bluuebunny/crossref_metadata_2025 huggingface dataset:
Interferon induction in marrow‐derived macrophages: Regulation by L cell conditioned medium
Authors: Fleit Howard B. , Rabinovitch Michel
Published: February 2005

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti‐CSF serum to LCM medium also reduced IFN titers in response to poly I:C but had no effect on NDV induction. The inhibitory effect of anti‐CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with poly I:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with poly I:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.
DNA synthesis in primary cultures of adult rat hepatocytes in a defined medium: Effects of epidermal growth factor, insulin, glucagon, and cyclic‐AMP
Authors: McGowan Joan A. , Strain Alastair J. , Bucher Nancy L. R.
Published: February 2005

Epidermal growth factor (EGF) especially in combination with insulin and glucagon, has been shown to stimulate DNA synthesis in liver cells, both in the whole animal and in cell cultures. As a further development we have found that in primary monolayer cultures of freshly isolated adult rat liver parenchymal cells, in which contamination with nonparenchymal cells was negligible, DNA synthesis was substantially stimulated by these substances. In control cultures, incorporation of [3H]thymidine into DNA and labeling of nuclei in autoradiographs was low. The stimulation by EGF was enhanced by insulin and glucagon, whereas these hormones by themselves exhibited only limited activity. These observations were made in cultures of hepatocytes that were never exposed to serum, even during cell isolation and plating. Hence for stimulation of DNA synthesis under these conditions neither serum factors nor interactions with other types of cells or their products were required. The effects of glucagon were reproduced by substances that elevate intracellular concentration of cyclic‐AMP, including cholera toxin, isoproterenol, and methylisobutylxanthine. These various substances, expecially EGF, glucagon, or cyclic‐AMP, altered the morphological characteristics of the cultures during early stages, promoting cellular spreading and aggregation.
Nucleoside requirements for the in vitro growth of bovine aortic endothelial cells
Authors: Fenselau Allan , Kaiser Donald , Wallis Katheleen
Published: February 2005

The nutritional needs of cultured fetal bovine aortic endothelial cells were studied with regard to their nucleotide metabolism. When Medium 199 containing calf serum was supplemented with up to 5μg/ml of the deoxyribo‐ or ribonucleosides found in DNA or RNA, the rate of endothelial cell growth increased. The effect was entirely attributable to the pyrimidine nucleosides. The combination of deoxythymidine and deoxycytidine was much more effective than either deoxyribonucleoside used alone or than the combination of uridine and cytidine. Addition of deoxythymidine and deoxycytidine (each at 1 μg/ml) to the medium supported the growth of endothelial cell cultures from initially sparse populations (ca. 50 cells/cm2), even at low concentrations (1%) of fetal bovine serum. The pyrimidine deoxyribonucleosides on their own were unable to stimulate cell growth; other bonafide growth stimulatory factors, such as those present in serum, serum dialysates, or retinal extracts, were needed in the medium to signal the initiation of DNA synthesis and cell replication. The significance of these findings with respect to improving cell performance under in vitro conditions and controlling endothelial cell growth in vivo are discussed.
Collagen synthesis in human fibroblasts: Effects of ascorbic acid and regulation by hydrocortisone
Authors: Russell Shirley B. , Russell James D. , Trupin Kathryn M.
Published: February 2005

The effects of hydrocortisone and ascorbic acid on collagen and noncollagen protein synthesis, and on growth were examined in fibroblasts derived from normal human dermis. When the medium was supplemented with 0.28 mM ascorbic acid, the apparent rate of collagen production increased 2‐‐3 fold over the culture cycle. Ascorbic acid also caused a small increase in the apparent rate of synthesis of noncollagen protein and an elevation in growth rate and maximum cell density. Growth was not required for the increase in collagen production since addition of ascorbate to confluent cultures induced a similar increase. Hydrocortisone (1.5 μM) blocked the ascorbate‐related increase in collagen production during growth and in confluent cultures. The hormone simultaneously increased the apparent rate of noncollagen protein production and maximum cell density, suggesting that the effect on collagen synthesis was specific. Inhibition of collagen production by hydrocortisone was observed only in the presence of ascorbate, while the increase in growth and noncollagen protein production occurred in the presence and absence of the vitamin.
Folate polyglutamate and monoglutamate accumulation in normal and SV40‐transformed human fibroblasts
Authors: Hoffman Robert M. , Coalson Dennis W. , Jacobsen Stephen J. , Erbe Richard W.
Published: February 2005

Folate polyglutamate and monoglutamate accumulation was measured in normal diploid and SV40‐transformed human fibroblasts by Sephadex G‐10 gel filtration chromatography. The cells were first depleted of folates and then provided with limiting amounts of [3H]‐folic acid in order that the cells would accumulate only forms of folate necessary for proliferation. Both the normal and the transformed cells accumulated monoglutamate and polyglutamate forms, but by 72 hours of labeling the transformed cells contained 3–10 times more polyglutamate than the normal cells. The growth rates for the normal and transformed cells were similar at this limiting folic acid concentration. Thus, if folate polyglutamates are more important for the proliferation of SV40‐transformed cells than the normal cells, then inhibition of polyglutamate formation may be an important potential target for chemotherapy.
Lipid composition of Friend leukemia cells following induction of erythroid differentiation by dimethyl sulfoxide
Authors: Rittmann Lana S. , Jelsema Carole L. , Schwartz Edward L. , Tsiftsoglou Asterios S. , Sartorelli Alan C.
Published: February 2005

The effects of dimethyl sulfoxide (DMSO)‐induced differentiation of Friend leukemia cells in vitro on the lipid composition of these cells have been examined. DMSO had no early effect on the incorporation of either [14C] glycerol or [3H] methyl choline chloride into the total lipids or individual phospholipids of Friend cells up to 240 min after addition of the inducer. Examination of DMSO‐diferentiated Friend cell phospholipids revealed a percentage composition which was similar to control cells, with phosphatidylcholine and phosphatidylethanolamine in both uninduced and differentiated cells accounting for over 75% of the total phospholipid. Sphingomyelin levels were significantly lower in Friend cells than in normal adult mouse erythrocytes, and differentiation of murine erythroleukemia cells resulted in a further lowering of this phospholipid. In contrast, a significant increase in the level of phosphatidylethanolamine occured as a result of maturation. Fatty acid analysis of major lipid classes of differentiated Friend cells showed significant reduction in saturation, but no alteration in chain length in comparison to undifferentiated cells. A pronounced decrease in the cellular content of both free and esterified cholesterol, which resulted in a 45% decrease in the ratio of cholesterol/phospholipids, occurred in cells differentiated by the polar solvent. The findings indicate that erythrodifferentiation induced by DMSO results in a variety of changes in the lipid composition of the membranes of Friend leukemia cells.
Glutamine synthetase induction by glucocorticoids in the glucocorticoid‐sensitive human leukemic cell line CEM‐C7
Authors: Harmon Jeffrey M. , Thompson E. Brad
Published: February 2005

Treatment of CEM‐C7 cells with glucocorticoids produces a 2.5‐fold increase in the activity of the enzyme glutamine synthetase (GS). This increase is specific for steroids with glucocorticoid activity and occurs over a range of steroid concentrations consistent with a receptor‐mediated mechanism. Half‐maximal and maximal inductions by dexamethasone (dex) occur at 2 × 10−8M and 2 × 10−7 M dex, respectively, concentrations approximately equal to those necessary to produce half and full occupancy of glucocorticoid receptors. GS activity began to increase 1 hour after dex treatment and was complete by 12 hours. This is well before any of the growth inhibitory or cytolytic effects of dex on this cell line occur. This increase was dependent on the presence of glucocorticoid receptors and required both RNA and protein synthesis. Removal of dex following stimulation to maximal levels resulted in a decrease of GS activity to preinduced levels with a half‐time of 5 hours. Glutamine deprivation of cells resulted in increased GS activity. However, even in the total absence of glutamine, dex treatment elicited a 2.0–2.5‐fold increase in GS activity, ruling out inhibition of glutamine uptake as a mechanism for the dex‐induced increase. Experiments with 5′‐bromodeoxyuridine (BrdU) demonstrated that GS elevation was sensitive to BrdU substitution of DNA, while dex‐induced growth inhibition was not. Therefore GS elevation and growth inhibition in this cell line appear to be independently expressed steroid responses.
Deoxycytidine excretion by mouse peritoneal macrophages: Its implication in modulation of immunological functions
Authors: Chan Teh‐Sheng , Lakhchaura Bala D.
Published: February 2005

Pyrimidine excretion by macrophages was studied in order to identify the potential immunoregulatory effector molecules. Deoxycytidine was found in the culture medium of thioglycollate‐elicited mouse peritoneal macrophages, along with thymidine, which was shown by others to be a possible immunoregulatory substance. The identification of deoxycytidine was based on: (1) cochromatography with the authentic compound in four different solvents, (2) UV absorption spectral analysis, and (3) the enzymatic peak shift method. Phagocytosis of nucleated chicken erythrocytes, but not enucleated sheep erythrocytes, increased deoxycytidine excretion. The macrophages lacked both deoxycytidine kinase and deoxycytidine deaminase, which is consistent with their excretory pattern. Since it has been known that deoxycytidine can protect cells against cytotoxic effects of thymidine, we propose that deoxycytidine has a role in preventing immunosuppression by thimidine. In patients with adenosine deaminase deficiency, however, immunosuppression caused by combined toxicity of thymidine and deoxyadenosine may not be adequately prevented by deoxycytidine.
Autoradiographic detection and characterization of a Chinese hamster ovary cell mutant deficient in fucoproteins
Authors: Hirschberg Carlos B. , Perez Mary , Snider Martin , Hanneman Wendy L. , Esko Jeffrey , Raetz Christian R. H.
Published: February 2005

Autoradiography of colony replicas immobilized on filter paper was used to isolate a Chinese hamster ovary cell line deficient in incorporation of radiolabeled fucose into a trichloroacetic acid‐insoluble fraction. This cell line, called 62.1, has the same growth rate at 37°C as wild‐type cells, but incorporates five times less fucose into acid‐insoluble radioactivity. Chemical analysis of fucose bound to macromolecules also showed a fivefold reduction in the mutant. The fucoproteins of the mutant cell line differ qualitatively from those of wild‐type cells as visualized by SDS gel electrophoresis fluorography; no differences were detected between total proteins as visualized by coomassie blue staining. The macromolecular sialic acid content of the mutant was somewhat higher than the wild type (20%).

Studies of the synthesis of the glycoprotein of vesicular stomatitis virus in mutant and wild‐type cells showed that the mutant is unable to synthesize complex‐type N‐linked oligosaccharides. Enzyme assays show that this defect in the mutant is due to reduction in UDP‐N‐acetylglucosamine‐glycoprotein N‐acetyl‐glucos‐aminyltransferase, a key enzyme in the assembly of complex glycopeptides.

Hybridization studies have shown that mutant 62.1 has common mutations belonging to the same complementation group as mutant PHaR1‐1. This latter mutant was previously isolated using lectin resistance by Stanley et al. (1975) and was also deficient in the above N‐acetyl‐glucosaminyltransferase.
The chemical mimicking of the mechanical stimulation, photoinhibition, and recovery from photoinihibition of bioluminescence in the marine dinoflagellate, Gonyaulax polyedra
Authors: Hamman J. P. , Seliger H. H.
Published: February 2005

Mechanically stimulable bioluminescence and photoinhibition of sensitivity to mechanical stimulation in the marine dinoflagellate Gonyaulax polyedra can be mimicked by a number of cations, proportional to the logarithm of their external concentrations. The data are consistent with mechanical stimulability as a membrane depolarization resulting in an increase in H+ions at bioluminescence sites and with photoinhibition as a hyperpolarization of the cell membrane.
Binding of phorbol esters to high‐affinity sites on murine fibroblastic cells elicits a mitogenic response
Authors: Collins Mary K. L. , Rozengurt Enrique
Published: February 2005

The level of occupancy of a single class of high‐affinity (3H)‐phorbol 12, 13‐dibutyrate (PDBu) bindings sites (Kd = 26 nM) in quiescent Swiss 3T3 cells was correlated with the dose of PDBu required to stimulate rapid (increase in 86Rb uptake, decrease in epidermal growth factor receptor affinity) and long‐term (induction of 2‐deoxyglucose uptake, initiation of DNA synthesis) events of the proliferative response. Further, structural analogues of PDBu showed identical relative potencies in the inhibition of (3H)‐PDBu binding and stimulation of DNA synthesis. Finally, prolonged (24‐hour) incubation of Swiss 3T3 or whole mouse embryo fibroblasts with 400 nM PDBu led to a decrease in the number of (3H)‐PDBu binding sites (down‐regulation) with a parallel loss of rapid and long‐term responses of the cells to PDBu (desensitization). These findings indicate that the high‐affinity (3H)‐PDBu binding sites mediate the mitogenic effects of phorbol esters in fibroblastic cells.
Hormonal responsiveness of a preadipose cell line derived from newborn mouse calvaria
Authors: Kodama Hiro‐Aki , Amagai Yuji , Koyama Hideki , Kasai Shiro
Published: February 2005

We established a clonal preadispose cell line from newborn mouse calvaria. Cells of this cell line, designated MC3T3‐G2/PA6, had the capacity to convert to adipose cells, to accumulate triglycerides in their cytoplasm, and to mature to differentiated fat cells in a resting state. This adipose conversion was markedly accelerated by addition of dexamethasone, which was the most potent inducer among the steroid hormones tested. The presence of dexamethasone was needed during logarithmic growth phase for maximal conversion. The frequency of adipose conversion was dependent on exposure time to the hormone, but cells already committed to differentiation continued to accumulate lipid and developed into mature adipose cell even in its absence. This indicates that the hormone accelerates the initiation of the adipose conversion, but is not required for the ongoing conversion process. In fact, it was rather inhibitory for the process of fat accumulation. Insulin alone slightly inhibited the adipose conversion, but its combination with dexamethasone neutralized the above inhibitory effect of dexamehasone. The responsiveness of this cell line is consistent with that observed for mouse bone marrow preadipocytes in primary culture but differs from that for preadipose cell lines derived from extramedullary tissues. These results strongly suggest that the MC3T3‐G2/PA6 cell line was derived from bone marrow.
A new preadipose cell line derived from newborn mouse calvaria can promote the proliferation of pluripotent hemopoietic stem cells in vitro
Authors: Kodama Hiro‐Aki , Amagai Yuji , Koyama Hideki , Kasai Shiro
Published: February 2005

A clonal preadipose cell line MC3T3‐G2/PA6, established from newborn mouse calvaria, responds to glucocorticoids and converts to adipose cells in a fashion similar to bone marrow preadipocytes. We investigated the effect of the cells on in vitro hemopoiesis of mouse bone marrow cells by cocultivation. When bone marrow cells were inoculated into confluent cultures of MC3T3‐G2/PA6 cells (104–106 cells/25‐cm2 flask), the number of hemopoietic stem cells (CFU‐S) significantly increased during 7‐day cultivation in proportion to inoculum size. Under these conditions, active replication of CFU‐S was maintained for several weeks until MC3T3‐G2/PA6 cell layers detached from the substratum. This capacity of the MC3T3‐G2/PA6 line was unique because other established cell lines, including the MTF preadipose line, failed to support CFU‐S growth. When bone marrow cells were not allowed to contact the MC3T3‐G2/PA6 cell layer, only a small number of CFU‐S survived for 7 days. Moreover, MC3T3‐G2/PA6 cell‐conditioned medium did not show any growth‐promoting activity for CFU‐S. These results indicate that the MC3T3‐G2/PA6 cell line has the ability to promote the proliferation of CFU‐S through a short range cell‐to‐cell interaction by providing an in vitro microenvironment probably similar to that for in vivo hemopoiesis.
Energy metabolism in cultured human fibroblasts during aging in vitro
Authors: Goldstein S. , Ballantyne S. R. , Robson A. L. , Moerman E. J.
Published: February 2005

To explore the relationship between energy metabolism and the limited replicative life span of cultured human fibroblasts, we studied several bioenergetic parameters in normal fibroblasts at early passage (young cells) and at late passage (old cells) and early passage cells from a subject with the Hutchinson‐Gilford (progeria) syndrome. Old cells consumed more glucose and produced more lactate during growth, but O2 consumption, both basal and following maximum uncoupling of oxidative phosphorylation by SF‐6847, was the same as in young cells. Progeria cells produced the most lactate but did not consume more glucose, while their basal and uncoupled O2 consumption was similar to that of young and old cells during both log and confluent states. Consumption of glutamine, a source of both oxidative energy and lactate, was approximately the same in all three cell types as was 14CO2 production from 2‐14C‐pyruvate and 5‐14C‐glutamate. ATP and ADP concentrations were similar in all cell types with a rise in the ATP/ADP ratio during growth from log to confluent state. Thus, old and progeria cells, in contrast to young cells, produce more lactate during growth consistent with a rise in energy demand and/or inefficiency of oxidative phosphorylation. Although limitations in total energy output do not appear to be causal to the loss of replicative capacity in normal cells after serial passage, they could play a role in the curtailed replicative capacity of progeria cells.
Regulation of transferrin receptor expression in concanavalin a stimulated and gross virus transformed rat lymphoblasts
Authors: Hamilton Thomas A.
Published: February 2005

Expression of the cell surface receptor for the serum glycoprotein transferrin has been correlated with cellular proliferation in normal lymphocytes undergoing mitogen or antigen induced proliferative responses. In the present study, the expression of transferrin receptor in Concanavalin A stimulated rat lymphocytes or Gross virus transformed lymphoma cells has been examined with respect to the following questions: (1) is expression of receptor activity related to blastogenesis or to the subsequent IL‐2 dependent DNA synthetic activity, and (2) is transferrin receptor expression regulated in similar fashion in both normal and malignant lymphoblasts? Scatchard analysis of saturation binding data illustrated that binding site number increased and subsequently decreased during the response while the receptor affinity for transferrin remained constant. These findings were confirmed by SDS‐polyacrylamide gel electrophoretic analysis of radiolabeled cell surface proteins which specifically interact with transferrin. Examination of nonproliferating normal lymphoblasts (96 hr post Con A stimulation) compared with the same population of cells stimulated to reinitiate DNA synthesis with a partially purified preparation of Interleukin 2 (IL‐2) showed that transferrin receptor expression was tightly linked to the IL‐2 dependent stimulation of DNA replication. This coordinate regulation of receptor expression was markedly less stringent in retrovirus transformed thymic lymphoma cells.
The cellular uptake of horseradish peroxidase and its poly(lysine) conjugate by cultured fibroblasts is qualitatively similar despite a 900‐fold difference in rate
Authors: Ryser Hugues J.‐P. , Drummond Iain , Shen Wei‐Chiang
Published: February 2005

When horseradish peroxidase (HRP) is conjugated to poly(L‐lysine) of molecular weight (MW) 13,000, its transport into cultured L929 fibroblasts in 1 hour at 37°C is increased 918‐fold. The kinetics of uptake are linear with time and concentration, reflecting a process of nonreceptor‐mediated adsorptive endocytosis. Neither HRP‐poly(Lys) conjugate nor free poly(Lys) of any size cause any increase in fluid phase endocytosis. All evidence indicates that the covalently bound poly(Lys) increases the binding of HRP to the cell surface and that this adequately accounts for an increased internalization occurring at a steady rate. In the absence of Ca++, both surface binding and uptake of the conjugate, but not of free HRP, are decreased. Cytochalasin B does not significantly inhibit the transport of either form of HRP. The half‐lives of HRP and HRP‐poly(Lys) are 7.6 and 5.6 hours, respectively, when measured over a period of 12 hours. Electron microscopic analysis of cells that have ingested comparable amounts of HRP shows that the intracellular localization of free and conjugated HRP are comparable and that both are found inside the same array of structures, thus HRP, which binds very poorly to the cell surface and is considered a fluid‐phase marker for the “contents” of pinocytotic vesicles, and HRP‐poly(Lys), which binds very strongly to the cell surface and is considered a membrane marker for adsorptive endocytosis, are taken up along the same endocytotic pathway. Moreover, despite the fact that neither markers are transported by receptor‐mediated endocytosis, both are seen in structures that were described as receptosomes. It appears, therefore, that the pathways utilized in receptor‐mediated transport are available to all other forms of pinocytosis.
Small nuclear RNAs and translation
Authors: Smith James H. , Subbarao Makam N. , Eliceiri George L.
Published: February 2005

The possibility that small nuclear RNA species U1 might be involved in the inhibition of protein synthesis that occurs during mitosis has been explored. Upon exposure of mitotic HeLa cell extracts to 1% sodium deoxycholate, the majority of the rapidly sedimenting U1 RNA shifted to lower sedimentation rates. This suggests that it is associated with heterogeneous nuclear RNA ribonucleoprotein particles, instead of a ribosomal population. Erythrocyte ghost‐mediated microinjection of anti‐(U1)RNP antibodies into synchronized HeLa cells did not prevent the suppression of protein synthesis that is observed under mitosis. Examination of the published nucleotide sequences of U1 and U2 RNA suggests that these RNA species could potentially code for some short peptides. When purified U1 or U2 RNA were added to cell‐free polypeptide synthesizing systems, the synthesis of these peptides was not detected.
Taxol resistant mutants of Chinese hamster ovary cells: Genetic biochemical, and cross‐resistance studies
Authors: Gupta Radhey S.
Published: February 2005

The effects of the microtubule inhibitor taxol on the growth and viability of Chinese hamster ovary (CHO) cells have been examined. Stable mutants which are between seven to 11‐fold more resistant to taxol have been selected in a single step from ethyl methanesulfonate‐mutagenized CHO cells. The two taxol‐resistant mutants (TaxR‐1 and TaxR‐2) which have been studied in detail exhibit novel and strikingly different cross‐resistance/collateral sensitivity patterns to various microtubule inhibitors. For example, the TaxR‐1 mutant exhibits increased resistance to vinblastine, but in comparison to the parental cells, it shows enhanced sensitivity toward colchicine, colcemid, stegnacine, and griseofulvin. However, the sensitivity of this mutant toward other unrelated compounds, e.g., puromycin, daunomycin, etc., remained largely unaltered. The specific pattern of cross‐resistance/collateral‐sensitivity of this mutant toward various microtubule inhibitors suggests that the genetic lesion in this mutant may be affecting a microtubule‐related component. The TaxR‐2 mutant, in contrast, is highly resistant to various microtubule inhibitors including colchicine, colcemid, stegnacine, maytan‐sine, vinblastine, and podophyllotoxin. This mutant also exhibits greatly increased cross‐resistance to daunomycin, puromycin, ethidium bromide, and VM‐26 (compounds which do not inhibit microtubule assembly), and shows reduced cellular uptake of 3H‐daunomycin indicating that the genetic lesion in this mutant nonspecifically affects the membrane permeability of various drugs. The cell hybrids formed between TaxR‐1 (or TaxR‐2 mutant(s)) and a taxol‐sensitive cell line exhibit intermediate levels of resistance to the drug, indicating that the TaxR phenotypes of both these mutants behave codominantly under these conditions.
Opposing effects of 12‐o‐tetradecanoylphorbol 13‐acetate on human myeloid and lymphoid cell proliferation
Authors: Aye M. T. , Dunne J. V.
Published: February 2005

The effect of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) on human hematopoietic cells has been investigated. It was found that 1–10 ng/ml of TPA totally abrogated erythroid and granulocytic colony growth and, simultaneously in the presence of PHA, stimulated T‐lymphocyte colony formation. TPA concentrations insufficient to inhibit myeloid colony growth also failed to stimulate lymphocyte colony formation. Optimal culture conditions for the growth of these colonies required the presence of TPA, PHA, and leukocyte‐conditioned medium in the cultures. Cells within the colonies were 80–90% E‐rosette positive and by monoclonal antibody characterization contained 45–66% OKT3‐positive cells. Colony‐forming cells were found in both E‐rosette‐positive and‐negative fractions. Although by cell surface marker characterization the cells within the colonies had properties of T‐cells, the exact relationship of cells forming colonies under these conditions to those detected in other T‐cell colony assays remain to be determined.
Phosphatidyl choline and the growth in serum‐free medium of vascular endothelial and smooth muscle cells, and corneal endothelial cells
Authors: Fujii D. K. , Cheng J. , Gospodarowicz D.
Published: February 2005

Liposomes made by sonication of egg yolk phosphatidyl choline support the proliferation of low‐density bovine vascular and corneal endothelial cells, and vascular smooth muscle cells maintained on basement laminacoated dishes and exposed to a defined medium supplemented with transferrin. The optimal growth‐promoting effect of phosphatidyl choline was observed at concentrations of 25 μg/ml for low‐density cultures of vascular smooth muscle cells, and 100 μg/ml for vascular and corneal endothelial cells. The growth rate and final cell density of vascular endothelial cells exposed to a synthetic medium supplemented with transferrin and either high‐density lipoproteins or phosphatidyl choline has been compared. Although cultures exposed to phosphatidyl choline reached a final cell density similar to that of cultures exposed to high‐density lipoproteins, they had a longer average doubling time (17 h vs. 12 h) during their logarithmic growth phase and a shorter lifespan (17 generations vs. 30 generations). Similar observations were made in the case of vascular smooth muscle cells or bovine corneal endothelial cells maintained in medium supplemented with transferrin, fibroblast growth factor (FGF) or epidermal growth factor (EGF), and insulin and exposed to either high‐density lipoproteins or phosphatidyl choline. Since phosphatidyl choline can, for the most part, replace highdensity lipoproteins in supporting the proliferation of various cell types, it is likely that the growth stimulating signal conveyed by high‐density lipoproteins is associated with its polar lipid fraction, which is composed mostly of phosphatidyl cholines.
Binding, sequestration, and processing of epidermal growth factor and nerve growth facor by PC12 cells
Authors: Chandler C. E. , Herschman H. R.
Published: February 2005

The rat PC12 pheochromocytoma cell line exhibits biological responses to both nerve growth factor (NGF) and epidermal growth factor (EGF). The existence of receptors and biological responses on a common cell for these two well‐characterized polypeptide growth factors makes this an attractive system for comparison of ligand binding and processing. Both NGF and EGF are bound to PC12 cells in a competable form at 4°C. At 37°C both ligands are “sequestered”, but at different rates and to different extents. While sequestration happens rapidly and nearly quantitatively for bound EGF, the dissociation reation appears to compete favorably with NGF sequestration. Both EGF and NGF are degraded by PC12 cells. Sequestered EGF, however, is degraded to a greater extent than sequestered NGF.
Clonal growth of lymphoid cells in serum‐free media requires elimination of H2O2 toxicity
Authors: Darfler Frederick J. , Insel Paul A.
Published: February 2005

We have recently described the development of a serum‐free medium that contains casein, insulin, testosterone, transferrin, and linoleic acid and that supports the long‐term growth of a wide variety of lymphoid cells. A problem of culturing cells in this medium is the difficulty of cloning cells or growing cells at low density. We now describe the formulation of a chemically defined medium that supports the clonal growth of the murine S49 T lymphoma cell line. This medium contains catalase, insulin, transferrin, testosterone, Na2SeO3, and dilinoleoyl phosphatidylcholine and contains less than 50 μg/ml total protein. The two novel additions in this medium are catalase, which replaces casein and dilinoleoyl phosphatidylcholine, which substitutes for linoleic acid in this defined medium. In addition to S49 cells, the medium described above supports the long‐term growth of other lymphoid cells, including human and murine hybridomas. We propose that catalase functions to degrade H2O2 that is present in the cultures and that casein, bovine serum albumin, and other proteins commonly included in media for cultured cells may also scavenge H2O2. Na2SeO3 also partially protects against the death of cells at clonal density and this protection may, like catalase, be due to removal of H2O2. Our results suggest that H2O2 is an important cytotoxic agent that prevents growth of lymphoid cells during culture in serum‐free media and perhaps in serum‐containing media as well.
Segregation of mouse hemopoietic progenitor cells using the monoclonal antibody, YBM/42
Authors: Watt Suzanne M. , Gilmore David J. , Metcalf Donald , Cobbold Stephen P. , Hoang Trang K. , Waldmann Herman
Published: February 2005

A rat monoclonal antibody, YBM/42, directed against mouse leukocyte common antigen, was used for the analysis and separation of hemopoietic progenitor cells from mouse bone marrow and fetal liver. Cells were fractionated on a FACS‐II cell sorter and the resulting subpopulations examined for their morphology and ability to form colonies in agar (for day 7 colonies) and methylcellulose (for day 2 erythroid clones). The antibody bound to all leukocytes, including blast cells and day 7 hemopoietic progenitor cells (day 7 colony forming cells, CFC), but not to erythrocytes or nucleated erythroid cells. This antibody can be used to advantage to enrich for early progenitor cells from mouse fetal liver, in which the majority of cells (70%) are nucleated erythroid cells. In day 12 fetal liver, approximately 10% of all cells bind this antibody strongly and, of these approximately 70% are blast cells. Contained within this positive population are 95% of all day 7 CFC. In the most enriched fraction about 20% of the cells formed day 7 colonies. This represents a 25‐fold enrichment over unsorted fetal liver. The negative fractions contain 94% of all cells forming erythroid clones (≥8 cells) on day 2 of culture (day 2 CFU‐E). In the most enriched fraction, 20% of the cells are day 2 CFU‐E. Day 7 CFC can therefore be well separated from day 2 CFU‐E, with good recovery of both cell types, by use of a single label. Day 7 colony forming cells were classified as granulocyte (G‐CFC), macrophage (M‐CFC), mixed granulocyte/macrophage (GM‐CFC), pure erythroid (E), or mixed erythroid (Emix). A high enrichment for multipotential cells is achieved and constitues 3–5% of cells in the most enriched fraction. Most types of day 7 CFC could not be separated with YMB/42, but GM‐CFC and M‐CFC exhibit a broader distribution than the other CFC with regard to fluorescence intensity. This implicit heterogeneity in GM‐CFC and M‐CFC is further substantiated by the finding that myeloid progenitors in the different FACS fractions also share a differential reactivity to different sources of growth factors.
Glucocorticoids and prostaglandins inhibit the induction of macrophage DNA synthesis by macrophage growth factor and phorbol ester
Authors: Hamilton John A.
Published: February 2005

The tumor‐promoting phorbol ester, 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA), stimulates starch‐elicited mouse peritoneal macrophages to undergo DNA synthesis in vitro, apparently without the generation of an endogenous macrophage growth factor (MGF). No evidence was found for any synergistic interaction between TPA and exogenous colony stimulating factors (CSFs) for macrophage DNA synthesis. Low concentrations of glucocorticoids and also prostaglandins E1 and E2 suppress both the CSF‐1‐stimulated and the TPA‐stimulated macrophage DNA synthesis; these same drugs inhibit the CSF‐1‐mediated and TPA‐mediated enhancement of macrophage plasminogen activator (PA) activity. Thus glucocorticoids and prostaglandins E1 and E2 oppose the action of growth factors and the tumor promoter on macrophage and precursor cell function.
Culture of quiescent arterial smooth muscle cells in a defined serum‐free medium
Authors: Libby Peter , O'Brien Kathleen V.
Published: February 2005

An ideal medium for metabolic studies would maintain cultured vascular smooth muscle cells in a quiescent, viable state, as they are in normal arteries in vivo, and would be chemically defined so that the concentrations of hormones and nutrients could be manipulated precisely. In unsupplemented serum‐free media these cultures lose protein and DNA, indicating impaired viability. Addition of maximally effective concentrations of insulin (10−6 M) and transferrin (5 m̈g/ml) prevents loss of DNA and produces near neutral protein balance. Further addition of ascorbic acid (10−4 M) actually promotes net gain of protein with little or no increase in DNA. Ascorbate consistently increased noncollagen protein synthesis by cultured aortic smooth muscle cells. This novel action of the vitamin did not require insulin but was additive to the effect of this hormone, and was produced by isoascorbate, but not by a variety of other reducing agents. Thus, vascular smooth muscle cells can be maintained in a quiescent but noncatabolic state in simple chemically defined culture media. This finding should facilitate studies of the effects of nutrients and hormones on the metabolism of these cells under conditions that resemble those in the normal artery in vivo. Such an approach may also prove valuable for culture of other differentiated cell types that do not usually divide in the intact organism.
Stimulation of tyrosinase activity of cultured melanoma cells by lysosomotropic agents
Authors: Saeki Hisaaki , Oikawa Atsushi
Published: February 2005

The tyrosinase (EC 1.14.18.1) activity of cell‐free extracts (TyH) of B16 melanoma cells cultured in the presence of 5 to 10 mM ammonium chloride was considerably higher than that of cells from control cultures. This increase in TyH in the presence of ammonium chloride seemed to be due to de novo synthesis of the enzyme, because it was inhibited by 1 μg/ml of cycloheximide. In the presence of the latter, however, ammonium chloride did increase the tyrosinase activity of living cells in culture (TyC) resulting in about threefold increase in the TyC/TyH ratio, a measure of the extent of tyrosinase reaction exerted by the enzyme present in living cells. This higher TyC/TyH ratio induced by ammonium chloride was also observed in the absence of cycloheximide. Similar increases in TyH, TyC, and TyC/TyH occurred in the presence of methylamine or ethylamine instead of ammonium chloride, but not in the presence of tetraethylammonium chloride, and also in culture medium of higher pH. The apparently similar effects of lysosomotropic bases and medium of higher pH on the TyC/TyH ratio suggest that there are some mechanisms that control the intramelanosomal pH lower than the cytoplasmic pH.
Microtubules and lymphocyte responses: Effect of colchicine and taxol on mitogen‐induced human lymphocyte activation and proliferation
Authors: Cuthbert Jennifer A. , Shay Jerry W.
Published: February 2005

The role of microtubules in mitogen‐induced human lymphocyte activation and proliferation was examined. The effect of colchicine, a microtubule‐disrupting agent, was compared with taxol, a microtubule‐stabilizing drug, and with isaxonine (N‐isopropyl‐amino‐2‐pyrimidine orthophosphate), a proposed microtubular‐active drug. Lymphocyte proliferation, assessed by measuring the increase in the number of cells in mitogen‐stimulated cultures, was completely suppressed by both colchicine and taxol (100 nM) whereas significant inhibition by isaxonine required much higher concentrations (5 mM). In order to characterize the inhibition, initial lymphocyte blast transformation and subsequent DNA synthesis were investigated. Neither colchicine nor taxol inhibited lymphocyte blast transformation assessed by quantitating the change in volume of the stimulated cells after a 24‐hour incubation. In contrast, isaxonine (2–5 mM) suppressed blast transformation. Initial DNA synthesis, evaluated by measuring the cumulative incorporation of [3H]thymidine between 30 and 48 hours of culture, was inhibited in a concentration‐dependent manner by both isaxonine and colchicine but not by taxol. Electron microscopic studies confirmed that both taxol and colchicine (10 nM) arrested the responding lymphocytes in mitosis, and that isaxonine inhibited initial activation. These results suggest that normal microtubule function is only necessary for cell division and that drug effects on blast transformation and initial DNA synthesis are unrelated to microtubules.
Factors influencing calcium‐induced terminal differentiation in cultured mouse epidermal cells
Authors: Hennings Henry , Holbrook Karen A. , Yuspa Stuart H.
Published: February 2005

Mouse epidermal cells can be grown as a proliferating monolayer in medium containing 0.02‐0.1 mM calcium. Terminal differentiation of these cells with formation of cornified cells and cell death is induced by elevating calcium in the medium to >0.1 mM. A variety of agents were studied as potential modifiers of this calcium‐induced terminal differentiation. Other than calcium, no cation tested was active in inducing or preventing epidermal maturation. Modifiers of calcium or sodium fluxes, local anesthetics and protease inhibitors were also without effect. Modulators or analogues of cyclic nucleo‐tides did not influence epidermal differentiation, and cyclic nucleotide levels did not change significantly in the first 10 min after increasing calcium.

Effective inhibition of calcium‐induced differentiation, as estimated by morphology, ultrastructure and cornified envelope formation, was seen with the divalent cation ionophore A23187 and the Na+K+ATPase inhibitor ouabain. The well‐known effects of ouabain on intracellular sodium and potassium suggested the possible involvement of these ions in the program of calcium‐induced epidermal maturation. The increase in medium calcium produced an elevation of both intracellular sodium and potassium within 12–24 hours. The calcium‐induced increase in intracellular potassium appears to be the more relevant of these changes since the increase was blocked by both ouabain and A23187. Other inhibitors of calcium‐induced differentiation, including harmaline, 8(diethylamino) octyl 3,4,5‐trimethoxybenzoate hydrochloride (TMB‐8) and low potassium medium, also blocked the rise of intracellular potassium. The five inhibitors had no consistent effect on intracellular sodium. Thus, elevated intracellular potassium may be necessary for the later stages of epidermal differentiation. However, neither ouabain nor A23187 affected the assembly of desmosomes, the earliest ultrastructural change noted after increasing medium calcium. This rapid change in cell‐cell contact, beginning within minutes after calcium elevation, appears to be independent of changes in sodium and potassium, but may instead be modulated by increased calcium at the cell surface.
Deposition of fibronectin on material surfaces exposed to plasma: Quantitative and biological studies
Authors: Grinnell Frederick , Phan Trung V.
Published: February 2005

Experiments were carried out to characterize plasma fibronectin deposition onto material surfaces exposed to plasma solutions. Under nonclotting conditions, the amount of fibronectin adsorption on the surfaces, determined by an indirect radioactive antibody assay, was maximal at low plasma concentrations (0.1%). At higher concentrations of plasma, other plasma proteins appeared to compete with and inhibit adsorption of fibronectin. Biological activity (fibronectin‐promoted cell spreading) was also greatest at low plasma concentrations and decreased as the plasma concentration was raised. When surfaces were exposed to plasma under clotting conditions (i.e., addition of Ca2+ and thrombin), fibronectin deposition on the surfaces and biological activity remained constant or increased as the plasma concentration was raised. Based on indirect immunofluorescent antibody assays, the fibronectin deposited from clotting plasma appeared to be in a punctate distribution over the entire material surface and occasionally was associated with discrete fibrillar structures. The increased deposition of fibronectin from clotting plasma compared to nonclotting plasma (approximately a 10‐fold difference with 10% plasma) was partially a result of covalent crosslinking of fibronectin to fibrin based upon studies with putrescine added to inhibit crosslinking during clotting. On the other hand, the increase in biological activity that occurred if the surfaces were exposed to clotting plasma was completely inhibited by putrescine, indicating that fibronectin had to be crosslinked to fibrin to have biological activity under these conditions. Finally, fibronectin deposition also occurred on surfaces exposed to whole blood and was markedly enhanced when clotting occurred.
Modulation of cyclic 3′5′‐adenosine monophosphate in cultured renal (MDCK) cells by endogenous prostaglandins
Authors: Hassid Aviv
Published: February 2005

Cyclic AMP plays an important regulatory role in transport activity and proliferation of renal MDCK cells. This observation and the ability of MDCK cells to synthesize prostaglandins provided the impetus to assess the hypothesis that endogenous prostaglandins modulate cyclic AMP concentrations in MDCK cells. Three dissimilar cyclo‐oxygenase inhibitors: acetylsalicylate, 5, 8, 11, 14‐eicosatetraynoate, and meclofenamate significantly decreased cellular cyclic AMP levels and inhibited basal prostaglandin E2 synthesis. On the other hand, three dissimilar stimulators of prostaglandin synthesis: bradykinin, Ca2+‐ionophore A23187 and arachidonate, increased cellular cyclic AMP levels, and stimulated prostaglandin E2 synthesis. Acetylsalicylate inhibited the bradykinin‐ and A23187‐evoked increases of cyclic AMP as well as that of prostaglandin E2 synthesis. Prostaglandin E2, the major prostaglandin synthesized by MDCK cells, dose‐dependently increased cAMP levels when added exogenously. Acetylsalicylate did not significantly affect increases of cyclic AMP evoked by exogenous prostaglandin E2, documenting that acetylsalicylate inhibited cellular cyclic AMP levels by decreasing endogenous prostaglandin synthesis, rather than by a direct effect on cyclic AMP metabolism. Other prostaglandins synthesized by MDCK cells, i.e., prostaglandins I2, 6‐keto‐F1α, and F2α added exogenously did not significantly affect MDCK cyclic AMP levels, suggesting that they were probably ineffective as endogenous modulators of cyclic AMP. Moreover, endogenous prostaglandin E2 appeared four‐ to eightfold more potent as a stimulator of cyclic AMP levels than exogenous prostaglandin E2. The results support the concept that prostaglandin E2 is an endogenous cellular mediator that acts between an extracellular effector such as bradykinin and a second endogenous mediator of hormone action: cyclic AMP.
The centriolar antigen expression in TC7 cells is dependent on growth conditions and occurs at a particular time point in G1
Authors: Segarini P. R. , Shyamala M. , Atcheson C. L. , Kasamatsu H.
Published: February 2005

The correlation between growth conditions and centriolar antigen (Cag) expression in TC7 cells, a subline of African green monkey kidney cells, was studied. TC7 cells became quiescent when their number reached a high cell density, or when serum factors were depleted from media containing a low concentration of fetal bovine serum (FBS). There was a stoichiometric relationship between the concentration of serum present and the number of new cells produced. During proliferation, the projected cell area decreased as a function of cell density with two abrupt transitions. The first transition appeared to be independent of cell‐cell contact. However, the second transition seemed to occur mainly as a result of the limitation of the available substratum surface on which the cells could grow. The appearance of Cag in TC7 cells was found to be associated with the cells' growth conditions as well as with the particular phase in cell cycle. In an exponentially growing culture of cells with 10% FBS and in cells that were growth restricted due to a high density (above 2–3 × 105 cells/cm2), the incidence of cells with Cag‐positive staining was about 10–20%. It increased, however, to about 40–60% at cell densities between 2 × 104 cells/cm2 and 1 × 105 cells/cm2. The frequency of cells with positive Cag staining was as high as 80% in TC7 cells that were growth restricted by depletion of serum factor(s). Thus, the quiescent states attained by the two different growth restrictions seem to be different in their ability to express Cag. The frequency of Cag could be further increased by stimulating the quiescent cell population by FBS. In mitotically selected TC7 cells, Cag staining appeared about 4 hr after mitosis and about 2–4 hr before the onset of DNA synthesis. Thus, expression of Cag in TC7 cells is related to their growth conditions, and is characteristic of a part of the G1 phase of the cell cycle.