bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibronectin inhibits morphological changes in cultures of vascular smooth muscle cells
Authors: Brennan Michael J. , Millis Albert J. T. , Fritz Katherine E.
Published: February 2005

In culture, vascular smooth muscle cells proliferate until they form a confluent sheet of cells. At that time the morphology of the culture becomes altered and the cells form multilayered regions that eventually develop into nodular aggregations. We now demonstrate that the transition from monolayer culture to nodular culture is influenced by the presence of components in conditioned media. The development of nodules is enhanced by conditioned medium made from nodular cultures but is either inhibited or unaffected by monolayer culture‐conditioned medium. Examination of the two types of conditioned media using NaDodSO4‐polyacrylamide gels reveals many similarities and one major difference. Nodular‐conditioned medium contains a prominent 42 kilodalton polypeptide which is not present in monolayer‐conditioned medium. Further, we demonstrate that although both nodular and monolayer cultures produce fibronectin the transition to nodular culture does not occur in the presence of exogeneously added plasma fibronectin.
Thrombin‐mediated mitogenesis: The role of secreted protease nexin
Authors: Baker Joffre B. , Low David A. , Eaton Dan L. , Cunningham Dennis D.
Published: February 2005

Protease nexin (PN) is a cell‐secreted protein that links to thrombin (Th) and certain other serine proteases. PN mediates the binding, internalization, and degradation of these proteases by cells (Baker et al., 1980; Low et al., 1981). Here we show that binding of Th‐PN complexes to human foreskin fibroblasts (HF cells) accounted for 90% of the specific cellular Th binding at certain mitogenic doses of the protease. However, cell‐associated Th‐PN complexes were likely to be inactive mitogenically because heparin (170 units/ml) inhibited cellular binding of 125‐Th‐PN by about 95% (a reduction from 1.3 × 105 to 6 × 103 125I‐Th‐PN complexes per cell) but did not influence Th‐mediated mitogenic stimulation. In experiments with mouse embryo cells, heparin also markedly decreased cellular binding of 125I‐Th‐PN without changing the mitogenic response to Th. The lack of mitogenic activity of cell‐associated Th‐PN complexes suggested that PN might inhibit the mitogenically essential proteolytic activity of Th. This possibility is supported by the following findings. First, amounts of serum‐free conditioned culture medium that contained enough PN to complex a large fraction of added Th inhibited the clotting activity of Th. Second, heparin increased the formation of 125I‐Th‐PN complexes and also increased this inhibitory effect of conditioned medium. We conclude that PN acts as a negative modulator of thrombin mitogenic activity.

It is shown that like other fibroblastic cells HF cells bound free 125I‐Th specifically (although with relatively low affinity, Kass < 108 M‐1). Specific binding of free 125I‐Th to HF cells increased fourfold in the presence of heparin (50 IU/ml).
Pig reticulocytes. IV. In vitro maturation of naturally occurring reticulocytes with permeability loss to glucose
Authors: Zeidler Robert B. , Kim Hyun Dju
Published: February 2005

Naturally occurring reticulocytes of week old piglets were used to characterize the maturation process under in vitro conditions. When the reticulocytes were suspended in tissue culture medium fortified with metabolic substrates, nearly all cells were viable after 24 hours incubation and usually more than 85% of the initial cell population survived after an 80 hour period. In cells maintained as long as a week in incubation, an adequate level of total adenine nucleotide with a large accumulation of IMP was found. In most cases, reticulocytes lose their reticular materials within two days and assume normal erythrocyte configuration. Concomitant with the morphological change, the cell volume decreases toward normal erythrocyte size, the extent of which can be accounted for by the intracellular loss of salt and accompanying water. As in the in vivo reticulocyte maturation process, reticulocytes undergoing in vitro maturation lose their membrane permeability to glucose. These findings suggest that the process of reticulocyte maturation occurring in cell culture approaches that which naturally occurs in vivo. Thus, these cells may be used to delineate the mechanism of the loss of membrane transport of glucose which normally occurs in the adult pig cells.
Electrophysiological and pharmacological properties of cultured rat thyroid cells
Authors: Sinback Christopher Nelson , Coon Hayden G.
Published: February 2005

The electrophysiological properties of a hormone‐dependent, differentiated thyroid epithelial cell strain were studied using intracellular microelectrodes. The average membrane potential of solitary, isolated cells was –78.4 ± 1.3 mV. The membrane potential depolarized 55 mV per tenfold increase in extracellular potassium concentation. Weak electrical coupling was recorded between contiguous cells. Like tyroid cells in vivo, these cells did not generate action potentials. In some cells a spontaneous, slow transition in the membrane potential from –80mV to –30 mV was accompanied by an increase in input resistance. Membrane potential transitions could be induced by perfusing cells with isotonic Hanks solutions saturated with CO2 (pH = 5.5) or by perfusing cells with hypotonic Hanks solutions (190–290 mOsm/kg). Membrane potential transitions were due to a decreased potassium permeability. Noradrenaline elicted both a fast depolarization and a slow depolarization. The fast depolarization was due to an increase in conductance of Na+ channels and of Cl– channels. Intracellular injection of Ca++ elicited the fast depolarization. Intracellular injection of EGTA or cobalt abolished the fast depolarization. Replacemnt of extracellular Ca++ by Mg++ did not affect the fast depolarization. Thus, the fast depolarization was due to accumulation of intracellular Ca++. The fast depolarization was abolished by the alpha adrenergic blocker phentolamine (10–6 M), and was not abolished by the beta adrenergic blocker propranolol (10–5 M).
Murine cell surface transferrin receptor: Studies with an anti‐receptor monoclonal antibody
Authors: Trowbridge Ian S. , Lesley Jayne , Schulte Roberta
Published: February 2005

A rat monoclonal antibody against the murine transferrin receptor has been identified. The receptor is a 95,000 molecular weight species that exists in the cell membrane as a disulphide‐bonded dimer. Whereas 29 of 29 murine hematopoietic tumor cell lines express detectable numbers of transferrin receptors, less than 1% of adult thymocytes or spleen cells and only 5% of bone marrow cells are positive. However, fetal liver and neonatal spleen contain substantial numbers of transferrin receptor‐positive cells. Induction of Friend cells in vitro with dimethyl‐sulphoxide leads to an overall increase in the expression of transferrin receptors on the cell surface. The anti‐transferin receptor antibody we have obtained partially blocks iron uptake from 59Fe‐transferrin by a variety of murine cell lines and inhibits the growth of a murine myeloma cell line in vitro.
Effects of retinoic acid on protein synthesis in cultured melanoma cells
Authors: Lotan Reuben , Fischer Itzhak , Meromsky Leonid , Moldave Kivie
Published: February 2005

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G1 phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell‐free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell‐free system prepared from retinoic acid‐treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome‐associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two‐dimensional electrophoresis anlaysis of L‐[35S]methionine‐labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid‐induced suppression of protein synthesis activity may be the cause for growth inhibition.
Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation
Authors: Klip Amira , Logan William J. , Gagalang Enriqueta
Published: February 2005

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with α‐aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid‐deprived cells and that into amino acid‐supplemented cells differed in several chemical properties: (1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. (2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N‐ethylmaleimide (1 mM) or by 50 μM p‐chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation‐induced stimulation of transport, bringing the rate of proline uptake to the “basal” uptake level observed in amino acid‐supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation‐deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4°C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis‐dependent and that the stimulated transport had chemical properties distinct from the “basal” transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the “basal” transport system.
Evidence that a critical threshold of DNA polymerase‐alpha activity may be required for the initiation of DNA synthesis in mammalian cell heterokaryons
Authors: Pendergrass William R. , Saulewicz Anthony C. , Burmer Glenna C. , Rabinovitch Peter S. , Norwood Thomas H. , Martin George M.
Published: February 2005

The specific activity of DNA polymerase (90% alpha) was determined in nine “neoplastoid” cell lines (Martin and Sprague, 1973) and in three different strains of HDF (human diploid fibroblast‐like cells), all examined in logarithmic phases of growth. This was compared to the ability of each cell type to “rescue” (reinitiate DNA synthesis in) senescent HDF cells subsequent to polyethylene glycolmediated cell fusions. A sharp “threshold” value of DNA polymerase activity was observed below which reinitiation of DNA synthesis in heterokaryons with senescent HDF does not occur. This threshold was especially obvious when the specific activity of DNA polymerase (p moles dTTP incorporated per mg protein or per cell) was divided by the percent of S‐phase cells present in each culture as determined by flow microfluorometry. Our results indicate that the specific activity of DNA polymerase‐alpha (or some other factor tightly coregulated with it) in “recessive” cell types (those unable to rescue senescent cells) is only about two times this theoretical “threshold” value, and that fusion of recessive cell types to senescent HDF cells reduces the specific activity in the heterokaryon to below this minimum, thus preventing the cells from entering S phase.
Cells transformed by rous sarcoma virus release transforming growth factors
Authors: Krycève‐Martinerie Cécile , Lawrence David A. , Crochet Janine , Jullien Pierre , Vigier Philippe
Published: February 2005

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage‐independent colony formation in gelified medium, at the restrictive temperature (41°5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.
Growth stimulation of Rous sarcoma virus‐transformed BHK cells by biotin and serum lipids
Authors: Cheng David K. S. , Moskowitz Merwin
Published: February 2005

Biotin or a serum lipid extract stimulated proliferation of G1 arrested Rous sarcoma virus‐transformed BHK cells in modified Eagle's MEM (BM). The cells could be maintained continuously in BM plus biotin (BMB), but not in BM plus serum lipid extract (BM · L). Avidin inhibited growth stimulation when added to BMB, but did not inhibit growth when added to BM · L.

14C‐acetate incorporation into total cellular lipids was stimulated in BMB, but not in BM. Thin‐layer chromatography of the labeled cellular lipid extract indicated that relatively large amounts of 14C‐acetate were incorporated into phosphatidylserine and little into the other major phospholipids. In the neutral lipids, the largest amount of incorporation was in cholesterol.

G1 arrested cells multiplied rapidly in BM supplemented with dialyzed serum (BM · DS), but they did not multiply in BM with delipidized serum (BM · DLS). The addition of biotin or serum lipid extract to BM · DLS stimulated growth. Growth stimulation in BM · DLS by biotin was inhibited by avidin, but avidin had no effect on growth stimulation by serum lipid extract. Biotin stimulated additional multiplication in BM · DS and avidin inhibited this additional growth stimulation.

These results suggest that growth stimulation requires lipids supplied by serum lipids or by de novo synthesis stimulated by biotin. In the absence of serum, the stimulation of the synthesis of growth factor(s) by biotin are also required for continuous multiplication.
Are factors originating from serum, plasma, or cultured cells involved in the growth‐promoting effect of the extracellular matrix produced by cultured bovine corneal endothelial cells?
Authors: Gospodarowicz D. , Gonzalez R. , Fujii D. K.
Published: February 2005

The possibilities that the growth‐promoting effect of the extracellular matrix (ECM) produced by cultured bovine corneal endothelial (BCE) cells could be due to: (1) adsorbed cellular factors released during the cell lysis process leading to the denudation of the ECM; (2) adsorbed serum or plasma factors: or (3) adsorbed exogenous growth factors have been examined. Exposure of confluent BCE cultures to 2 M urea in medium supplemented with 0.5% calf serum denudes the ECM without cell lysis. The ECM prepared by this procedure supports cell growth just as well as ECM prepared by denudation involving cell lysis. Thus, it is unlikely that the growth‐promoting properties of ECM are due to adsorbed cellular factors. When the ECM produced by BCE cells grown in defined medium supplemented with high‐density lipoprotein, transferrin, and insulin was compared to the ECMs produced by cells grown in the presence of serum‐ or plasma‐supplemented medium, all were found to be equally potent in stimulating cell growth. It is therefore unlikely that the growth‐promoting ability of the ECM is due to adsorbed plasma or serum components. When fibroblast growth factor (FGF)‐coated and ECM‐coated plastic dishes were submitted to a heat treatment (70°C, 30 min) which results in the inactivation of FGF, the growth‐supporting ability of FGF‐coated dishes was lost, while the comparable ability of ECM‐coated dishes was not affected significantly. This observation tends to demonstrate that the active factor present in the ECM is not FGF. Nor is it platelet derived growth factor (PDGF), since treatment known to destroy the activity of PDGF, such as exposure to dithiothreitol (0.1 M, 30 min, 22°C) or to β‐mercaptoethanol (10%) in the presence or absence of 6 M urea for 30 min at 227°C, does not affect the growth‐promoting activity of ECM. It is therefore unlikely that the growth‐promoting effect of ECM is due to cellular growth‐promoting agents or to plasma or serum factors adsorbed onto the ECM.
Reactivation of NAD(H) biosynthetic pathway by exogenous NAD+ in nil cells severely depleted of NAD(H)
Authors: Mandel Kenneth G. , Lively Mary K. , Lombardi Donald , Amos Harold
Published: February 2005

The culture of Nil hamster fibroblasts in MEM lacking nicotinamide (NAm− MEM) leads to: (1) the rapid loss of intracellular total nicotinamide adenine dinucleotide (NAD(H)) content in these cells from a level of 150–200 pmoles/105 cells to less than 20 pmoles/105 cells; (2) the cessation of cell division and inhibition of DNA synthesis; and (3) a reduction of glucose consumption and lactic acid production. In most situations, following nicotinamide starvation, the restoration of intracellular NAD(H) follows rapidly the readdition of NAD+ (oxidized), nicotinamide mononucleotide (NMN), nicotinamide, or nicotinic acid. Resumption of cell division occurs after only a lag of about 24 hours. Nil cells subcultured for three consecutive times in the absence of nicotinamide (3° NAm‐ cells) exhibit different behavior. These severely starved cells are incapable of quickly restoring their intracellular NAD(H) content to normal levels when provided with any pyridine ring compound except NAD+. One‐hour exposure of such cells to NAD+ allows utilization of nicotinamide to rapidly restore intracellular NAD(H). This short incubation with NAD+ does not result in any significant restoration of intracellular NAD(H) or lead to the accumulation of an intracellular pool of some precursor. This function of NAD+ as a stimulatory signal to the NAD(H)‐biosynthetic pathway in severely starved Nil cells is a previously unreported role of NAD+, and does not require protein synthesis.
Cell hybrids between SV40‐transformed macrophage cell lines and a Chinese hamster cell line: Growth responsiveness and induction of colony‐stimulating factor
Authors: Ohki Kazunori , Nagayama Ariki
Published: February 2005

Three cell lines from resident macrophages of BALB/c mice and four from activated macrophages of the same strain were isolated by infection with simian virus 40 (SV40). A majority of these cells showed dependency on L cell‐conditioned medium (LCM), which is necessary for proliferation of normal macrophages in vitro. Somatic cell hybridization was applied in the study of macrophage growth responsiveness. A macrophage cell line (BR 15) with strict dependency on LCM for growth was fused to a Chinese hamster cell line (hs222‐16); it was found that dependency on LCM was a dominant trait in the hybrids. Following fusion fo a macrophage cell line (BAM3) which grew without LCM to hs222‐16, a large number of colonies appeared in the selection medium containing LCM . Four hybrids not requiring LCM for growth were selected in an LCM‐free culture, and their hybrid properties were examined. Three out of the four hybrids secreted colony‐stimulating factor (CSF) constitutively, whereas the fourth secreted no CSF. The level of acid phosphatase activity in the hybrids was higher than in the parent cells. Two peaks of CSF activity were observed after gel filtration chromatography of conditioned medium: One was eluted at molecular weight of 36,000 and the other at 17,000.
Modulation of microsomal glucose‐6‐phosphate translocase activity by free fatty acids: Implications for lipid domain structure in microsomal membranes
Authors: Hill David J. , Dawidowicz Eliezar A. , Andrews Mark L. , Karnovsky Morris J.
Published: February 2005

Recent studies from this laboratory have demonstrated differential effects of cis‐unsaturated (type A) free fatty acids (FFA) and trans‐unsaturated or saturated (type B) FFA on protein‐mediated surface phenomena, namely, (1) capping on surface immunoglobulin in lymphocytes, (2) receptor‐mediated aggregation of platelets, and (3) adhesion of BHK cells. These results were explained in terms of the FFA‐perturbing specific lipid domains of the plasma membrane with subsequent modulation of the function of proteins occupying those domains. We wanted to determine if a differential effect of type A and type B FFA could be measured in an isolated membrane system, and chose to study the glucose‐6‐phosphate (Glc‐6‐P) translocase:hexose phosphate phosphohydrolase complex of rat liver endoplasmic reticulum. It was found that in intact microsomes, hydrolysis of Glc‐6‐P was inhibited by linoleic acid and linolenic acid. When the permeability barrier of the microsome was disrupted inhibition of hydrolysis was abolished. These results suggested that the Glc‐6‐P translocase was effected by the type A FFA. Importantly, palmitic acid, stearic acid, and elaidic acid had no significant effect on either translocation or hydrolysis of Glc‐6‐P. In addition, other microsomal enzymes, including the serine ethanolamine base exchange protein, diacylglycerol CDP choline phosphotransferase, diacylglycerol CDP ethanolamine phosphotransferase, NAD(P)H cytochrome C reductase, and NADH ferricyanide reductase were not significantly effected by the FFA used in these experiments. The FFA used, although bound to microsomes, were apparently not incorporated into phospholipids, or cyclooxygenated into prostaglandins during the time course of these experiments. Based on previous results showing that cis‐unsaturated FFA exert their greatest perturbing effects in gel‐like lipid, we postulate that the transport protein occupies such a gel‐like lipid domain in the endoplasmic reticulum bilayer.
Microtubule‐disrupting agents can independently affect the prereplicative period and the entry into S phase stimulated by prostaglandin F2α and fibroblastic growth factor
Authors: Otto Angela M. , De Asua Luis Jimenez
Published: February 2005

Fibroblastic growth factor (FGF) and prostaglandin F2α (PGF2α) are two different growth factors in Swiss 3T3 cells: each stimulates the initiation of DNA synthesis after a lag phase of 14–15 hr. Colchicine, colcemid, and nocodazole have no mitogenic effect on their own, but each has a synergistic effect on the rate of initiation of DNA synthesis stimulated by either FGF or PGF2α. Independent of the growth factor, the microtubule‐disrupting drug had to be added before 8 hr of the lag phase to enhance the stimulation. Colchicine could be removed at 5 hr of the lag phase with no impairment of the synergistic effect. However, both colcemid and nocodazole had to remain present up to 10 hr of the lag phase to achieve the full synergy. This result can be accounted for by the fact that colchicine, in contrast to colcemid and nocodazole, binds almost irreversibly to microtubules, and it strengthens the interpretation that the state of microtubular organization plays a role in regulating the rate of entry into S phase. Preincubating quiescent Swiss 3T3 cells with colchicine, colcemid, or nocodazole before stimulation by FGF or PGF2α shortened the lag phase by about 3 hr. When the microtubule‐disrupting drug was removed prior to stimulation, the lag phase was also shortened, but there was no enhancement of the rate of initiation of DNA synthesis, except upon preincubation with colchicine. This suggests that microtubule disruption can independently affect the length of the lag phase and the rate of initiation of DNA synthesis, and that these two phenomena can be uncoupled.
Hyperthermia‐induced increase in potassium transport in Chinese hamster cells
Authors: Stevenson Anita P. , Galey William R. , Tobey Robert A. , Stevenson Michael G. , Jett James H.
Published: February 2005

The effect of hyperthermia (42°C) on K+ transport has been examined by monitoring the uptake of 42KCl by Chinese hamster (line CHO) cells at both 37° and 42°C as well as 37°C following a 30‐min treatment at 42°C. Early studies showed that CHO cells exhibit two components of uptake at both 37° and 42°C. Consequently, it was necessary to determine rate equations describing a three‐compartment system (i.e., the bathing medium and two cell‐associated compartments) and apply them to this system. Using the equations describing the behavior of the tracer in each compartment as well as the general solutions given by Robertson et al (1957), a method was developed for obtaining the two components of influx. The two transport rates were determined from equations arising from mathematical analysis and requiring only measurable quantities for their solutions. The results of this study showed that (1) both components of influx at 42°C are increased over influx at 37°C; (2) the increase is significant (by an average 68% for the rapid component and 55% for the slow component); and (3) the increase is due entirely to an increase in active transport. The increased active transport is both immediate and quickly reversible when cells are returned to 37°C; resumption of cell division is delayed for several additional hours, indicating that changes in potassium transport are not directly responsible for the prolonged delay in cell division. Rather, the results suggest that increased activity of the membrane pump may be the initial event in a series of events resulting from heat treatment.
Induction of thermotolerance and enhanced heat shock protein synthesis in chinese hamster fibroblasts by sodium arsenite and by ethanol
Authors: Li Gloria C.
Published: February 2005

Synthesis of a family of proteins called “heat shock” proteins is enhanced in cells in response to a wide variety of environmental stresses. This suggests that these proteins may have functions essential to cell survival under stressful conditions. A causative relationship between heat shock protein synthesis and development of thermotolerance would imply that agents known to induce heat shock protein synthesis, such as sodium arsenite, also induce thermotolerance. Conversely, agents known to induce thermotolerance, such as ethanol, would also enhance heat shock protein synthesis. To test this hypothesis, I have examined the effect of sodium arsenite or ethanol treatment on protein synthesis and cell survival in Chinese hamster ovary HA‐1 cells. After either sodium arsenite or ethanol treatment, the synthesis of heat shock proteins was greatly enhanced over that of untreated cells. In parallel, cell survival was increased as much as 104‐fold when cells exposed to either agent were challenged by a subsequent heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis of 70,000 and 87,000 molecular weight proteins most closely mirrored the development of thermotolerance. The results, therefore, strongly reinforce the hypothesis that a causal relationship exists between the enhanced synthesis of heat shock protein and cell survival under specific stresses.
The nature of 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA)‐stimulated hemopoiesis, colony stimulating factor (CSF) requirement for colony formation, and the effect of TPA on [125I]CSF‐1 binding to macrophages
Authors: Guilbert L. J. , Nelson D. J. , Hamilton J. A. , Williams N.
Published: February 2005

The tumor‐promoting phorbol diester, 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) was found to act both independently of and synergistically with the mononuclear phagocyte specific colony stimulating factor (CSF‐1) to stimulate the formation of macrophage colonies in cultures of mouse bone marrow cells. In contrast, TPA did not synergize with other CSF subclasses that stimulate the formation of eosinophil, eosinophil‐neutrophil, neutrophil, neutrophil‐macrophage, and macrophage colonies, nor with either of the two factors required for megakaryocyte colony formation, megakaryocyte CSF, and megakaryocyte colony potentiator. In serum‐free mouse bone marrow cell cultures TPA retained the ability to independently stimulate macrophage colony formation. However, TPA‐stimulated colony formation was suboptimal and delayed in serum‐free cultures that could support optimal colony formation in the presence of CSF‐1. In addition, TPA did not directly compete with [125I]CSF‐1 at 4°C for its specific, high‐affinity receptor on mouse peritoneal exudate macrophages. However, a 2‐hour preincubation of the cells with TPA at 37° caused almost complete loss of the receptor. Thus, TPA is able to mimic CSF‐1 in its effects on CSF‐1 responsive cells in some aspects (the spectrum of target cells, the morphology of resulting colonies, and the ability to down‐regulate the CSF‐1 receptor) but it is not able to mimic CSF‐1 in other ways (TPA alone cannot stimulate the full CSF‐1 response, TPA does not stimulate the most primitive CSF‐1 responsive cells, and TPA does not bind to the CSF‐1 receptor).
The effects of thyroparathyroidectomy and 1,25 dihydroxyvitamin D3 on changes in the activities of some cytoplasmic and nuclear protein kinases during liver regeneration
Authors: Sikorska M. , Whitfield J. F. , Rixon R. H.
Published: February 2005

Partial hepatectomy (HPX), which proliferatively activates the remaining liver cells, triggered two transient prereplicative surges in the total activities of cytoplasmic types I and II cyclic AMP‐dependent protein kinase holoenzymes, and of nuclear catalytic subunits from cyclic AMP‐dependent protein kinases. It also induced a transient prereplicative increase in the activities of a nuclear Ca2+‐calmodulin‐stimulable, protamine‐phosphorylating protein kinase, and a nuclear poly(L‐lysine)‐phosphorylating, 105 kDa protein kinase. Thyroparathyroidectomy (TPTX) delayed and reduced the first surge and completely eliminated the second surge of both of the cytoplasmic cyclic AMP‐dependent protein kinases, reduced the rises in the activity of nuclear catalytic subunits, and completely eliminated the surge of the Ca2+‐calmodulin‐stimulable protein kinase, but did not affect the surge of the nuclear 105 kDa protein kinase. The impairment of the responses of the two cyclic AMP‐dependent protein kinases to HPX in TPTX rats was not accompanied by a rise in the level of heat‐stable inhibitor of cyclic AMP‐dependent protein kinase activity. One intraperitoneal injection of 1,25‐dihydroxyvitamin D3 into TPTX rats immediately after HPX completely restored the post‐HPX surges in the activity of type I cyclic AMP‐dependent protein kinase, but the hormone, even in high doses, had little or no effect on the type II isoenzyme or the nuclear Ca2+‐calmodulin‐stimulable, protamine‐phosphorylating enzyme.
Effects of Zn2+ ions on protein phosphorylation in epithelial cell membranes
Authors: Alitalo Kari , Keski‐Oja Jorma , Bornstein Paul
Published: February 2005

The regulation of protein phosphorylation by Zn2+ ions and by other divalent cations was studied in membrane vesicles from a normal mouse epithelial cell line, MMC‐E (Mus musculus castaneous). Four major phosphoacceptor polypeptides were found in these membranes. Micromolar concentrations of Zn2+ ions inhibited the phosphorylation of the epidermal growth factor (EGF) receptor and of threonine residues in a 47,000‐dalton polypeptide. In contrast, two polypeptides with molecular weights of 54,000 and 57,000 showed increased phosphorylation, mainly of serine residues, in the p. esence of Zn2+ ions. These results were not obtained using similar concentrations of other divalent cations and were apparently not due to an effect of Zn2+ ions on phosphoprotein phosphatases. Thus, the effects of Zn2+ ions on protein phosphorylation in membrane vesicles are complex and are not restricted to an inhibition of a single protein phosphatase or kinase.
The role of calmodulin in the proliferation of transformed and phenotypically normal tsASV‐infected rat cells
Authors: Durkin Jon P. , Whitfield James F. , MacManus John P.
Published: February 2005

NRK cells infected with a temperature‐sensitive, transformation‐defective mutant of avian sarcoma virus (ASV), tsLA23, behaved as if nontransformed at a nonpermissive 40°C and were rendered quiescent by serum deprivation. These serum‐deprived cells were stimulated to start entering S phase about 7 hours after serum addition at 40°C or about 9 hours after shifting the cultures to 36°C, a temperature allowing the production of active viral pp60src and expression of the transformed phenotype. The transit of both serum‐and temperature‐stimulated tsLA23‐NRK cells through later G1 was inhibited by the unrelated calmodulin antagonists W7 and R24571. The former drug was found to block the cells at a point in the cell cycle no more than 2 hours from the G1/S transition. The weaker calmodulin antagonist, W5, was less effective in impairing progression. Thus, calmodulin is likely required for the transit of both transformed and phenotypically normal tsLA23‐NRK cells through the later stages of their G1 phases. Cells neoplastically transformed by ASV contain more calmodulin than uninfected, non‐neoplastic cells. At the nonpermissive 40°C, the calmodulin content of the tsLA23‐NRK cells dropped to the non‐neoplastic level. When these phenotypically non‐transformed cells were enabled to reenter the cell cycle while still in low‐serum medium by a 40 to 36°C shift, they passed through the G1 and S phases and divided without a concomitant rise in the total calmodulin content. Thus, a calmodulin rise does not appear to be required for the expression of one characteristic of transformed cells, i.e., reduced requirement for exogenous growth factors.
An estradiol‐responsive mouse endometrial cell strain with inducible aryl hydrocarbon hydroxylase activity
Authors: Iannaccone Philip M. , Stols Lucy , Hollenberg Paul F. , Gurka David P.
Published: February 2005

A mouse endometrial cell population has been isolated by mild tryptic digestion of the uterine lining. The cells were morphologically similar to endometrial gland cells in the intact mouse endometrial gland. The endometrial cells had a modal chromosome number of 66. The cells were adherent to glass as well as plastic and contained numerous large refractile, osmophilic, non‐membrane‐limited granules which stain with periodic acid‐Schiff reagent but do not stain with oil red O, Sudan black, or Alcian blue. Cell growth was responsive to 17β‐estradiol; cell number increased 1.34‐fold in 4 days in the presence of 10−8 M estradiol. The cells are not tumorigenic. The cells showed induction of aryl hydrocarbon hydroxylase (AHH) activity when 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) was added to the growth media for 24 h. AHH activity and its induction were investigated with cells grown in the presence and absence 10−8 M estradiol. Cells grown in media containing estradiol exhibited a 6.2‐fold induction by TCDD; cells grown without estradiol gave an 8.4‐fold induction of AHH activity. AHH activity and its induction by TCDD were demonstrated in cells grown with fetal calf serum that had been pretreated with dextran‐coated charcoal to remove endogenous steroids. Benzanthracene failed to induce AHH activity significantly.
Fibroblast contraction of collagen lattices in vitro: Inhibition by chronic inflammatory cell mediators
Authors: Ehrlich H. Paul , Wyler David J.
Published: February 2005

Fibroblast‐populated collagen lattices (FPCL), prepared in petri dishes with serum‐containing culture medium and incubated at 37°C, undergo progressive and symmetric contraction (reduction in size) over a period of days. The in vitro contraction process requires viable cells with intact cytoskeletal elements, is associated with cell elongation, and is believed to represent a fibroblast function which also occurs in vivo during wound healing and tissue fibrosis. We report that soluble mediators elaborated by chronic inflammatory cells cultured in vitro, when added to FPCL, inhibit lattice contraction. Granulomas, isolated from the liver of Schistosoma mansoni‐infected mice, secrete a factor(s) with an estimated molecular weight between 13,700 and 43,000 daltons (gel filtration: Sephadex G‐200) and pl = 6 (preparative isoelectrofocusing in granular gel) which inhibits lattice contraction but is not toxic to fibroblasts. Supernatants (cell‐free conditioned culture medium) of cultured macrophages isolated from these granulomas also contain this activity. The contraction inhibitory activity present in granuloma culture supernatants is abrogated by the addition of indomethacin to the lattices, while the addition of prostaglandin E2 (PGE2) alone to lattices inhibits contraction. Furthermore, culture supernatants interfere with fibroblast elongation in lattices. We propose that the ability of fibroblasts to contract collagen lattices in vitro and a fibrotic mass in vivo may be regulated by soluble products of chronic inflammatory cells, including macrophages. This process may be mediated by fibroblast‐derived prostaglandins which alter cytoskeletal functions and has implications for understanding regulation of tissue fibrogenesis in a variety of diseases.
Cytotoxic effects of dexamethasone restricted to noncycling, early G1‐phase cells of L1210 leukemia
Authors: Smets Lou A. , Bout Bram , Brouwer Marjoleine , Tulp Ab
Published: February 2005

The cytostatic and cytolytic effects of dexamethasone were studied as functions of cell cycle position in mouse L1210 leukemia cells. To this end, the cells were separated according to size by sedimentation at unit gravity in a specially designed sedimentation chamber. The fractions were analyzed by radioautography and flow cytophotometry. The size‐distributions obtained by 1g sedimentation coincided with cell‐cycle age distribution. With increasing fraction number, samples highly enriched in G1, S, and G2/M cells, respectively were obtained: the smallest cells being in early G1 and the largest in mitosis. In the presence of dexamethasone (10−6‐10−5 M), growth slowed down after a few cell cycles and the cells accumulated in early G1 phase. Lytic cell kill by continued exposure to the drug was confined to the fractions containing the small, early G1‐phase cells. These fractions were also enriched in noncycling cells that were not labeled by prolonged exposure to 3H‐thymidine. After removal of dexamethasone, the cells in S and G2/M phase completed cell cycle traverse but were retarded again in the G1 and early S phase of the next division cycle. The data suggest a memory effect for previous drug exposure. It is concluded that the cytostatic and cytolytic effects of dexamethasone are separate, though not unrelated events. Cytolysis is confined to the noncycling cells that in untreated populations can exit from the dividing compartment during a transitional phase of about 60 minutes subsequent to mitotic division. The cytostatic effects potentiate cytolysis by accumulating the cells in the early G1 phase and thus increasing the probability of their transit to the G0 compartment, sensitive for drug‐mediated cytolysis.
Cell membrane potential changes follow epstein‐barr virus binding
Authors: Rosenthal Ken S. , Shapiro Howard M.
Published: February 2005

Upon binding to receptor‐bearing target cells, viruses cause cell membrane potential changes. Epstein‐Barr Virus causes a biphasic membrane potential change in receptor‐bearing B lymphocytes but not receptor‐negative T lymphocytes, as measured by flow cytometry of cyanine dye uptake. Membrane potential changes upon EBV binding to receptor‐bearing cells resemble electrical responses of other cells following ligand binding to transmembrane receptors.
Changes in the levels of viral and cellular gene‐transcripts in the cell cycle of SV40 transformed mouse cells
Authors: La Bella Franca , Brown Eric H. , Basilico Claudio
Published: February 2005

We have analyzed the regulation of transcription of integrated SV40 DNA and of five cellular genes during the cell cycle of two lines of SV40 transformed mouse 3T3 cells. These cells (ts SV3T3) are temperature sensitive for the expression of the transformed phenotype and at the nonpermissive temperature (39°C) become arrested in G1 at low serum concentrations. SV40 specific RNAs are not detected either in the nuclear or in the cytoplasmic poly(A+)RNA of quiescent cells, suggesting control at the level of transcription. After serum stimulation, however, viral transcription increases and reaches its maximum during S‐phase. The expression of a group of selected housekeeping genes has received parallel analysis to determine whether other cellular genes, beside the integrated SV40, are shut off in G1 arrested cells or are expressed in restricted periods of the cell cycle. We have found that, while the mRNAs for collagen, adenosinphosphoribo‐siltransferase (APRT) and the mouse major histocompatibility complex (H2) are present throughout the cell cycle, the genes coding for the multifunctional protein CAD and dehydrofolate reductase are cell‐cycle regulated.
Phosphatidylethanolamine synthesis in ethanolamine‐responsive and ‐nonresponsive cells in culture
Authors: Kano‐Sueoka T. , Errick J. E. , King D. , Walsh L. A.
Published: February 2005

Mammalian cells can be classified into two types based upon whether or not they show growth response to ethanolamine (Etn) in culture. The content of phosphatidylethanolamine (PE) in phospholipid and incorporation of radioactive Etn into the cells were examined in the Etn‐responsive and ‐nonrespponsive cells in order to elucidate the mechanisms of growth stimulation by Etn. In all Etn‐responsive cells tested, 5 μM Etn significantly altered the composition of cellular phospholipid compared to that grown without Etn, while Etn‐nonresponsive cells had a similar phospholipid composition whether the growth medium contained Etn or not. Using two rat mammary carcinoma cell lines, 64–24 (responsive type) and 22‐1 (nonresponsive type), further studies were carried out. In 64–24 cells there was a proportional increase in PE content as the dosage of Etn in the medium was increased. The increase in PE content leveled off at 10 μM. Further, the increase in PE content was correlated with increased rate of growth. In contrast, PE content or growth rate did not change at all in 22‐1 cells. In 64–24 cells radioactive Etn (0.1–50 μM) was incorporated four‐ to five‐fold more efficiently into phospholipid, and the aqueous pool of precursors of PE was ten times less as compared to 22‐1 cells, indicating that Etn‐responsive cells utilize Etn supplied in the medium to synthesize PE far more efficiently than Etn‐nonresponsive cells. De novo synthesis of PE must not be sufficient to support optimum growth in Etn‐responsive cells.
Transforming growth factors released from Kirsten sarcoma virus transformed cells do not compete for epidermal growth factor membrane receptors
Authors: Chua Chu Chang , Geiman Deborah , Ladda Roger L.
Published: February 2005

Kirsten murine sarcoma virus (KiMSV)‐transformed rat kidney cells (KNRK) release small polypeptides (M, 12,500–15,300) into the culture medium that are capable of stimulating normal rat kidney cells (NRK) to form colonies in soft agar. The transforming growth factors (TGFs) did not compete with epidermal growth factor (EGF) for its receptor and did not induce specific phosphorylation of EGF receptor on NRK cell membranes. These properties differ from the TGFs isolated by other investigators. Our data further establish the heterogeneity of the materials produced by transformed cells that induce transformation‐specific changes in normal cells.
Differential effect of αdifluoromethyl‐ornithine on the proliferation of balb 3T3 and chemically transformed 3T3 cells
Authors: Medrano Estela E. , Goldemberg Sara H. , Algranati I. D.
Published: February 2005

In order to study the effect of polyamine depletion on growth and proliferation of untransformed and chemically transformed cells, α‐difluoromethyl‐ornithine (DFMO) was added to cultures of 3T3 cells and their benzo[a]pyrene derivative BP‐3T3. Both types of cells stopped their proliferation after 72 hr of treatment with the inhibitor. When DFMO was removed and cells were cultivated afterwards in fresh medium without the drug, untransformed cells resumed growth after a lag period, whereas transformed cells were unable to proliferate unless exogenous polyamine was added. These alterations showed a strict correlation with intracellular polyamine pools, since after removal of DFMO from the culture medium, polyamine concentrations increased to almost normal values in 3T3 cells, but remained at low levels or decreased even more in the transformed cells BP‐3T3. The analysis by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of labeled proteins of both cell extracts has indicated that the described control of cell proliferation by intracellular polyamine levels might be related to the synthesis of at least two proteins with molecular weights of about 36,000 and 55,000 daltons.
Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease‐binding factor
Authors: Eaton Dan L. , Baker Joffre B.
Published: February 2005

Four criteria were used to examine serum‐free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS‐stable ∼77 K Da complexes between a medium component and [125l]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 μg/106 cells), rat embryo heart muscle cells (0.13 μg/106 cells), mouse myotubes (0.1 μg/106 cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV‐40)‐transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus‐transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells.

Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin‐binding factors. Lysates from all of these cell types contained a factor that formed ∼60–65 K Da sodium dodecyl sulfate (SDS)‐stable complexes with [125l] thrombin. This MW is significantly lower than that of [125l] thrombin‐PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI‐26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease‐binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.