the thrombin receptor.
Production and characterization of interferon from endothelial cells
Authors: Einhorn Stefan , Vlodavsky Israel , Fuks Zvi , Eldor Amiram , Panet Amos
Published: February 2005

The capacity of cultured bovine aortic, capillary, and corneal endothelial cells as well as of human umbilical cord endothelial cells to produce interferon (IFN) was investigated. The endothelial cells of the two species produced significant amounts of IFN in response to various viruses and poly (I) poly (C). The IFN produced by human umbilical cord endothelial cells was a mixture of α‐ and β‐IFN, as determined by neutralization with antibodies directed against these two types of IFNs as well as by measuring the antiviral activity on heterologous cells. Bovine endothelial cells also produced a mixture of at least two IFN subspecies, one of them labile at pH 2 and active on human cells and the other stable at pH 2 and inactive on human cells.
Induction of stress proteins in chicken embryo cells by low‐level zinc contamination in amino acid‐free media
Authors: Whelan Sandra A. , Hightower Lawrence E.
Published: February 2005

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat‐shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid‐free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half‐maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.
Induction of endoreduplication in temperature‐sensitive mutants of rat 3Y1 fibroblasts
Authors: Yamada Koji , Kimura Genki
Published: February 2005

Four temperature‐sensitive mutants of rat 3Y1 fibroblasts belonging to separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly with a 2C DNA content, when cells proliferating at 33.8°C are shifted up to 39.8°C (Ohno et al., 1984). Zaitsu and Kimura (submitted for publication) showed that 3Y1tsF121 cells synchronized in the early S phase were arrested with a 4C DNA content at 39.8°C. We studied the traverse through the S and G2 phases at 39.8°C in the four ts mutants synchronized at the early S phase and found that 3Y1tsG125 and 3Y1tsH203 cells were arrested with a 4C DNA content as 3Y1tsF121, while 3Y1tsD123 cells went through S and G2 phases and underwent mitosis. When 3Y1tsF121 and 3Y1tsG125 mutants arrested at 39.8°C were shifted down to 33.8°C, a substantial fraction of the cells with a 4C DNA content started, with a certain lag period, DNA synthesis without intervening mitosis and underwent the first mitosis with a lag period similar to that in the cells arrested with a 2C DNA content. The tetraploid cells thus generated had a proliferating ability lower than that of diploid cells.
Lineage specific receptors used to identify a growth factor for developmentally early hemopoietic cells: Assay of hemopoietin‐2
Authors: Bartelmez Stephen H. , Sacca Rosalba , Stanley E. Richard
Published: February 2005

A new approach, based on the occurrence of receptors for the mononuclear phagocyte lineage specific hemopoietic growth factor (HGF) colony stimulating ractor‐1 (CSF‐1) on developmentally early multipotent cells, is utilized to detect and assay rapidly another HGF, hemopoietin‐2. This method is also used to determine the relative maturity of hemopoietin‐2 target cells, to investigate synergism between hemopoietin‐2 and CSF‐1, and to measure CSF‐1 receptor levels on maturing cells. While the target cell specificities of hemopoitin‐2 and CSF‐1 overlap, hemopoietin‐2 causes the appearance of developmentally earlier 125I‐CSF‐1 binding cells de novo in the absence of CSF‐1. Increased CSF‐1 receptor densities are observed on cells incubated with either HGF, consistent with acquisition of the capacity for increased expression of the receptor by mononuclear phagocyte progenitor cells just prior to their differentiation to adherent mononuclear phagocytes. Together, both HGFs have a synergistic effect on the generation of 125I‐CSF‐1 binding cells with elevated CSF‐1 receptor densities. Preliminary characterization of hemopoietin‐2 from medium conditioned by WEHI‐3 cells indicates that it is very similar to, if not identical with, interleukin‐3 (IL‐3) and the HGF(s) acting on multipotential cells and cells giving rise to erythroid cells, granulocytes, mononuclear phagocytes, and megakaryocytes. Purified IL‐3 was shown to possess hemopoietin‐2 activity.
FACS analysis of a hyperthermia‐induced alteration in Hoechst 33342 permeability and direct measurement of its relationship to cell survival
Authors: Rice Glenn C. , Gray Joe W. , Dewey William C.
Published: February 2005

Heat‐induced alterations in CHO‐10B cell Hoechst 33342 (Ho342) permeability in vitro were analyzed by flow cytometry. Immediately after 45.5°C heating, uptake was decreased in a dose‐dependent manner with cytotoxicity. Kinetic analysis indicated that a treatment that reduced cell survival to approximately 10%, reduced the maximal velocity, Vmax, to 53% of control and increased the dissociation constant, Km, to 156% of control. Also, little change in Ho342 efflux was found to occur from control up to 90 min after heating. Upon incubation at 37°C after the heat treatment from 1 to 24 hr (depending on the severity of the dose) diffuse heterogeneity of Ho342 staining developed which was not evident immediately after heating. The altered staining was not due to the presence of trypan blue staining cells. Membrane permeabilization and nuclei isolation studies indicated that the lesion responsible was most likely a plasma membrane event. Induction of the heterogenous staining was not inhibited by either actinomycin D or hydroxyurea but was inhibited by incubation at 4°C. An inverse correlation existed between Ho342 permeability and clonogenicity, with nearly a 10‐fold difference in survival between the high and low fluorescence intensity sorted cells. Also, larger fractions of heatsensitive S and G2M‐phase cells were found in the highly fluorescent sorted fractions. These results are discussed in terms of the putative molecular events that may be involved in hyperthermic modulation of Ho342 permeability.
Uptake of transferrin and iron by cultured rat placental cells
Authors: McArdle H. J. , Douglas A. J. , Morgan E. H.
Published: February 2005

This paper describes a method for the culture of rat placental cells. The method involved separation of the basal layer from the labyrinth and sequential digestion of the cells. The cells were demonstrated not to be fibroblasts and are described in terms of their appearance under the light and electron microscopes. Transferrin and iron uptake by the cells was examined and compared with results achieved using other methods of study. The results showed that transferrin bound to receptors on the cell surface and that the transferrin, once bound, was taken into the cell. Only this internalized transferrin was capable of donating iron to the cells. The iron was accumulated within the cells and did not appear to be released to the incubation medium. The apparent dissociation constant (Ka) for transferrin was found to be 6.96 × 106 M−1, a value similar to that described by earlier workers. The placental cells had 3.4 × 1011 binding sites/μg DNA, equivalent to approximately 1 × 106 sites/cell. From these data, and from the rate of accumulation of iron by the cells, the receptor turnover time was estimated as being between 5 and 10 min.