bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of differentiation and proliferation of haemopoietic cells in vitro
Authors: Dexter T. M. , Allen T. D. , Lajtha L. G. , Schofield R. , Lord B. I.
Published: February 2005

A liquid culture system, for haemopoietic cells, has been developed using bone marrow cells alone, or co‐cultures of thymus and bone marrow cells, inoculated into four ounce medical bottles. After several days growth, such cultures consisted of an attaching population of cells, forming discrete colonies, and a non‐attaching population. In the (co‐cultures) there was a 2 X enhancement of monolayer colony development compared with the combined total present in the (marrow alone) plus (thymus alone) cultures. Also, better maintenance of non‐attaching cells was seen in the (co‐cultures). Normal CFUS and CFUC were present in both the (marrow alone) and the (co‐cultures) for at least 14 days.

In the (marrow alone) cultures, granulocytes in all stages of development were present for the first week, but by 12 days the culture consisted mainly of mono‐nuclear cells. In the (co‐cultures), however, at 12 days more than 60% of the cells were granulocytes, in all stages of differentiation. (Co‐cultures) established using lethally irradiated thymus cells were not able to support this prolonged myeloid differentiation.

By feeding the (co‐cultures) it was possible to maintain production of (granulocytic) cells for at least ten weeks, although no fully mature granulocytes were observed. After the second feeding, no CFUS were detectable, but variable numbers of agar colony forming cells (not classical CFUC) were present at least for ten weeks.
Inactivation of protein synthesis stimulating activity in serum by cells
Authors: Kaminskas Edvardas
Published: February 2005

When Ehrlich ascites cells were cultivated in serum‐free media their cellular protein synthetic rate declined to a new steady‐state level and the cells stopped multiplying. On addition of serum the cellular protein synthetic rate increased to the level before serum starvation and cells resumed multiplication. The activity in serum stimulating protein synthesis was inactivated on incubation with cells. At cell concentrations of the usual culture conditions this inactivation took several hours; at very high cell concentrations it was complete in ten minutes. Serum‐starved cells inactivated low serum (2%–6%) media in the same length of time. Studies of inactivation of high serum media demonstrated that cells had a limted capacity to inactivate. Cells grown in 10% serum were unable to inactivate. Inactivation was not due to accumulation in the medium of either low molecular or macromolecular cell products. Inactivation was strongly inhibited at 4° or by treatment of cells with fluoride or cycloheximide (long exposure): less inhibited by treatment with 2‐deoxyglucose or glutaraldehyde; and slightly inhibited by treatment with cyanide or cycloheximide (short exposure). Inactivating ability was unaffected by trypsinization. These findings are best explained by the hypothesis that cells take up the serum activity by endocytosis.
Influence of the explantation milieu on intranuclear [Na], [K] and [Mg] of Chironomus thummi salivary gland cells
Authors: Kroeger Heinrich , Trösch Walter
Published: February 2005

Intranuclear Na, K and Mg concentrations were determined in cells of salivary glands incubated for 1h in selected NaCl/KCl/MgCl2 media. By variation of the external milieu beyond “physiological” limits the intranuclear electrolytes can be shifted between ca 100 and 280 mM [K]i, between ca 8 and 100 mM [Na]i and between ca 5 and 75 mM [Mg]i. No significant competition or interactions of the 3 ionic species are apparent. The relationships [K]e : [K]i and [Na]e : [Na]i can best be described by a positive and linear, that between [Mg]e : [Mg]i by a negative and exponential function. Regression parameters are given which permit a computation of intranuclear [Na], [K] and [Mg] as induced by NaCl/KCl/MgCl2 in any binary or triple combination that is tolerated by the explanted gland without visible damage.
Nucleoside diphosphate kinase at the cell surface of neoplastic human cells in culture
Authors: Ågren G. , Pontén J. , Ronquist G. , Westermark B.
Published: February 2005

Strong evidence is given that a nucleoside diphosphate kinase is present to some extent on the surface of intact neoplastic cells in culture. Experiments could be performed with cells cultured in a few plates to which an incubation medium was added. The cells were firmly attached to the supporting medium and remained viable during the incubation procedure.

Determinations of lactate dehydrogenase were carried out to rule out any possible contamination from the culturing medium as well as from the cell interior. From these analyses, a procedure was developed which easily removed the last traces of the culturing medium and which showed that there was no leakage of intracellular lactate dehydrogenase during the incubation procedure. There was a rather insignificant diffusion of nucleoside diphosphate kinase into the incubation medium. In common with other nucleoside diphosphate kinases, the glioma cell surface enzyme seemed to be nonspecific with regard to nucleotide substrates.
Reduced permeability in CHO cells as a mechanism of resistance to colchicine
Authors: Ling Victor , Thompson Larry H.
Published: February 2005

Colchicine resistant (CHR) lines of stable phenotype have been isolated from cultured Chinese hamster (CHO) cells. Successive single‐step selections for increasing resistance were performed by isolating resistant colonies at each step. Two complementary assays involving [3H] colchicine uptake by whole cells and binding of [3H] colchicine by cytoplasmic extracts were developed to test for altered permeability and altered intracellular target protein, respectively. All clones isolated appeared to have decreased permeability to the drug while their colchicine‐binding ability was not reduced. The amount of reduction in colchicine uptake correlated strongly with cellular resistance. The CHR lines were also cross resistant to other drugs such as actinomycin D, vinblastine and Colcemid; furthermore, the degree of cross resistance was positively correlated with the degree of colchicine resistance. The non‐ionic detergent Tween 80 potentiated the cytotoxic action of colchicine on mutant cells as well as its rate of uptake into whole cells.
Establishment of a clonal strain of hepatoma cells which maintain in culture the five enzymes of the urea cycle
Authors: Richardson U. Ingrid , Snodgrass Philip J. , Nuzum C. Thomas , Tashjian Armen H.
Published: February 2005

A clonal strain of epithelial cells has been established from the transplantable Morris hepatoma 7800 and is designated 7800C1. The cells grow with a population doubling time of about three days in serum‐supplemented synthetic medium. Cells of the 7800C1 strain have maintained measurable activities of all the enzymes of the urea cycle during 17 months in continuous culture. The activity of argininosuccinate lyase is approximately that found in normal rat liver, while argininosuccinate synthetase, carbamoyl phosphate synthetase, arginase and ornithine carbamoyl transferase activities are, respectively, 40%, 28%, 6%. and 1% of normal values. Treatment of 7800C1 cells with glucagon, dibutyryl 3′,5′‐cyclic adenosine monophosphate or hydrocortisone did not increase the activity of any of the five enzymes.
Effect of cAMP on nucleoside metabolism. I. Effect on thymidine transport and incorporation in monkey cells (CV‐1)
Authors: Roller Barbra , Hirai Kanji , Defendi Vittorio
Published: February 2005

The effect of cAMP on a monkey kidney cell line (CV‐1) noninfected and infected by SV40 was studied. No effect was found on either growth rate or cell morphology when concentrations up to 1 mM of 3′5′ cAMP were used. However, cAMP was found to increase the incorporation of 3H‐thymidine into both cellular and viral DNA without a net increase in DNA synthesis. This increased incorporation was found to be related to an enhanced uptake of thymidine into the nucleotide pool which is reflected in an increase of phosphorylated nucleotides. This, coupled with a lack of effect of cAMP on endogenous deoxyribonucleotide production, produces an increased specific activity of the deoxyribonucleotide triphosphates, with a resultant increase in specific activity of DNA.
Concanavalin A‐induced alterations in sodium and potassium content of Ehrlich ascites tumor cells
Authors: Aull Felice , Nachbar Martin S.
Published: February 2005

Net fluxes of sodium and potassium were studied in Ehrlich mouse ascites tumor cells during contact with the agglutinating protein, concanavalin A. This lectin altered cation transport markedly at concentrations of 20–105 μg/ml (6–47 μg/mg cell protein). Whereas control cells extruded sodium and maintained or accumulated potassium against electrochemical gradients, in the presence of concanavalin A there was rapid net sodium entry and potassium loss. After 10–20 minutes in concanavalin A, sodium extrusion began and potassium loss diminished but these events were prevented by ouabain. The alterations in cation content induced by concanavalin A are unlikely to be the result only of agglutination since soybean agglutinin caused much smaller changes although it agglutinated the cells equally well.
Pyrimidine excretion by cultured fibroblasts: Effect of mutational deficiency in pyrimidine salvage enzymes
Authors: Chan Teh‐Sheng , Meuth Mark , Green Howard
Published: February 2005

In cultured fibroblasts, a mutation resulting in deficiency of a pyrimidine salvage enzyme leads to excretion of related pyrimidines. For example, absence of thymidine kinase led to loss of thymidine and deoxyuridine, and absence of deoxycytidine kinase to loss of deoxyuridine. Both wild type and mutant cells excreted uracil; if established lines are representative in this respect, a fully adequate salvage system for uracil does not seem to be present in the fibroblast.
Serum and insulin initiation of DNA synthesis in mammary gland epithelium in vitro
Authors: Hsueh H. W. , Stockdale Frank E.
Published: February 2005

These experiments demonstrate that serum, like insulin, can initiate DNA synthesis in mouse mammary gland epithelium, resulting in a three‐ to four‐fold increase in the rate of DNA synthesis and number of cells synthesizing DNA. Both serum and insulin also increase the tritiated thymidine counts in the acid soluble material of these cells, suggesting that each alters thymidine transport. When combined at any concentration, these agents produce an additive effect on DNA synthesis, number of cells synthesizing DNA and thymidine transport. The factor(s) in serum responsible for these effects is associated with high molecular weight material. These experiments suggest that DNA synthesis and thymidine transport are affected independently by serum and insulin in mammary gland epithelium.
Erythroid colony formation in cultures of mouse and human bone marrow: Analysis of the requirement for erythropoietin by gel filtration and affinity chromatography on agarose‐concanavalin A
Authors: Iscove N. N. , Sieber F. , Winterhalter K. H.
Published: February 2005

Assay of red cell progenitors by colony formation in culture is expected to allow study of early events in erythroid differentiation in animal models and in man. In this communication, a simplification of the culture method for mouse bone marrow cells originally reported by Stephenson et al. ('71) is described. In the modified procedure, plasma clot is replaced by methyl cellulose, and scoring of erythroid colonies is done directly in the plates without staining. With additional modification the system proved applicable to the culture of erythroid colonies from human bone marrow as well. The development of both mouse and human erythroid colonies was dependent on “erythroid colony stimulating activity” (E‐CSA) supplied by extracts of anemic sheep plasma, or by extracts of urine from anemic patients. Over a certain range, the number of colonies was a function of dose of E‐CSA.

In addition to E‐CSA, these extracts also possessed erythropoietin activity as measured in plethoric mice. The possible chemical equivalence of urinary E‐CSA and erythropoietin was suggested by their similar behavior on both gel filtration and affinity chromatography on agarose‐concanavalin A. The finding further suggests that the culture method might prove useful for the bioassay of erythropoietin.

Granulocyte colony stimulating activity (G‐CSA) was also present in the urine extracts, as detected in cultures of mouse bone marrow. Virtually all of this activity was bound on agarose‐con A, whereas only a small fraction of E‐CSA was retained on this material. Agarose‐con A may thus be useful for the purification of erythropoietin.
Calcium transport in isolated bone cells II. Calcium transport studies
Authors: Dziak Rosemary , Brand John S.
Published: February 2005

Calcium transport was studied in bone cells isolated from fetal rat calvaria. 45Ca uptake experiments revealed an active component of calcium exchange. Calcium uptake was inhibited by iodoacetamide, DNP, CCCP and oligomycin and appeared to be dependent on medium phosphate concentration. Initial influx values exhibited saturation kinetics from 0.6 mM to 1.5 mM extracellular calcium.

Efflux of 45Ca from loaded cells increased in the presence of iodoacetamide, DNP and CCCP. Incubation of the cells af 4° C inhibited both influx and efflux of calcium.

Parathyroid hormone had no consistent effect on calcium uptake although characteristic increases in cyclic AMP levels were seen with the hormone. Calcitonin appeared to cause a transient increase in calcium uptake.
Some biological characteristics of a pituitary growth factor (CGF) for cultured lapine articular chondrocytes
Authors: Malemud Charles J. , Sokoloff Leon
Published: February 2005

A recently described pituitary chondrocyte growth factor (CGF) is a contaminant of several related glycoprotein hormones (TSH, LH and HCG). Chondrocytes were cultured from rabbits two to three months old. Bovine TSH (NIH) 69.5 μg/ml, used as the source of CGF, reduced the generation time from 16 to 10 hours through a virtual effacement of the G1 period. Incorporation of 3H‐thymidine declined rapidly after 48 hours from maximal values (control 300 cpm/μg DNA; CGF, 679). Total DNA accumulated thereafter until 116 hours when the figures were 36 and 98 μg/flask, respectively. Little growth response occurred in spinner cultures. CGF lowered plating efficiency from 4.5 to 2.3%. The stimulatory effect diminished when CGF was removed from the medium. The treated cells were smaller and contained less protein and RNA than controls. They synthesized smaller quantitites of sulfated mucopolysaccharides (chondroitin sulfates 4,6 and doubly sulfated chondroitin as well as some dermatan sulfate) but hyaluronate production was not diminished.
Formation of eosinophilic‐like granulocytic colonies by mouse bone marrow cells in vitro
Authors: Metcalf D. , Parker J. , Chester H. M. , Kincade P. W.
Published: February 2005

Formation of granulocytic and macrophage colonies in agar cultures of mouse marrow or spleen cells was stimulated by the addition of medium from pokeweed mitogen‐stimulated cultures of mouse spleen cells (PKW‐CM). Approximately 5% of the colonies developing were large, dispersed granulocytic colonies (DG‐colonies) composed of cells with eosinophilic cytoplasmic granules. The capacity to stimulate DG‐colonies was shown by media conditioned by PKW‐treated lymphoid and peritoneal cells but not by other cells or organ fragments.

Velocity sedimentation studies indicated that cells generating DG‐colonies were separable from cells generating regular granulocytic or macrophage colonies. DG‐colonies did not survive if transfered to cultures containing other forms of CSF. The active colony stimulating factor in pokeweed mitogen‐conditioned medium which stimulates DG‐colony formation was antigenically distinct from the factor stimulating granulocytic and macrophage colony formation, was separable electrophoretically from the latter factor and on gel filtration had an apparent molecular weight of 50,000.

Although the cells in DG‐colonies have not been established to be eosinophils, DG‐colonies represent an interesting new system for analysing further aspects of the control of growth and differentiation in hemopoietic populations.
Cell cycle dependent modulations of the surface membrane of normal and SV40 virus transformed 3T3 cells
Authors: Smets L. A. , De Ley L.
Published: February 2005

Agglutinability with Concanavalin was studied as function of cell cycle transition in normal and SV40 virus transformed 3T3 cells. In synchronized cultures of normal cells, agglutinbility was high during mitosis and disappeared rapidly. Agglutinability of transformed cells remained high in G1 phase but diminished gradually upon entering S phase and reached minimum in G1 phase. Decreased agglutinability a the end of the cell cycle was also observed in synchronous SV3T3 cultures by a combined technique of haemadsorption and density gradient centrifugation. In normal 3T3 cells, similar variations in agglutin ability during interphase could not be observed.
The role of the electrogenic sodium pump in modulation of pacemaker discharge of Aplysia neurons
Authors: Willis James A. , Gaubatz Gary L. , Carpenter David O.
Published: February 2005

The discharge of Aplysia pacemaker neurons varies with temperature over the range 10 to 22°C. Three types of frequency‐temperature plots are found, with maximal discharge at lowest, intermediate or highest temperatures. In the presence of ouabain, however, all cells show maximal discharge at the highest temperature, suggesting that the steady state activity of an electrogenic sodium pump is an important determinant of membrane excitability. The average magnitude of pump current, as indicated by the applied current necessary to restore discharge to control values after ouabain application, was about 4 namps at 20°C but near zero at 10°C. These neurons may be excellent models of mammalian thermoreceotprs.
Growth control in primary fetal rat liver cells in culture
Authors: Paul Dieter , Walter Stephanie
Published: February 2005

Primary fetal rat liver cells cultured in medium deficient in, but not free of, arginine in the presence of dialyzed fetal calf serum grow until the final cell density is attained and cells become quiescent in the G0 phase of the cell cycle. When growing cells are transferred into arginine free medium, cells become reversibly arrested in G0. Fetal rat liver cells can be induced to synthesize DNA by addition of high levels of arginine to serum free medium. Low arginine levels in the culture medium do not induce cell growth unless serum is present. Serum stimulates arginine uptake in fetal rat liver cells suggesting that serum growth factor(s) act by increasing intracellular arginine levels high enough to initiate the growth cycle. Fractionation of fetal calf serum by gel filtration on G‐200 Sephadex yields a partially purified arginine uptake stimulating activity which is eluted from the column in the same fractions that contain fetal rat liver cell growth promoting activity. Insulin induces DNA synthesis in quiescent fetal rat liver cells. Glucagon reverses the stimulatory effects of insulin. N6,O2‐Dibutyryl adenosine 3′:5′‐cyclic monophosphoric acid (But2c‐AMP) (10−4 M) and theophilline (10−3 M) inhibit arginine uptake and the initiation of DNA synthesis by serum. The role of arginine in the control of DNA synthesis in fetal rat liver cells and the mechanism of action of serum growth factors are discussed.
Cell to substrate adhesion and spreading: Inhibition by cationic anesthetics
Authors: Rabinovitch Michel , Destefano Maryjo
Published: February 2005

The plasma membrane is the postulated site of action of anesthetics on nerve or muscle. The drugs may be useful in the analysis of membrane phenomena in other cells. We show here that cationic anesthetics and tranquilizers inhibit cell adhesion and spreading, metabolically dependent processes that involve membrane motility and changes in cell shape. Adhesion was measured by layering 51Cr labeled Sarcoma I (Sa I) cells on glass coverslips for 20 minutes at 34°C, rinsing and estimating the glass‐associated radioactivity. Spreading was evaluated microscopically. Both cell adhesion to untreated glass and the Mn2+ dependent adhesion to serum‐coated coverslips were inhibited by the drugs, in the following order of increasing activity: tetracaine, promethazine, cyclomethycaine, chlorpromazine and fluphenazine. Similar ranks of drug activity have been reported for nerve blocking, inhibition of cell fusion and inhibition of induced spreading of macrophages. Microscopic observations showed the drugs also inhibited Mn2+ induced spreading of Sa I. Drug treated cells were rounded, refractile, devoid of cell processes or ruffles visible by light microscopy. The effects of the drugs on adhesion and spreading were reversible upon washing of the cells. We postulate that the inhibition of adhesion and spreading are a consequence of the inhibition of cell surface motility by the anesthetics.
Immunological characterization of hypoxanthine‐guanine phosphoribosyl transferase mutants of mouse L cells: Evidence for mutations at different loci in the HGPRT gene
Authors: Wahl G. M. , Hughes S. H. , Capecchi M. R.
Published: February 2005

A large collection (105) of mouse L cell mutants lacking hypoxanthine‐guanine phosphoribosyl transferase activity (HGPRT; E. C. 2.4.2.8) were analyzed for the presence of serologically cross reacting material (CRM). Antibody directed against highly purified mouse liver HGPRT was used for detecting CRM activity by two methods: (1) the standard precipitation‐inhibition assay; and (2) a radioimmune‐precipitation assay. The latter assay proved to have far greater sensitivity for the detection of altered forms of HGPRT. Approximately 40% of the HGPRT− cell lines contain CRM activity (i.e., were CRM+). This indicates that a minimum of 40% of the HGPRT− clones arose as a result of mutations in the HGPRT structural gene. The CRM+ cell lines were shown to contain different levels of CRM activity. Measurements of the heat sensitivity of CRM in the different HGPRT− cell lines showed a broad spectrum of CRM heat inactivation kinetics. These latter two observations provide strong evidence that the mutations giving rise to the HGPRT−CRM+ phenotype occurred at different sites in the HGPRT structural gene.
The role of sulfhydryl groups in cellular adhesiveness
Authors: George John V. , Rao K. Vasudeva
Published: February 2005

Aggregation of chick embryonic liver and kidney cells was completely abolished by a treatment with carboxypyridine disulfide which binds –SH groups. The effect could be reversed by a subsequent treatment with some thiols. Inhibition of RNA synthesis or respiratory metabolism did not prevent cell aggregation. Cell adhesion is discussed in the light of these observations.
Galactose and glucose metabolism in galactokinase deficient, galactose‐1‐P‐uridyl transferase deficient and normal human fibroblasts
Authors: Friedman Thomas B. , Yarkin Rhoda J. , Merril Carl R.
Published: February 2005

Despite the genetic interruption of the Leloir pathway both galactosemic patients and galactosemic fibroblasts can convert galactose to CO2 and TCA precipitable products, although at less than the normal rate. These observations stimulated investigations into the identity of the alternative metabolic routes which allow for galactose metabolism in the absence of in vitro galactose‐1‐P‐uridyl transferase. Four lines of galactosemic cells, each without detectable gal‐transferase, produced 14CO2 from [1‐14C]‐galactose (0.094 μmoles in 20 cc of medium) at approximately 39% ± 16% the rate of transferase positive cells over a 48‐hour period. However, galactokinase deficient fibroblasts produced 14CO2 and TCA precipitable products from [1‐14C] ‐galactose or [U‐14C] ‐galactose at only 3% to 9% the rate of normal fibroblasts. Therefore it seems likely that galtransferase deficient fibroblasts must first synthesize galactose‐1‐P for further metabolism of galactose.
Sodium and calcium transport in cat red cells
Authors: Sha'afi R. I. , Naccache P.
Published: February 2005

The transport of radioactive sodium and calcium in high sodium low potassium cat red blood cells has been studied under various experimental conditions. In these cells, calcium uptake was found to increase by 25‐fold when cell volume was decreased from 1.0 normal to 0.55. Increasing cell volume from 1.0 to 1.1 normal was found to decrease calcium‐uptake by 30%. N‐ethylmaleimide, NEM, phloretin, colchicine, and vinblastine were found to inhibit Na‐uptake by these cells. Among these four agents, phloretin was the most potent inhibitor of Na‐influx. It appears that phloretin produces its effect through its interaction with the cell membrane and not with cell interior.
Variations in the cell‐free translating apparatus of cultured animal cells as a function of time during cell growth
Authors: Engelhardt Dean L. , Sarnoski Joseph
Published: February 2005

Vero M3 cells, a line derived from the kidney of an African Green Monkey, display certain alterations in their protein synthetic apparatus as a function of time during a growth cycle. (Growth cycle here refers to exponential growth of unsynchronized cells in culture and their subsequent passage into the stationary phase.) The capacity of cytoplasmic extracts of these cells to promote endogeneous mRNA‐mediated polypeptide synthesis or poly U‐mediated polyphenylalanine synthesis declines from the second day after the initiation of the growth cycle. The ribosome sedimentation profile indicates that after the second day of growth a decrease also occurs in the total amount of ribosomes per cell, and that a shift occurs from predominantly polyribosome structures to predominantly subunits and monoribosomes structures. The activity of the translation factor, elongation factor 1, also progressively decreases after the second day of growth. Furthermore, when crude factor preparations from cells in the second day of growth (Exponential phase) and from cells in the fifth day of growth (Stationary phase) are compared for leucyl‐tRNA synthetase and prolyl‐tRNA synthetase activities, it is found that the extracts from fifth‐day cells have significantly less activity. The activity of another enzyme, acid phosphatase, remains relatively unaffected as a function of time during the cell growth cycle. When HeLa S3 plating cells are grown under the same conditions, they do not display the same responses.
The effect of serum on the transport and phosphorylation of 2‐deoxyglucose by untransformed and transformed mouse 3T3 cells
Authors: Hassell John A. , Colby Clarence , Romano Antonio H.
Published: February 2005

Serum starvation of growing and nongrowing (density‐inhibited) mouse 3T3 cells resulted in decreased phosphorylation of 2‐deoxy‐D‐glucose. while the time course of transport of this sugar remained unchanged. Serum starvation of SV40 transformed 3T3 cells (SV101) and spontaneously transformed 3T6 cells did not alter either the time course of transport, or phosphorylation of the sugar. Treatment of SV101 cells with 10−4 M dibutyryl adenosine cyclic 3′:5′ monophosphate and 10−3 M theophylline did not restore the capacity to regulate 2‐deoxy‐D‐glucose phosphorylation when these cells were serum deprived. We conclude that serum factors are involved in the modulation of phosphorylation of 2‐deoxy‐D‐glucose in 3T3 cells rather than its transport. This regulation is operative both in growing as well as nongrowing 3T3 cells. In contrast, transformed cells do not respond to this regulation of 2‐deoxy‐D‐glucose phosphorylation.
The role of cyclic AMP in myogenesis
Authors: Epstein Charles J. , De Asua Luis Jiminez , Rozengurt Enrique
Published: February 2005

The effect of exogenously administered cyclic AMP derivatives and of endogenously elevated cyclic AMP levels on the spontaneous fusion of skeletal muscle myoblasts has been investigated. Contrary to earlier reports, cAMP does not appear to have a direct inhibitory effect on the fusion of an established line (L8) of rat myoblasts. Similarly, cAMP did not block the fusion of primary chick myoblasts. However, fusion of the rat myoblasts was prevented when the cAMP induced inhibition of growth prevented the cells from reaching the “critical” cell density necessary for fusion.
Preparation and neutralization characteristics of an anti‐CSF antibody
Authors: Shadduck Richard K. , Metcalf Donald
Published: February 2005

Nine of ten rabbits immunized with a partially purified L‐cell CSF had demonstrable titers of anti‐CSF activity. In vitro the antibody was markedly inhibitory to both post‐endotoxin mouse sera and several mouse tissue extracts. CSF containing conditioned media prepared from a number of sources showed variable inhibition suggesting that murine CSF's may be characterized by marked antigenic differences. Human sources of CSF were also inhibited thus indicating a degree of cross‐reactivity between murine and human factors. These studies may provide the initial steps toward definition of the role of CSF in vivo.
Failure of tritiated thymidine incorporation into DNA to reflect the autoradiographically demonstrable calcium‐induced increase in thymic lymphoblast DNA synthesis
Authors: Youdale T. , Macmanus J. P.
Published: February 2005

Increasing the extracellular calcium concentration in thymic lymphocyte suspension from 0.6 to 1.8 mM stimulated the proliferation of the lymphoblast subpopulation as measured by increases in the proportion of cells autoradiographically labeled with 3H‐TdR and in mitotic activity. However it was not possible to show this increased DNA synthesis by scintillometric measurement of the amount of 3H‐TdR incorporated into extracted DNA. On the other hand, calcium did raise the incorporation of 14C‐formate into the thymine residues of DNA, and increased the activity of isolated thymocyte thymidylate synthetase. In contrast to the mitogenic calcium ion, a thymidylate synthetase inhibitor, methotrexate, actually increased the incorporation of 3H‐TdR into DNA.

It is concluded that calcium increases the endogenous synthesis of thymidylate which in turn prevents the amount of incorporation of exogenous 3H‐TdR from accurately reflecting the true level of DNA synthesis.
Polyamine metabolism in a rat brain tumor cell line: Its relationship to the growth rate
Authors: Heby Olle , Marton Laurence J. , Wilson Charles B. , Martinez Hugo M.
Published: February 2005

To investigate whether the metabolism of the polyamines putrescine, spermidine and spermine is related to cellular growth rate, we have measured the activities of L‐ornithine decarboxylase and S‐adenosyl‐L‐methionine decarboxylase as well as the levels of the polyamines in rat brain tumor cells at various stages of a 7‐day in vitro growth period and correlated them with the continuous changes in specific growth rate ([dN[t]/dt]/N[t]). L‐Ornithine decarboxylase and S‐adenosyl‐L‐methionine decarboxylase both exhibited their maximal activities at the time of most rapid growth. A high positive correlation between the activities of these enzymes and the specific growth rate of the tumor cells during the entire growth period was demonstrated statistically. The pattern of fluctuation of the spermidine content during the culture cycle was similar to those of the enzyme activities and likewise showed a high positive correlation with the specific growth rate of the tumor cells during the entire growth period. The putrescine content exhibited a low positive correlation, whereas the spermine content exhibited a somewhat higher, but negative correlation with the specific growth rate.

The high correlation between the specific growth rate of the tumor cells and the synthesis of the polyamines indicates that these events are primarily associated with processes involved in cell replication. Putrescine and spermidine are thought to participate in the regulation of cellular growth rate; a high content may augment, and a low content may restrain, cellular growth rate.
A mechanical dissociation method for preparation of muscle cell cultures
Authors: Tepperman Katherine , Morris Glenn , Essien Francine , Heywood Stuart M.
Published: February 2005

A cell preparation method by which large numbers of embryonic chick skeletal muscle cells may be obtained is described. The procedure requires fewer manipulations and much less time than standard trypsinization. By the criteria used, both methods are comparable with respect to percent viable cells and survival of plated cells. However, in addition to the ease of preparation, the mechanical dissociation method offers the significant advantage that the cell suspension is greatly enriched for myoblasts without the necessity of an additional preplating step.
Effects of prolonged quiescence on nuclei and chromatin of WI‐38 fibroblasts
Authors: Rossini Mara , Lin Jung‐Chung , Baserga Renato
Published: February 2005

DNA synthesis and cell division are markedly reduced in confluent mono‐layers of WI‐38 diploid fibroblasts, but resume again if the depleted medium is replaced by fresh medium containing 10% fetal calf serum. If the cells are kept quiescent for prolonged periods of time after confluence (1 or 2 weeks), the fraction of cells that can be stimulated to proliferate by fresh serum decreases and the length of the prereplicative phase increases. The template activity of isolated nuclei decreases with increasing time of quiescence, and parallel changes occur in chromatin as evidenced by circular dichroism spectra and capacity to bind the intercalating dye, ethidium bromide.

When WI‐38 cells are stimulated to proliferate after prolonged quiescence, the increase in template activity of nuclei is delayed by several hours in comparison to cells stimulated after short periods of quiescence. Two distinct steps, both requiring serum, can be identified in the prereplicative phase of cells stimulated to proliferate after prolonged quiescence.

We interpret the results as indicating that, during prolonged quiescence, WI‐38 fibroblasts go into a deeper G0 state from which they can be rescued only after prolonged stimulation. In this respect, prolonged quiescence may bear some resemblance to the process of aging.
Interrelationships between water and cellular metabolism in Artemia cysts IV. Adenosine 5′‐triphosphate and cyst hydration
Authors: Clegg James S. , Cavagnaro Joy
Published: February 2005

The concentration of ATP in cysts of the brine shrimp, Artemia salina, has been studied as a function of cyst hydration. Cysts dried over CaSO4 contain 0.02 gH2O/g cysts and 0.54 ± 0.05 (S.D.) m̈moles of ATP/g dried cysts. Addition of water up to 0.05 g/g cysts produced no net change in the level of ATP during incubation. Hydration levels between 0.05 and 0.62 g/g cysts resulted in a net loss of ATP, whereas above 0.65 g/g cysts a net increase was observed with incubation time. No net change in the amount of ATP, compared with dried cysts, was detected between the latter two hydrations. These results, when integrated with those from previous work, indicate that conventional aerobic energy metabolism does not begin until cyst hydrations of about 0.65 g/g are achieved. The fate of ATP in cysts hydrated to levels lower than 0.65 g/g was discussed.
Studies on the intranuclear distribution of human and mouse genomes and formation of human‐mouse hybrid cells
Authors: Rechsteiner Martin , Parsons Bruce
Published: February 2005

Autoradiographic examination of early hybrid cells formed by the fusion of 3H‐thymidine labeled D98/AH2 cells and 3T3‐4E cells revealed that human and mouse chromosomes are often separated within metaphase and interphase nuclei. Although the marked separation of human and mouse chromosomes progressively disappeared with succeeding cell division, the occurrence of sectored nuclei in 16‐cell hybrid colonies and the labeling pattern of human chromosomes within separated metaphases indicate that separation of human and mouse chromosomes may persist through several mitoses.

Fusion of 3H‐thymidine labeled D98/AH2 cells and 3T3‐4E cells was coupled with aminopterin selection to study other aspects of hybrid cell formation. Hybrid cells arose from heterokaryons by cell division. Nuclear fusion may have occurred in two out of several thousand heterokaryons examined. However, these could reflect the close apposition of adjacent nuclei. A large fraction (> 0.5) of D98/3T3 heterokaryons underwent at least one cell division. However, the number of hybrid colonies containing more than eight cells at ten days following fusion was about 0.03 of the total number of heterokaryons. Many hybrid colonies arrested growth before the 8‐cell stage, and the cells in such colonies exhibited nuclear abnormalities.
Development of a clonal myogenic cell line with unusual biochemical properties
Authors: Brandt Bruce L. , Kimes Brian W. , Klier F. George
Published: February 2005

The morphological, ultrastructural, biochemical and electro‐physiological properties of B104‐F, a clonal cell line derived from a nitrosoethylurea‐induced neoplasm in a rat, were studied as a function of the growth phase of the culture. Cells in exponentially growing cultures are mononucleate and produce action potentials when stimulated electrically. Stationary phase cultures contain three types of cells: cells of the first type are mononucleate and have long processes containing microfilaments and many parallel microtubules; cells of the second type are mononucleate but contain no microtubules and few microfilaments; and cells of the third type have ultrastructural features typical of multinucleate, striated myotubes. Multinucleate cells generate action potentials with both sodium and calcium components and are depolarized by acetyl‐choline. The acetylcholine response is blocked by d‐tubocurarine. The specific activity of creatine phosphokinase is nine times higher in stationary phase cultures than in exponentially growing ones while the myokinase specific activity is unchanged. The gamma‐aminobutyric acid content of the cells is 3.5‐ to 26‐fold higher in stationary phase than in exponentially growing cultures, depending on the degree of fusion of the culture. The properties of B104‐F are discussed in relation to the properties of developing skeletal muscle and of central nervous system cell lines.
Establishment in culture of a multihormone‐secreting cell strain derived from the mtT/F4 rat pituitary tumor
Authors: Richardson U. Ingrid
Published: February 2005

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin‐releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.
Cellular responsiveness to stimulation in vitro: Increased responsiveness to colony stimulating factor of bone marrow colony‐forming cells treated with surface‐active agents and cyclic 3′5′ AMP
Authors: Fleming W. A. , McNeill T. A.
Published: February 2005

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte‐macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10−8 to 10−10 M) also enhanced colony numbers although concentrations above 10−5 M were inhibitory. Enhancement was found when marrow cells were pre‐treated with these agents and cultured in their absence.

The agents did not stimulate colony development in the absence of colony‐stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony‐stimulating factor which was sub‐optimal in terms of maximum colony development.

There was no indication of increased colony‐stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony‐forming cells to colony‐stimulating factor.
Transport and countertransport of thymidine in ATP depleted and thymidine kinase‐deficient novikoff rat hepatoma and mouse L cells: Evidence for a high Km facilitated diffusion system with wide nucleoside specificity
Authors: Plagemann Peter G. W. , Marz Richard , Erbe John
Published: February 2005

Incubation of cultured Novikoff rat hepatoma and mouse L cells in a glucose‐free basal medium containing 5 mM KCN and 5 mM iodoacetate for about 10 minutes resulted in a complete depletion of the cells of ATP. ATP‐depleted wild type cells or thymidine kinase‐deficient sublines of Novikoff or L cells took up thymidine rapidly from the medium without concentrating it intracellularly, and exhibited countertransport of thymidine. Thus uptake was by facilitated diffusion. This transport system differs from the substrate‐specific, low‐Km (0.5 μM) thymidine transport system previously described for various types of cultured cells in that it exhibits an at least 100‐fold higher Km and transports equally well various ribo‐ and deoxyribonucleosides. The results suggest that the rate‐limiting step in thymidine incorporation into the nucleotide pool by wild type cells is phosphorylation rather than transport, or that the cells possess two transport systems, a facilitated diffusion system with low substrate specificity and a second system which involves substrate phosphorylation by thymidine kinase.
Density‐dependent changes in the amount of sulfated glycosaminoglycans associated with mouse 3T3 cells
Authors: Underhill C. B. , Keller J. M.
Published: February 2005

The relative amount of sulfated glycosaminoglycans associated with the cell layer of parent and SV40‐transformed Swiss mouse 3T3 cells was determined from the incorporation of labeled sulfate (35SO4) into macromolecular material. In cultures of SV40‐transformed cells, the glycosaminoglycan content per cell was constant over a wide range of densities. In cultures of parent 3T3 cells, the glycosaminoglycan content per cell increased directly with density, the highest values being found in contact‐inhibited cultures. At high cell densities, the glycosaminoglycan content of 3T3 cells was several‐fold higher than that for SV40‐transformed cells. Most of the density‐dependent increase in glycosaminoglycans of 3T3 cells was accounted for by chondroitin sulfate (dermatan sulfate) which was over 6‐fold higher in confluent cultures than in low density cultures.
Ornithine decarboxylase inactivation in HeLa cells
Authors: Prouty W. F.
Published: February 2005

Ornithine decarboxylase (ODC) can be induced up to 100‐fold over basal levels four hours after addition of glutamine to the medium of HeLa cells growing in suspension culture. As demonstrated in several other cell types, ODC is inactivated very rapidly in HeLa cells, and the rate of inactivation is seen to vary with a half life of 9–15 minutes in uninduced cells and rises to ca. 60 minutes at the peak of induction. Quantitatively, the change in rate of inactivation cannot completely account for the observed rise in activity, thus synthesis or activation of ODC must also be involved in the induction process. The inactivation process requires metabolic energy and it can be sustained by glycolytic derived energy. Other factors which are known to inhibit protein breakdown in mammalian cells, such as sodium fluoride, insulin, or tosyl phenylalanyl chloromethyl ketone, had no effect on the rate of inactivation of ODC. Attempts to demonstrate ODC inactivation in a cell free system at neutral pH were unsuccessful.
Growth of in vitro colony‐forming cells from normal human peripheral blood leukocytes cultured in diffusion chambers
Authors: Hoelzer D. , Harriss E. B. , Slade M. , Kurrle E.
Published: February 2005

The growth of granulopoietic progenitor cells (CFU‐C) in diffusion chambers during culture of peripheral blood leukocytes from 10 normal subjects has been studied. At various times after initiation of diffusion chamber culture, cells harvested from the chambers were transferred to agar culture for measurement of CFU‐C concentration. Under these conditions colonies could be grown successfully in agar culture provided pronase, necessary for the chamber harvesting procedure, was first removed by careful washing.

A marked increase in the number of CFU‐C, up to 25‐fold the initial value, was observed in 8 out of 10 subjects. Here the growth pattern was similar, independent of the initial CFU‐C values, with an immediate rise to a maximum between 6 and 13 days of culture followed by a decrease. In the other two subjects the growth of CFU‐C throughout the diffusion chamber culture period was very poor. The growth of CFU‐C from a given individual's blood was shown to be reproducible in repeated studies in 2 subjects, one of whom showed a proliferative and the other a non‐proliferative pattern.

Evidence suggests that the increase in CFU‐C in diffusion chambers is the result of both self‐renewal of these cells and influx from a more primitive compartment, although the present data do not allow an estimate of the relative magnitude of each.
Cell cycle dependent changes in potassium transport
Authors: Mills Barry , Tupper Joseph T.
Published: February 2005

K transport has been investigated during progression of cultured Ehrlich ascites tumor cells through the cell cycle. Using a double thymidine block technique, Ehrlich cells carried in continuous culture have been synchronized, as verified by simultaneous monitoring of cell number, cell volume, 3H‐thymidine incorporation and mitotic index. Unidirectional influx, efflux and cell content of K have been monitored throughout the cell cycle. The nature of the pump mediated, ouabain‐sensitive K flux and the furosemide‐sensitive component of K flux, presumably representing K‐K exchange, have also been evaluated. In early S period the ouabain sensitive component, representing the Na‐K pump, comprises 52% of the total unidirectional K influx. During S period the pump activity increases to a maximum of 65% of the unidirectional K influx and subsequently declines during G2 period to a minimum of 40% in mid G2. During M and early S the activity again rises. As the ouabain sensitive component becomes maximal in late S period, the furosemide sensitive component diminishes from approximately 30% of the total influx to approximately 10%. The same pattern is observed in the G2 period. As the pump component diminishes, the furosemide sensitive component increases. Furosemide sensitive K efflux has also been monitored and the pattern is equivalent to that observed in the influx studies. No change in net K flux is observed in the presence of furosemide. This indicates that the furosemide sensitive component represents an exchange component for K. These results are consistent with the conclusion that the alterations in exchange and pump fluxes are physiological events associated with progression of the cell cycle.