bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissociation of plasminogen activator from the transformed phenotype in a 5‐Bromodeoxyuridine dependent mutant of syrian hamster melanoma cells
Authors: Rosenthal S. L. , Zucker D. , Davidson R. L.
Published: February 2005

The relationship between plasminogen activator levels and the expression of the transformed phenotype was studied in a 5‐bromodeoxyuridine (BrdUrd) dependent mutant of Syrian hamster melanoma cells. In terms of cell morphology and cellular interactions, the BrdUrd dependent cells resemble transformed cells when grown in the presence of BrdUrd but resemble untransformed cells when grown in the absence of BrdUrd. It was found that the BrdUrd dependent cells release significant levels of plasminogen activator only when cultured in the absence of BrdUrd. In the presence of BrdUrd, the release of plasminogen activator by the dependent cells is suppressed, and the decreased level of plasminogen activator released in the presence of BrdUrd seems to be due to decreased production of active enzyme. Growth tests revealed that the BrdUrd dependent cells, when attached to a substrate, required BrdUrd in order to attain high densities. Furthermore, the cells are able to grow well in soft agar only in the presence of BrdUrd. These results suggest that the production and release of high levels of plasminogen activator are not related (either as cause or effect) to the expression of the transformed phenotype in the BrdUrd dependent cells

The effect of dog serum (as a plasminogen source) on the BrdUrd dependent cells also was tested. It was found that cells cultured in medium containing dog serum exhibit a morphological alteration, but only in the absence of BrdUrd. The morphological response of the cells to dog serum resembles that previously observed with virus‐transformed cells. In the BrdUrd dependent cells, the morphological response to dog serum appears correlated with the release of plasminogen activator but separated from other transformed characteristics.
Microtubule inhibitors alter the secretion of β‐glucuronidase by human blood platelets: Involvement of microtubules in release reaction II
Authors: Kenney Dianne M. , Chao Francis C.
Published: February 2005

The effects of the antimicrotubular drugs colchicine and vinblastine on the blood platelet release reaction were studied by measuring release of 14C‐5‐hydroxytryptamine (14C‐5‐HT, release I) and β‐glucuronidase (release II) from gel‐filtered human platelets. β‐glucuronidase release induced by thrombin was significantly inhibited by colchicine (0.01‐1 mM) or vinblastine (0.05–0.1 mM). Release of 14C‐5‐HT, however, was unaffected at low concentrations of colchicine and only slightly inhibited at higher concentrations. Inhibition of β‐glucuronidase release depended on colchicine or vinblastine concentrations and decreased with longer time intervals (1′, 5′, 20′) after thrombin stimulation. Levels of the cytoplasmic enzyme, lactic acid dehydrogenase, in supernatants of colchicine treated platelets were not significantly different from controls. Colchicine also inhibited β‐glucuronidse release, but not 14C‐5‐HT release, induced by trypsin and sodium arachidonate. Binding of 14C‐colchicine by platelets was measured and it was found that platelet aggregation and release of 5‐HT induced by adenosine diphosphate, epinephrine and collagen proceeded without any alteration in colchicine binding. However, significant increases in the rate and degree of colchicine binding were observed when platelets were stimulated by thrombin, trypsin and arachidonic acid which induced aggregation, release of both 5‐HT and β‐glucuronidase. The results suggest that an alteration in platelet microtubules is correlated with the physiologic response resulting in release II and that the cellular mechanisms effecting release I and II by platelets differ qualitatively in that the microtubules may facilitate release II.
Neoplasms produced from C3H/10T 1/2 cells attached to plastic plates; saturation density, anchorage dependence and serum requirement of in vitro lines correlated with growth aggressiveness in vivo
Authors: Paranjpe Meera S. , del Ande Eaton Sol , Boone Charles W.
Published: February 2005

The C3H/10T 1/2 embryo cell line, which is nontumorigenic when inoculated subcutaneously in saline suspension, produces tumors when implanted subcutaneously attached to 1 × 5 × 10 mm plastic plates. Under these in vivo conditions there is direct selection for “spontaneous” transformants that have undergone the specific cellular alterations required for neoplastic behavior. This is in contrast to the conventional situation where transformants are obtained in vitro and are only secondarily tested in vivo for neoplastic behavior. Early passages of cell lines from four different C3H/10T 1/2 tumors explanted back in culture were quantitatively examined for tumorigenicity and for alteration in the properties of density inhibition, anchorage dependence, serum requirement, and plasminogen activator production. A fairly consistent quantitative relationship was found between the degree of growth aggressiveness in vivo and the degree of expression of these phenotypic markers of the transformed state in vitro during early passages of the cell lines after tumor explantation.
Spontaneous mutation rate to thioguanine resistance is decreased in polyploid hamster cells
Authors: Morrow John , Stocco Douglas , Barron Emily
Published: February 2005

The mutation rate to thioguanine resistance was 3.11 × 10−6 in a near diploid V79 hamster cell line and 7.58 × 10−8 in a near tetraploid derivative produced with colchicine. The specific activities of glucose‐6‐phosphate dehydrogenase and phosphoglycerate kinase of the tetraploid line were greater than that of the diploid which suggests that twice the number of active X chromosomes were present in the tetraploid. These results are compatible with the hypothesis that spontaneous variants resistant to thioguanine arise through mutation and chromosomal segregation, as has been suggested for induced mutations in tetraploid hamster cells.
Effect of phosphate esters, nucleotides and nucleosides on 5′‐nucleotidase of cultured mouse macrophages
Authors: Lazdins Janis , Karnovsky Manfred L.
Published: February 2005

Mouse peritoneal macrophages elicited by intraperitoneal injection of sodium caseinate exhibit low levels of ecto‐5′‐nucleotidase (E. C. 3.1.3.5) activity in contrast to macrophages obtained by peritoneal lavage. When elicited cells were cultured under standard conditions in the presence of serum, a 2.5‐fold increase in 5′‐nucleotidase activity was observed over a period of 48 hours. Addition of adenosine monophosphate to the culture medium led to an augmented (5‐fold) increase in the specific activity (per unit cell protein) as well as an absolute increase (per culture plate) of 5′‐nucleotidase. Other adenosine‐containing compounds also had stimulatory effects. The levels of this enzyme thus appear to be regulated by the extracellular levels of adenosine nucleotides. The product of the enzymatic reaction–adenosine–when added to the medium exhibited a toxic effect on these cells–as did adenosine monophosphate. However, the former substance did not augment the increase in enzyme activity during culture. The toxic effect could be suppressed when the cells were cultured in the presence of uridine 5′‐monophosphate. The latter substance also depressed the stimulation of enzyme activity due to AMP.
Peritoneal exudate cells. V. Influence of age, sex, strain and species on the induction and the growth of macrophage colony forming cells
Authors: Lin Hsiu‐San , Kuhn Charles , Stewart Carleton C.
Published: February 2005

We investigated the effects of age, sex and strain in the induction of peritoneal exudate colony‐forming cells (PE‐CFC) in mice. Sex and age (ranging from 3 weeks to 12 months) had no significant effect on the induction of PE‐CFC. However, we found significant difference between strains in terms of the number of nucleated cells and the proportion of PE‐CFC in exudate cells. When we investigated the mechanisms behind this strain difference, we found that it was due to neither the type of colony‐stimulating factor employed in culture, nor the type of stimulants used to induce the exudate, nor the difference in the kinetics of appearance of PE‐CFC in the peritoneal cavity.

We also studied the induction of PE‐CFC in rats and hamsters and the growth of these cells in vitro. Unlike mouse cells, PE‐CFC from rats and hamsters could also use media conditioned by cells from other species as a source of colony‐stimulating factor.
Binding and internalization of 125I thrombin in chick embryo fibroblasts: Possible role in mitogenesis
Authors: Martin Bernice M. , Quigley James P.
Published: February 2005

Human α thrombin acts as a mitogen for cultures of resting chick embryo fibroblasts (CEF) in serum free medium. The use of 125I‐labeled thrombin shows that thrombin specifically binds to CEF and that after a lag of approximately 30 to 60 minutes it can not be removed by subsequent exposure to trypsin. The entry of 125I thrombin into the trypsin‐insensitive domain is not inhibited to any great extent by excess unlabelled thrombin. The cell‐associated thrombin retains its native molecular weight and its catalytic activity toward synthetic amide substrates. It appears to be located in the crude nuclear fraction of homogenized CEF cells. The association of thrombin with CEF is specific, since the non‐mitogenic serine protease chymotrypsin is internalized to a much lesser extent than thrombin. The data are discussed in terms of a possible intracellular site for thrombin's mitogenic action.
Culture pH, CO2 tension, and cell division in Euglena gracilis Z
Authors: Jones C. R. , Cook J. R.
Published: February 2005

Growth characteristics of Euglena gracilis Z as functions of culture pH, CO2 tension, temperature, and lighting regime were investigated. The results are consistent with the possibility that cell division is preceded by a lowered intracellular pH. Also consistent with this possibility is the finding that division rhythmicity can be induced by periodic changes in CO2 tension. It is suggested that the rhythmicity is induced by changes in intracellular pH produced by carbonic acid.
Amphibian cells in culture. II. Isolation of drug‐resistant variants and an asparagine‐independent variant
Authors: Chinchar Giovina D. , Sinclair John H.
Published: February 2005

With L‐15 as the base medium, drug‐resistant variants were isolated from two amphibian tissue culture strains: the Xenopus laevis A8 diploid cell line and the ICR 2A cell line of Rana pipiens. Four different classes of variants were obtained: (1) A8 cells resistant to chloramphenicol, an inhibitor of mitochondrial protein synthesis; (2) A8 cells resistant to ouabain, an inhibitor of the Na+/K+‐activated ATPase of the plasma membrane; (3) ICR 2A cells resistant to low (20 μg/ml) and high (300 μg/ml) levels of bromodeoxyuridine (BUdR), a thymidine analog which interferes with the pyrimidine salvage pathway; and (4) ICR 2A cells resistant to 2,6‐diaminopurine (DAP), an adenine analog which interferes with the purine salvage pathway.

Unlike the other variants, isolation of BUdR resistant cells is a 2‐step process. Resistance to low levels of BUdR is phenotypically expressed by a reduction in thymidine transport activities while resistance to high levels of this compound is evidenced by greatly reduced levels of thymidine kinase activity.

DAP‐resistant cells, which are characterized by reduced levels of adenine phosphoribosyl transferase (APRT) activity, do not die in AAT (adenine, aminopterin, thymidine) selection medium. This suggests that these cells utilize adenine efficiently.

With MEM as the base medium, an asparagine independent clone was isolated from the ICR 2A cell line. When compared with the wild type, this variant exhibited a slightly reduced growth rate in the presence or absence of asparagine.
Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture
Authors: Loskutoff David J.
Published: February 2005

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus‐transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell‐mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid‐treated FBS, their normal counter‐parts did not. Apparently, acidification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.
Glucocorticoid induction of proline oxidase in LLC‐RK1 cells
Authors: Kowaloff Edward M. , Phang James M. , Granger Alnora S. , Downing Sylvia J.
Published: February 2005

Dexamethasone induced proline oxidase in cultured LLC‐RK1 cells, an epithelial cell line derived from rabbit kidney. The dexamethasone‐mediated increase in enzyme activity was concentration and time dependent. Although the effect could be dissociated from cell growth and cell density, it was dependent on protein and RNA synthesis. A comparison of the enzyme isolated from control and dexamethasone‐treated cells showed that the increased activity was not due to an alteration in the affinity of the enzyme for proline. These findings suggest that glucocorticoids induce the synthesis of proline oxidase in mammalian cells.
Amino acid transport in thymic‐ and spleen‐derived lymphocytes
Authors: Wise W. C.
Published: February 2005

We have examined the transport of amino acids by the sodium‐dependent “A” and “ASC” system in thymic‐ and splenic‐derived lymphocytes from the Long‐Evans rat. Lymphocytes derived from the thymus transport amino acids by both the “A” and “ASC” systems, whereas lymphocytes from the spleen transport amino acids by the “ASC” system only. Thymic lymphocytes are capable of establishing a steady state distribution ratio of 7.9 for 2‐aminoisobutyric acid, but splenic lymphocytes can attain only 3.5. The steady state distribution ratio of alanine was the same in both cell types. Sodium‐independent transport is also different in splenic and thymic lymphocytes. But both cells move amino acids by a Na+‐independent system for mediated exchange‐diffusion. The studies show that lymphocytes derived from the spleen and thymus transport amino acids differently, and that the “T” lymphocytes from the spleen have membrane transport systems different from “T” lymphocytes from the thymus.
Isolation of mutant mammalian cells altered in polyamine transport
Authors: Mandel Jean‐Louis , Flintoff Wayne F.
Published: February 2005

Chinese hamster ovary and rat myoblast cells resistant to the toxic action of methylglyoxal bis guanylhydrazone (MGBG), an antimitotic agent and inhibitor of polyamine synthesis, have been isolated by single step selection. Mutagenesis with ethyl methanesulfonate increases the recovery of the variants at least 30‐fold. Intracellular accumulation of MGBG is greatly reduced in resistant cells. This property is accompanied by a 99% decrease in the uptake of all three naturally occurring polyamines. Both the resistant phenotype and the defect in polyamine transport behave recessively in somatic cell hybrids.
Cell surface‐associated ribonuclease activities
Authors: Fuller Frederick J. , Marcus Philip I.
Published: February 2005

The ribonuclease (RNase) activity associated with the surface of cells (primarily the Vero line of Green monkey kidney) was assayed under conditions where all cells remained viable as members of colonies or monolayers. RNase activity associated with individual clones was revealed as clear zones of hydrolyzed (acid‐soluble) RNA against an opaque background of acidprecipitated RNA in an isotonic agarose‐yeast RNA overlay. High resolution assays for single‐ and double‐stranded RNase were achieved by measuring the loss in activity of infectious Sindbis virus RNA, and of the antiviral state induced by poly(rI). poly(rC), respectively.

Experiments using these assay procedures revealed that virtually all of the RNase activity associated with viable (intact) vertebrate cells in culture was contributed by the serum in the growth medium, and was superficially adsorbed to the cell surface, rendering it relatively easy to remove by extensive washing.

A confluent monolayer of 2 × 106 unwashed Vero cells contained the equivalent of 500 ng of pancreatic RNase, representing about 5% of the total activity initially present in the serum of the growth medium. In terms of serum nuclease activity, only 0.45 ng equivalents of pancreatic RNase in 500 μl were required to destroy 50% of the activity of an infectious Sindbis virus RNA preparation in 15 minutes at 37°C.

Cells grown in the absence of serum and cells washed about ten times had comparably low levels of RNase activity–about 100‐fold less than unwashed cells grown in the presence of 6% calf serum.

The isotonic yeast RNA:agarose overlay procedure may be useful to identify and isolate cell mutants with different endogenous levels, or binding capacities, of surface RNase activity.

Operationally, these studies describe the assay of cell surface‐associated RNase activity under conditions where all cells remain viable as members of individual colonies or monolayer populations. Specifically, the assay measures the solubilization of yeast RNA in an isotonic agarose mixture overlaying the cells. We describe its use to determine how much of the total RNase activity in viable cell monolayers or colonies is contributed by serum in the growth medium. In addition, we describe the results of two biological assays used as higher resolution probes for cell surface ribonuclease activity: (i) single‐strand (ss) RNA, in the form of infectious RNA from Sindbis virus, and (ii) double‐stranded(ds)‐RNA, in the form of poly(rI). poly(rC) as an inducer of an interferon‐mediated antiviral state.
Growth and electron microscopic studies on an experimentally established bacterial endosymbiosis in amoebae
Authors: Ahn T. I. , Jeon K. W.
Published: February 2005

A strain of nonsymbiotic A. proteus was infected with endosymbiotic bacteria isolated from another strain of amoeba which had become dependent on the symbionts after a few years of spontaneously established symbiosis. In the newly infected amoebae, the bacteria avoided digestion and multiplied at a faster rate than the hosts, reaching the maximum carrying number (about 42,000 per amoeba) in fewer than ten cell generations of the hosts. The experimentally infected amoebae were also examined under the electron microscope, and the development of bacteria‐containing vesicles was followed. The results show that the infective bacteria that were initially harmful to host amoebae have become harmless and that they have changed in their mode of multiplication during the course of establishing a stable symbiosis with their hosts.
Effect of amino acid deprivation on DNA synthesis in BHK‐21/C13 cells
Authors: Melvin William T. , Burke Julian F. , Slater Alison A. , Keir Hamish M.
Published: February 2005

Removal of serum from BHK‐21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cells.
Lipid‐saccharide intermediates and glycoprotein biosynthesis in a temperature‐sensitive Chinese hamster cell mutant
Authors: Tenner Andrea J. , Scheffler Immo E.
Published: February 2005

The characterization of a temperature‐sensitive Chinese hamster cell mutant has been continued with the aim of localizing the apparent defect in glycoprotein synthesis (Tenner et al., 1977). Although the mutation is lethal, a demonstration of the ability of the mutant cells to support proliferation of Mengo virus at the nonpermissive temperature indicates that the general metabolic processes of the cells remain intact at a time when glycoprotein synthesis is severely depressed. A quantitative study of protein synthesis on membrane‐associated polysomes suggests that the synthesis of the polypeptide portion of the glycoproteins at 40.8°C may be normal. The investigation of lipidsaccharide molecules which have been implicated in the formation and transfer of the oligosaccharide „core„ to polypeptide acceptors shows that mutaant cells at the nonpermissive temperature are capable of synthesizing these lipid saccharides normally, and that the pool of the dolichyl oligosaccharide is maintained at a constant level independent of the temperature. The rate of formation of the lipid‐oligosaccharide, however, is reduced in intact mutant cells at the nonpermissive temperature. Further investigations show this decreased rate to be the result of an increased half life of the lipid‐oligosaccharide at 40.8°C. These data indicate that the temperature‐sensitive step in glycoprotein biosynthesis is the transfer of the oligosaccharide core from the lipid‐oligosaccharide intermediates to the nascent polypeptide chain. The data presented also provide evidence that the lipid‐saccharide intermediates, previously described mainly in in vitro systems, are in fact involved in the glycosylation of a majority, if not all, of the mannose‐containing glycoproteins in intact, growing hamster cells.
In Vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation
Authors: Veldhuijzen J. P. , Bourret L. A. , Rodan G. A.
Published: February 2005

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L‐cells ODC. The dose‐response pattern of the IC effect indicated an all‐or‐none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The km of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The Vmax, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non‐competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in 3H‐thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum‐deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G1 phase. Exposure to IC early in G1 (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on 3H‐thymidine incorporation in L‐cells.
Colony formation in agar by multipotential hemopoietic cells
Authors: Metcalf D. , Johnson G. R. , Mandel T. E.
Published: February 2005

Agar cultures of CBA fetal liver, peripheral blood, yolk sac and adult marrow cells were stimulated by pokeweed mitogen‐stimulated spleen conditioned medium. Two to ten percent of the colonies developing were mixed colonies, documented by light or electron microscopy to contain erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. No lymphoid cells were detected. Mean size for 7‐day mixed colonies was 1,800‐7,300 cells.

When 7‐day mixed colonies were recloned in agar, low levels of colony‐forming cells were detected in 10% of the colonies but most daughter colonies formed were small neutrophila and/or macrophage colonies. Injection of pooled 7‐day mixed colony cells to irradiated CBA mice produced low numbers of spleen colonies, mainly erythroid in composition. Karyotypic analysis using the T6T6 marker chromosome showed that some of these colonies were of donor origin. With an assumed f factor of 0.2, the mean content of spleen colony‐forming cells per 7‐day mixed colony was calculated to vary from 0.09 to 0.76 according to the type of mixed colony assayed.

The fetal and adult multipotential hemopoietic cells forming mixed colonies in agar may be hemopoietic stem cells perhaps of a special or fetal type.
Fast and slow fractions of K+ flux in human lymphocytes
Authors: Negendank William , Shaller Calvin
Published: February 2005

Potassium influx and efflux were studied in human peripheral blood lymphocytes equilibrated over a wide range of external K+ levels. The absence of a net ion movement throughout the flux study was established, trapped space was measured with polyethylene glycol, and cells were separated from incubation media without exposure to any washing solution. There are both rapid and slow cellular fractions of 42K influx and efflux, with half‐times of exchange of around 2 minutes, and 400 minutes, respectively. The rapid component is identical in magnitude to the smaller non‐saturable component of cell K+, while the slow component is identified with the larger, sigmoidal, saturable component of cell K+ that was previously shown to follow a cooperative adsorption isotherm. These results support the association‐induction hypothesis, which predicts (a) a rapid fraction of K+ flux due to equilibration of ion within cell water existing in a state of polarized multilayers, and (b) a slower component of K+ flux limited by adsorption onto, or desorption from, fixed anionic sites existing throughout the cell. K+ influx, as a function of external K+, showed a triphasic relation with a peak around 1 mM K, then a trough around 4 mM K, and then a gradual rise. This relation was readily explained, in terms of the association‐induction hypothesis, by the cooperative interaction between, and ion occupancy of, fixed anionic sites that absorb K+ or Na+.
Glucocorticoids enhance the mitogenic action of insulin in serum‐free cultures of chick embryo cells
Authors: Baker Joffre B. , Simmer Robert L. , Glenn Kevin C. , Cunningham Dennis D.
Published: February 2005

Addition of insulin to nonproliferating serum‐free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10−10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA.

The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased.

In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.
Growth of human embryonic fibroblasts at clonal density: Concordance with results from mass cultures
Authors: Smith James R. , Braunschweiger Karen I.
Published: February 2005

In the past, it has been difficult to grow human diploid fibroblast cells at clonal densities. Newly devised cell culture media and rigorously controlled environmental conditions have greatly increased the ease with which such cells can be cloned. The present work was undertaken to determine whether, under appropriate conditions, diploid fibroblast cells from human embryonic lung, grow as well at clonal densities as in mass culture. The parameters studied were: (1) population doubling time, (2) in vitro proliferative capacity, (3) attachment, (4) percentage of non‐dividing cells. In all cases essentially the same results were obtained for cultures at clonal densities and mass cultures. These results indicate that the behavior of these types of cells in clonal culture can be used to infer the behavior of individual cells and clones within a mass culture.
Effects of hydrostatic pressure in the range 100–300 atmospheres on cell division and protein synthesis in synchronized Tetrahymena pyriformis: A comparison with cycloheximide and emetine
Authors: Walker Eric , Wheatley Denys N.
Published: February 2005

Heat‐synchronized Tetrahymena pyriformis have been subjected to 5‐, 15‐ and 30‐minute pulses of hydrostatic pressure in the range 100–300 atm, without being simultaneously subjected to significant heats of compression. The pressure‐induced division delays depend on (1) the level of pressure used, (2) the length of pressure pulse and (3) the time after the synchronizing treatment at which the pressure is applied. A pressure‐dependent inhibition of 3H‐leucine incorporation into protein was also measured. Comparison of the effects of pressure with those of pulse treatments of cycloheximide and emetine on cell division and protein synthesis revealed that the physical agent produced characteristically different responses from those of the chemical agents. Of particular interest was the fact that the division delays induced by pressures of 200 atm and above were greater than those observed after treatments with cycloheximide and emetine which produced comparable levels of protein synthesis inhibition. Pressure also delayed cells if it was applied at a time when addition of chemical inhibitors had little effect on the first synchronous division. The results show that inhibition of protein synthesis by pressure cannot entirely account for pressure‐induced effects on cell division. The possibility that pressure may also directly affect other processes, such as the assembly of proteins into structures required for division, is discussed.
Amino acid transport in normal and Rous sarcoma virus‐transformed chicken embryo fibroblasts
Authors: Nakamura Kenji D. , Weber Michael J.
Published: February 2005

A study was made of the transport of a variety of amino acids by uninfected and Rous sarcoma virus‐infected chicken embryo fibroblasts. Following a period of amino acid starvation, transformed, but not normal cells, showed increased levels of transport for α‐aminoisobutyric acid, proline and alanine, three amino acids which are transported primarily by the A transport system. There was no starvation‐induced increase in the transport of leucine, phenylalanine, lysine, or cycloleucine. In the absence of starvation, normal and transformed cells exhibited comparable rates of amino acid transport. Cycloheximide was able to block the increase in uptake. The enhanced uptake was characterized by an increase in Vmax for transport and little change in Km.

The data demonstrate that an alteration in the regulation of the A amino acid transport system is an early event in malignant transformation by Rous sarcoma virus. However, since this alteration is made manifest only following a period of starvation, our findings suggest that increased amino acid uptake does not play a role in generating the other manifestations of the transformed state seen in cell culture.
Effect of phagocytosis by human polymorphonuclear leukocytes and rabbit alveolar macrophages on 2‐deoxyglucose transport
Authors: Tsan Min‐Fu
Published: February 2005

2‐Deoxyglucose transport was characterized in human polymorphonuclear leukocytes (PMN) and rabbit alveolar macrophages (AM). The Km was 1 mM for human PMN and 1.6 mM for rabbit AM, and the Vmax was 0.66 × 10−3 μmoles/45 sec/106 PMN and 5.09 × 10−4 μmoles/45 sec/106 AM. The rate of 2‐deoxyglucose transport was the same before and after phagocytosis in PMN from normal individuals and three patients with chronic granulomatous disease, as well as rabbit AM. Studies of the kinetics of 2‐deoxyglucose transport and intracellular fate of 2‐deoxyglucose in human PMN indicate that the nature of the membrane transport system is not altered by phagocytosis. The results support the concept that the plasma membrane is mosaic in character with geographically separate transport and phagocytic sites.
Defective transient endogenous spleen colony formation in S1/S1d mice
Authors: Wiktor‐Jedrzejczak W. , Ahmed A. , Sharkis S. J. , McKee A. , Sell K. W.
Published: February 2005

WCB6F1 mice of the genotype S1/S1d did not form transient 5‐day endogenous spleen colonies following midlethal irradiation, either spontaneously or in response to postirradiation bleeding. Their hematologically normal (+/+) littermates produced colonies equivalent in number and morphologic type to a normal strain (D2B6F1), as evaluated by both macroscopic and microscopic criteria. Bone marrow cells from S1/S1d mice, when transplanted into lethally irradiated +/+ mice, were able to generate equivalent numbers of transient endogenous spleen colonies (TE‐CFUs), as compared to that obtained when syngeneic +/+ marrow cells were injected into lethally irradiated +/+ recipients. A defective growth of an early class of hematopoietic progenitor cells, resulting in the clinical course of the S1/S1d anemia is suggested and confirms previous reports on the microenvironmental nature of this abnormality.
Selective inhibition of preribosomal RNA synthesis by puromycin aminonucleoside in transformed human fibroblasts: Studies of the nature of the inhibition in isolated nuclei and nucleoli
Authors: Albanese Ernest A. , Studzinski George P.
Published: February 2005

Puromycin aminonucleoside selectively inhibits the synthesis of ribosomal RNA in human lung fibroblasts transformed by the oncogenic virus SV40. The mechanism of this inhibition was studied utilizing nuclei and nucleoli isolated from cells treated for 18 hours with 100 μg/ml of this compound. It was established that for a limited period of time nuclei and nucleoli isolated from the fibroblasts continue synthesis of RNA of size classes seen in intact cells, and that the inhibitory effect of aminonucleoside persists after isolation of these organelles. The inhibition was shown to be directed primarily to the activity of RNA polymerase I.

Studies of the mechanism of this inhibition have indicated that the decreased rate of the polymerase reaction is not due to the impairment of the template function of nucleolar chromatin, and that unbound, as well as chromatin‐bound, RNA polymerase I is present in both control and treated nucleoli. Analysis of the size distribution of the products of cell‐free RNA synthesis showed that aminonucleoside pretreatment results in marked reduction in the synthesis of preribosomal 45S RNA, abnormal accumulation of 32S RNA, and reduced formation of mature ribosomal RNA species in the in vitro system. The data suggest that the inhibitory effect of aminonucleoside on ribosomal synthesis is due in part to a lower rate of transcription by RNA polymerase I of preribosomal RNA, and in part to its impaired maturation.
The kinetics of chick cell population aging in vitro
Authors: Ryan J. M.
Published: February 2005

We have examined the kinetics of chick cell population aging in vitro using the percentage of labeled nuclei, the number of colonies formed from a low density inoculum and the number of cells/colony to monitor culture age. The results from these studies showed a gradual age‐associated decline in each of the parameters which was first detected early in the culture lifespan and well in advance of changes in total cell number at confluency. Our results also indicated that each of the above parameters, in addition to the calendar time cells had been in culture, could be used to estimate the percentage of lifespan completed by the culture. A comparison of the methods used to estimate the remaining culture lifespan indicated that the percentage of labeled nuclei was the most accurate in describing cell age.
Rapid separation of mouse T and B lymphocytes using wheat germ agglutinin
Authors: Bourguignon Lilly Y. W. , Rader Richard L. , McMahon James T.
Published: February 2005

A separation procedure has been developed for mouse splenic T and B lymphocytes which is based on their differential agglutination by wheat germ agglutinin (WGA). In the presence of 50‐100 μg/ml of WGA, multicellular aggregates are formed which are enriched in B cells. These aggregates can be separated from monodisperse T cells by gravity sedimentation and subsequently dissociated into single cells by treatment with N‐acetylglucosamine (NAG). Immunocytochemical analyses and mitogenic assays indicate approximately 10‐15% cross contamination of the resultant B and T cell fractions. The separation procedure is not only convenient and rapid but also allows the simultaneous recovery of viable T and B cells from the same spleen preparation.
Regulation of epidermal growth factor (EGF) receptor activity during the modulation of protein synthesis
Authors: Carpenter Graham
Published: February 2005

The capacity of cultured human fibroblasts to bind 125I‐labeled epidermal growth factor (EGF) was measured during protein synthesis inhibition and reinitiation. Protein synthesis was inhibited by incubation of human fibroblasts in histidine‐free medium supplemented with L‐histidinol to produce a stringent amino acid starvation. Under these conditions 125I‐EGF binding activity decreased with a half‐life of 14.5 hours. Protein synthesis could be rapidly reinitiated by the addition of L‐histidine to human fibroblasts which had been preincubated in histidinol containing media for 36 to 48 hours. 125I‐EGF binding activity rapidly increased upon the reinitiation of protein synthesis. In the presence of serum 100% of the original binding capacity was recovered ten hours after the renitiation of protein synthesis, while 70% of the binding capacity was recovered in 12 hours in serum‐free media. The recovery of 125I‐EGF binding activity after the reinitiation of protein synthesis, was not blocked by the presence of Actinomycin D, indicating that the messenger RNA for the EGF receptor may accumulate during the period of histidinol‐mediated inhibition of protein synthesis. The time course of recovery of 125I‐EGF binding activity after the reinitiation of protein synthesis is very similar to that observed during the recovery of receptor activity following “down regulation” of EGF receptor activity. Recovery from down regulation, however, was markedly sensitive to Actinomycin D.
Ultrastructural localization of wheat germ agglutininbinding sites on surfaces of chick embryo cells during early differentiation
Authors: Sanders E. J. , Anderson A. R.
Published: February 2005

The objective of this work was to examine changes in a surface component of cells from the chick embryo during morphogenetic migrations of gastrulation. Two electron microscope techniques were used to localize cell‐bound wheat germ agglutinin (WGA), a lectin which specifically binds N‐acetyl glucosamine residues. One technique involved conjugation of peroxidase to WGA before reaction with the cells; the other technique used glucose oxidase to mark WGA which was already cell‐bound. In both cases, binding was revealed using diaminobenzidine. Before formation of the primitive streak, all surfaces of the two‐layered embryo bound WGA. After migration of cells through the streak, to form the three‐layered embryo, not all cell surfaces bound WGA equally. Epiblast cells generally bound WGA lateral to the primitive streak but not during passage through the streak. Mesenchyme cells, after passage through the streak, bound WGA increasingly as they migrated away from the streak. A WGA‐binding matrix was observed in the vicinity of the mesenchyme cells and on the dorsal surface of the endoblast. The ventral surface of the endoblast bound the lectin very poorly. In some instances, a peroxidase reaction product was consistently seen on certain surfaces which was not removable by addition of the simple hapten N‐acetyl glucosamine. In these cases, the density of the deposit was lessened by use of diacetyl chitobiose as a hapten. This result, together with the reduction of reaction product following certain hyaluronidase treatments, suggests that WGA may be binding to hyaluronic acid as well as membrane glycoproteins.
Regulation of DNA replication by serum and the transforming function in cultured rat fibroblasts transformed by Rous sarcoma virus
Authors: Magun Bruce E. , Thompson Richard L. , Gerner Eugene W.
Published: February 2005

The onset and rate of semiconservative DNA replication were measured in stimulated cultured rat fibroblasts and their Rous sarcoma virus‐transformed derivatives after a period of serum deprivation. Rat‐1 (tsLA24/RSV) cells initiated DNA synthesis following a shift to the permissive temperature or addition of serum at the non‐permissive temperature. Their rate of DNA replication was unaffected by the presence of serum at the permissive temperature, however, there was a serum requirement at the non‐permissive temperature. The transition probability was less at the permissive temperature, independent of serum, than at the non‐permissive temperature in the presence of serum. The amount of DNA induced to replicate by addition of serum at the non‐permissive temperature or by a shift to the permissive temperature was similar. Using the untransformed Rat‐1 cells and these cells transformed by wild‐type RSV (Rat‐1 (wt/RSV)), it was confirmed that the rate of entry into S phase (transition probability) was always lower in the transformed cell line at both 39° and 35°. In both cell lines the rate of DNA replication was independent of temperature, but the onset was delayed at the lower temperature. These results indicate that in the cell lines examined, (1) serum was able to commit the cells to replicate DNA (alter the transition probability) in both transformed and untransformed cells, but the transforming function was able to supplant a serum‐dependent process during G1 necessary for the initiation of DNA replication, and (2) the effects of the transforming function and serum factor(s) on the alteration of the transition probability are not additive, suggesting that the transforming function initiates a process which acts at the level of the commitment to DNA replication which may render the normal serum‐related control mechanisms ineffective in the regulation of growth.
Inosine di‐ and triphosphate synthesis in erythrocytes and cell extracts
Authors: Vanderheiden Bernardo S.
Published: February 2005

The ability to synthesize inosinetriphosphate was demonstrated in blood cells as well as in a variety of tissue extracts in spite of the presence of ITP pyrophosphohydrolase. At the expense of having sub‐optimal conditions, an assay system was selected that completely repressed the hydrolyzing enzyme, thus permitting the accumulation of ITP.

In an attempt to define the biosynthetic pathway of ITP, and since guanylate kinase has been implicated in the formation of ITP, the rate of synthesis of ITP and GTP in cell extracts was compared.

The comparison of the specific activities of the [14C]‐labeled hypoxanthine and guanine moieties of the inosine and guanosine phosphates formed during incubation with [8‐14C]‐inosine and [8‐14C]‐guanosine respectively, revealed striking differences in the relative rates of isotope incorporation. Tentative mechanisms are proposed to explain these differences.

The data obtained thus far does not discard the possibility that ITP may be formed by stepwise phosphorylation and (or) by direct pyrophosphorylation of IMP.
Early decrease of 2‐deoxy glucose and α‐amino isobutyric acid transport are among the first events in differentiating synchronized murine erythroleukemia cells
Authors: Gazitt Yair
Published: February 2005

Murine erythroleukemic cells were induced to differentiate along the erythroid pathway by Me2SO and HMBA. These inducers caused an early decrease in the transport of glucose and amino acids, both in non‐synchronized and in synchronized cultures. Careful analysis of the transport parameters in synchronized cultures showed a cyclic fluctuation of the Vmax but no significant change of the Km. In the presence of the inducers, however, a modification of the Km and Vmax of both carriers was observed which was not dependent on cell cycle. This modification is very early and precedes the transient arrest of the cells in G1 reported previously. In addition, a Me2SO‐resistant cell line (DR10) does not show any changes in the transport of glucose and amino acids when incubated with Me2SO. However, there is an effect on the transport when incubated with HMBA which induces differentiation of 50% of the cells.

These data support the hypothesis that an early effect of the inducers on the plasma membrane may be a necessary prerequisite for initiation of differentiation in murine erythroleukemic cells.
Sodium‐potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: Kinetic parameters, substrate specificity, and effects of phytohemagglutinin
Authors: Segel George B. , Kovach George , Lichtman Marshall A.
Published: February 2005

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood.

The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+‐ATPase activity were characterized. The Na+, K+‐ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by iptimal concentrations of Na+ and K+ (Na+, K+‐ATPase) was 1.5 nmol of Pi hydrolyzed, μ g protein−1, 30 min−1 (range 0.9‐2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The Km for K+ was approximately 1.0 mM and the Km for Na+ was approximately 15 mM.

Active Na+ and K+ transport and ouabain‐sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+‐ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+‐ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+‐ATPase when studied at the Km for ATP, Na+, K+ or Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.