Showing papers in "Journal of Cellular Physiology in 1969"

Separation of cells by velocity sedimentation.

Abstract: A system for fractionating populations of living cells by velocity sedimentation in the earth's gravitational field is described. The cells start in a thin band near the top of a shallow gradient of 3% to 30% fetal calf serum in phosphate buffered saline at 4°C. Cell separation takes place primarily on the basis of size and is approximately independent of cell shape. A sharply-defined upper limit, called the streaming limit, exists for the cell concentration in the starting band beyond which useful cell separations cannot be achieved. This limit, which varies with the type of cell being sedimented, can be significantly increased by proper choice of gradient shape. For sheep erythrocytes (sedimentation velocity of 1.6 mm/hour) it is 1.5 × 107 cells/ml. Measured and calculated sedimentation velocities for sheep erythrocytes are shown to be in agreement. The technique is applied to a suspension of mouse spleen cells and it is shown, using an electronic cell counter and pulse height analyzer, that cells are fractionated according to size across the gradient such that the sedimentation velocity (in mm/hour) approximately equals r2/4 where r is the cell radius in microns. Since cells of differing function also often differ in size, the system appears to have useful biological applications.

Formation and properties of thin‐walled phospholipid vesicles

Abstract: Large numbers of thin-walled vesicles, 0.5 to 10 μ in diameter, can be formed by permitting a thinly spread layer of hydrated phospholipids to swell slowly in distilled water or an aqueous solution of nonelectrolytes. Electron micrographs and phospholipid analyses indicate that the walls consist of a single or a few bilayers. The vesicles can be centrifuged and resuspended in another medium, making them a useful system for studying permeability. The osmolarity of the solution in the interior of the vesicles can be estimated by immersion refractometry. The osmolarity of the internal aqueous phase is linearly related to the osmolarity of the external medium.

Differentiation of a cell line of myeloid leukemia

Abstract: A cell line was established in vitro from a spontaneous myeloid leukemia of SL strain mice. This cell line was cytologically identified as myeloblast, and its normal differentiation appeared to be completely blocked in mass culture. When the line cells were seeded in soft agar with a conditioned medium from normal cells, either macrophages or neutrophil granulocytes appeared from a single clone. The rate of formation of colonies containing differentiated cells always increased with an increase in the concentration of conditioned medium. The conditioned medium from this line cell was not as effective as was that from normal cells in inducing differentiation.

Physical separation of hemopoietic stem cells from cells forming colonies in culture.

Abstract: Mouse bone marrow cells in suspension were separated into a number of fractions on the basis of cell density by equilibrium density gradient centrifugation, or on the basis of cell size by velocity sedimentation. After each type of separation, the cells from the various fractions were assayed for their ability to form macroscopic spleen colonies in irradiated recipient mice, and for their ability to form colonies in a cell culture system. The results from either separation technique demonstrate that cells in some fractions formed more colonies in vivo than in the culture system, while cells in other fractions formed more colonies in culture than in the spleen. The results of control experiments indicate that this separation of the two types of colony-forming cells was not an artifact of the separation procedures. From these experiments it was concluded that the population of cells which form colonies in culture under the conditions used is not identical to the population of cells detected by the spleen colony assay.

Genetics of somatic mammalian cells. IX. Quantitation of mutagenesis by physical and chemical agents

Abstract: The system previously described for inducing single gene mutations in Chinese hamster cells has been extended to produce additional auxotrophic mutants. An improved method for quantitating the efficiency of single gene mutation to specific auxotrophies has been developed. Mutagenesis in the forward direction has been measured after treatment of these cells with ethyl methanesulfonate, N-methyl-N1-nitro-N-nitrosoguanidine, hydroxylamine, an acridine mustard (ICR-191), caffeine and ultraviolet- and X-irradiation. For each agent, the single cell survival curve and the efficiency of chromatid breakage and rearrangement were measured. Similar measurements were also carried out with a water-soluble carcinogen N-nitrosomethylurea, which was shown to be effective in producing auxotrophic, somatic mutations. These results offer promise of illuminating the relationships between cell killing, chromosomal aberration, single gene mutations and carcinogenesis produced by various agents. The methods described can be used in routine testing of drugs, food additives, and environmental pollutants for mutagenic action in mammalian cells in vitro.

Viability of human diploid cells as a function of in vitro age.

Abstract: The fraction of cell capable of division was determined for (1) population of the human diploid cell strains, WI38 after different numbers of subcultivations in vitro and (2) a single population of WI38 cells at intervals throughout its entire in vitro lifespan. In both cases the percentage of cells capable of division decreased with increasing age in tissue culture. The rate and the magnitude of the decrease is sufficient to account for the limited in vitro lifespan reported by other investigators. Furthermore, the decrease in the fraction of cells capable of division in similar in some respects of senescence among human populations.

Early alterations in amino acid pools and protein synthesis of diploid fibroblasts stimulated to synthesize DNA by addition of serum.

Abstract: Diploid human fibroblasts in culture (WI 38) were allowed to reach a stationary phase and were then stimulated to reenter DNA synthesis and cell division by addition of serum to the culture medium. The rate of protein synthesis increased during the first hours after addition of serum reaching at three hours a plateau value that continued for at least 24 hours after serum addition. Inhibition of protein synthesis during the early hours after serum addition abolished the stimulation of DNA synthesis occurring 20 to 28 hours later. Increased protein synthesis was preceded by a rapid decrease in the intracellular pool size of most amino acids. These changes were independent of concomitant protein synthesis. They suggest that serum exerts an immediate effect on the function of the cell membrane.

Fluctuations of DNA‐dependent RNA polymerase and synthesis of macromolecules during the growth cycle of Novikoff rat hepatoma cells in suspension culture

Abstract: The transition of suspension cultures of Novikoff rat hepatoma cells from the exponential to the stationary phase is accompanied by decreases of over 90% in the rates of synthesis of RNA, DNA and protein, a 90% loss of the apparent DNA-dependent RNA polymerase activity of the cells, and a disaggregation of the polyribosomes with a concomitant accumulation of 80 S and 110 S ribosomal structures. The cells also attain a minimum content of DNA, RNA and protein and a minimum size. Upon dilution of stationary phase cultures with fresh medium, the rate of protein synthesis begins to increase immediately and this correlates with a rapid reformation of the polyribosomes. The initial re-formation of polyribosomes is little affected by the presence of actinomycin D. RNA polymerase activity also begins to increase immediately after dilution and an increase in rate of RNA synthesis becomes apparent shortly thereafter. The increase in polymerase activity is inhibited by treating the cells with puromycin or actidione. Cell division commences only 9–13 hours after dilution and the rate of DNA synthesis begins to increase about midway through the lag period. During the lag period the average cellular content of protein increases about 80% and that of RNA and DNA about 30%. These increases are accompanied by a marked increase in the average size of the cells. Upon continued incubation of stationary phase cultures, the cells become irreversibly damaged physiologically before gross morphological damage becomes apparent. The irreversible physiological damage is recognized by the fact that the cells fail to recover when suspended in fresh medium.

Studies on colony formation in vitro by mouse bone marrow cells. I. Continuous cluster formation and relation of clusters to colonies.

Abstract: Colony formation in vitro by mouse bone marrow cells following stimulation by human urine was analysed over a 7-day incubation period. There was a linear increase with time in the number of cell aggregates (clusters) developing in such plates. Early in the incubation period all clusters were granulocytic although later macrophage clusters developed. Although most fully developed colonies were composed of macrophages, mapping and transfer studies showed that at least half of these had initially arisen early in the incubation period as granulocytic clusters.

Impairment of deoxyribonucleic acid synthesis by dietary zinc deficiency in the rat.

Abstract: Dietary zinc deficiency has been shown to decrease the in vivo incorporation of thymidine into the nuclear DNA of liver parenchymal cells in the rat. A single injection of 100 μg of zinc was sufficient to rapidly reverse the effect of zinc deficiency on the synthesis of DNA.

Phenylalanine hydroxylase activity in mammalian cells.

Abstract: Survey of twelve mouse tissues revealed the presence of appreciable phenylalanine hydroxylase activity in the pancreas and kidney as well as the liver but in no other of the tissues tested. Single cell suspensions of mouse liver were prepared by use of tetraphenylboron. The enzyme activity of such suspensions was much more stable than that of liver extracts, and permitted determination of the Michaelis-Menten constant, the pseudo-first order reaction velocity constant on a cell-number basis, and the temperature coefficient and apparent activation energy of the enzyme activity. Possible applications of these methods to problems in cellular biology have been indicated.

Thymidine as a synchronizing agent. I. Nucleic acid and protein formation in synchronous HeLa cultures treated with excess thymidine.

Abstract: Synchronous cultures of HeLa cells obtained by selective detachment of mitoses were treated with high concentrations of thymidine. The inhibitor was added soon after completion of cell division and rates of cell enlargement and accumulation of DNA, RNA and protein were compared for untreated and thymidine-treated cultures at various points of the cell cycle. It was found that concentrations of thymidine which in randomly growing cultures inhibit the rate of cell division by more than 90% allowed a considerable degree of DNA synthesis and did not affect the rate of accumulation of RNA and protein, when applied to cells in the G1 phase of synchronous culture. Treated and untreated cells enlarged at the same rate throughout their life cycle. The results show that concentrations of thymidine commonly employed to produce cell synchrony do not arrest the cells at the G1-S boundary, but allow slow progress through S in respect to DNA synthesis, and near-normal progress towards G2 as regards RNA and protein accumulation and cell enlargement.

Transcriptional controls in developing bacteriophages

Abstract: The development of a virus is programmed by a series of negative and positive controls which determine the timing and the segment on either of the two DNA strands (l or r) to be transcribed into specific messenger RNA's. Bacteriophage λ provides one of the most deeply studied systems for following the development of lysogenic viruses. In the lysogenic repressed state, only 2–4% of the λ genome is expressed. This pc-cI-rex region is transcribed leftward to produce a repressor protein which prevents any further transcription by blocking the oL and oR operators flanking the cI-rex operon (figs. 1, 2). This negative control is relieved by destruction of the repressor, and the result is the “induction” of viral development. The earliest post-induction or postinfection events are the leftward transcription of the pLoL N region from strand l and the rightward transcription mainly of the pRoR-x segment from strand r. The N product acts as a positive control, permitting a leftward transcription beyond gene N and a rightward transcription of genes cII-O-P and also Q. The int-xis system controls the excision of the λ genome, whereas the act of rightward transcription and the products of genes O and P initiate the replication of λ DNA. The product of gene Q, still another positive control, stimulates rightward transcription of the late genes which control the synthesis and assembly of the phage heads and tails as well as cell lysis. Among other types of negative control are the possible competition between the two divergent transcriptions originating in region x, the “antirepressor” effect of the x product, and the interference between the two convergent transcriptions which collide in the central b2 region. The majority of controls are based on protein-DNA interactions and can be modified by mutations. For instance, transcription can be rendered independent of negative repressor control either by constitutive, v, mutations which decrease or abolish the affinity of the o operators for the repressor or by insertion of new promoters–e.g., c17 or ric- on the “downstream” side of the operator. The need for the positive N and Q controls may also be obviated by mutations in the N- or Q-dependent promoter or terminator elements. The specific DNA structure within the controlling sites is not known. However, a remarkable coincidence was observed; namely, the occurrence of pyrimidine-rich clusters in those segments of the individual DNA strands acting as templates for RNA synthesis. This observation, which pertains to all studied DNA's, including those of phages T2, T3, T4, T5, T6, T7, λ, and ϕ 80, formed the basis for a proposal that implicates pyrimidine-rich clusters in the initiation, control and/or termination of transcription, and also in the determination of the preferred strand and, consequently, the orientation of transcription. General considerations regarding the possible role of the structural singularities, especially those represented by the pyrimidine clusters, in the bipartite structure of the recognition regions in DNA are discussed.

Decreased neutrophils and megakaryocytes in anemic mice of genotype W/W.

Abstract: The concentration of neutrophils and megakaryocytes was determined in the marrow of anemic mice of genotype W/Wv and their normal (+/+) litter mates. In all groups studied, the humerus of W/Wv mice contained significantly less neutrophils and megakaryocytes than did normal animals. Blood neutrophil concentration was less in all groups of W/Wv mice but in only one group which was the youngest group studied, did this value differ significantly from normal. The blood and marrow neutrophil response to endotoxin was similar in W/Wv and “+/+” animals. This suggests that the neutrophilic system of W/Wv mice responds to this stimulus in a relatively normal manner, much as their erythroid system responds to hypoxia, and androgens.

Relationship between uridine kinase activity and rate of incorporation of uridine into acid‐soluble pool and into RNA during growth cycle of rat hepatoma cells

Abstract: Uridine kinase activity measured in cell-free extracts of Novikoff rat hepatoma cells grown in suspension culture fluctuates about 10 fold during the growth cycle of the cells. Maximum specific activity (units/106 cells) is observed early in the exponential phase and then decreases progressively until the stationary phase. The rate of incorporation of uridine into the acid-soluble pool by intact cells fluctuates in a similar manner and both the rate of uridine incorporation by intact cells and the uridine kinase actvity of the cells increase several fold before cell division commences following dilution of stationary phase cultures with freshmedium. Regardless of the stage of growth, uridine is rapidly phosphorylated to the triphosphate level by the cells. The rates of incorporation of uridine into the nucleotide pool and into RNA by intact cells fluctuate in a similar manner during the growth cycle. However, evidence is presented that indicates that alterations in the rate of incorporation of uridine into RNA are not simply due to changes in the rate of phosphorylation of uridine, but are regulated independently. Inhibition of protein synthesis by treating cells with puromycin or actidione causes a marked inhibition of incorporation of uridine into RNA, but has little effect on the phosphorylation of uridine to UTP for several hours. Thus the depression of incorporation of uridine into RNA probably reflects a decrease in the rate of RNA synthesis as a result of inhibition of protein synthesis. Inhibition of RNA synthesis by treating cells with actinomycin D does not affect the rate of conversion of uridine to UTP and thus results in the accumulation of labeled UTP in treated cells.

Changes in DNA polymerase activity associated with cell fusion in cultures of embryonic muscle.

Abstract: In cultures of differentiating chicken embryo muscle cells there is a steep decline in DNA polymerase activity which closely parallels the time of rapid cell fusion and the formation of multinucleated myotubes. The DNA polymerase activity remaining in the cultures is almost completely associated with single unfused cells. Cell fusion does not require a confluent culture and fusion capability appears to be severely reduced in the remaining single cells following an approximately ten hour time period during which the majority of fusion takes place. A model is presented to explain the observed kinetics of cell growth and cell fusion in vitro.

The calcium-mediated promotion of mitotic activity in rat thymocyte populations by growth hormone, neurohormones, parathyroid hormone and prolactin.

Abstract: Growth hormone, oxytocin, parathyroid hormone, prolactin and lysine vasopressin strongly stimulate mitotic activity in rat thymocyte populations maintained in vitro. These hormones have no mitotic effect on cells maintained in calcium-free medium. It is concluded that they stimulate mitosis only indirectly by sensitising the mitotically competent segment of a thymocyte population to the action of calcium. The stimulatory action of calcium itself is opposed by low concentrations of the mucopolysaccharide chondroitin sulphate. However, the inhibitory action of chondroitin sulphate can be overcome by growth hormone. A possible common mechanism of action of these hormones on mitotically competent cells is discussed.

The effect of polyamines on cell culture cells

Abstract: Growth of KB cells was inhibited by both spermine and spermidine, but the inhibition is reduced in conditioned medium. The amount of spermine required for 50% inhibition of plating varied according to the type of serum used with medium 199 (calf, fetal bovine, and horse; 0.55, 0.9, and 24 μg/ml respectively). The spermine oxidase activity of the three sera was calf > horse > fetal bovine, which is not the same ordering as was obtained for the inhibition. When the concentration of sera in the media was varied, the inhibition decreased as calf and fetal bovine sera concentration increased, whereas, with horse serum, an increase in serum concentration increased the inhibition. The opposite effects of increasing concentrations of the sera on the inhibition suggest that at least two factors are involved in the inhibition. A scheme which involves three factors (spermine oxidase, another enzyme and its activator) is postulated to account for the inhibitions and reversals observed. Spermine oxidase alone cannot account for the action of polyamine on cells.

Development of the mouse hematopoietic system. II. Estimation of spleen and liver "stem" cell number.

Abstract: Colony-forming cells (CFU), which have the general properties of hemopoietic “stem” cells, appear to be augmented in the mouse fetal liver from 12–18 days gestation and then decrease in the newborn. This finding suggests that few, if any, hemopoietic “stem” cells remain in the adult liver, an organ which appears to be unable to function erythropoietically, even at times of severe crises. In the spleen, and active adult as well as embryonic hematopoietic organ, the total number of CFU increases from 18 days gestation until at least 7 days after birth. Spleen and liver CFU augmentation seems to occur in cojunction with an analogous expansion of non-hematopoietic cells. The data suggests, in fact, that while there is an increase in the total number of liver CFU, there is also a dilution of liver CFU in the total cell population at successively later gestational ages.

The relationship of protein synthesis to cell division and oral development in synchronized Tetrahymena pyriformis GL‐C: An analysis employing cycloheximide

Abstract: The effects of cycloheximide on synchronized Tetrahymena pyriformis strain GL-C were investigated at concentrations ranging from 0.01 to 10 μg/ml. The initial inhibition of protein synthesis was nearly total (>85%) at 1 μg/ml and above, partial (50–80%) at 0.2 to 0.05 μg/ml, and slight (<30%) at 0.02 μg/ml. Eventual recovery of protein synthesis to a rate approaching that of the controls took place at concentrations of 1 μg/ml and less. When the drug was added before a “transition point” at 55 minutes after the end of the synchronizing treatment (EST), cell division was blocked by 10 μg/ml, and delayed at concentrations of 1 μg/ml or less. The duration of delay was related to the degree of initial inhibition, and to the time required for recovery of protein synthesis; it also depended on the time after EST at which the drug was added. At a given concentration, maximum division delay was observed just prior to the “transition point;” this maximum delay was correlated with resorption of differentiating oral primordia, followed by the appearance of new primordia. The lesser delays observed at earlier times were correlated with temporary blockage of development of primordia in the “stomatogenic field” stage. Resumption of oral primordium development was, in both cases, temporally correlated with a substantial recovery of protein synthesis. After the “transition point,” cell division, and completion of oral development, was delayed slightly at the lower concentrations, and more substantially at 1 and 10 μg/ml, with some division-arrest at the latter concentration. Except for the recovery phenomenon, the developmental responses elicited by cycloheximide were similar to those observed earlier with puromycin. The bearing of these findings on the mechanism of synchronization in Tetrahymena is considered in the Discussion.

Nucleic acids and polynucleotides

Abstract: The conformation of native double helical DNA is well-known, but it is possible that small regions occur within native DNA, undetectable by X-ray diffraction methods, which have different conformations. Model structures are the synthetic deoxypolynucleotides of defined sequence. Under the conditions used, DNA, poly d(A-T) • poly d(A-T), and poly d(T-G) • poly d(C-A) can all give similar X-ray diffraction patterns, whereas poly dA • poly dT, poly dI • poly dC, poly dG • poly dC, and poly d(T-C) • poly d(G-A) clearly differ from DNA. This led to the tentative hypothesis that those DNA's in which all purines are in one strand and all pyrimidines in the other differ in structure from those (such as native DNA) in which purines and pyrimidines alternate or are irregular. We now find that poly d(I-C) • poly d(I-C) does not fit the hypothesis and is a most unusual structure, having seven or eight base pairs per turn. Both molecular model building and circular dichroism studies suggest that it is a left-handed helix. A number of purified tRNA's have been crystallized. We have obtained, from unfractionated tRNA, crystalline “powder” X-ray diffraction patterns showing rings and spots to about 20 A resolution. It is not clear whether cocrystallization has occurred, or whether there is fractional crystallization, though preliminary evidence favors the latter. Determination of the structure of crystalline tRNA has many features in common with protein crystallography, but there are a number of distinct differences.

Phenotypically variant adrenal tumor cell cultures with biochemical lesions in the ACTH-stimulated steroidogenic pathway

Abstract: Four clonal adrenal tumor cell lines which exhibit biochemical lesions in the ACTH-stimulated steroidogenic pathway have been isolated Two of these cell lines, designated Y-6 and OS3, appear to contain their lesions at points proximal to cyclic AMP formation in the ACTH-stimulated steroidogenic pathway Growth of Y-6 and OS3 as tumors in isogenic mice results in a restoration of ACTH sensitivity in both cell lines by mechanisms which do not appear to involve selection or fulfillment of specific nutritional requirements Growth of Y-6 and OS3 as tumors in heterogenic mice results in restoration of ACTH sensitivity in Y-6 but not in OS3, suggesting that the biochemical lesions in these cell lines are at different loci Two other cell lines, designated OS1 and OS4, possess biochemical lesions in the steroidogenic pathway beyond the formation of cyclic AMP and before the formation of pregnenolone Growth of OS1 and OS4 as tumors in isogenic mice results in the repair of the biochemical lesions in these cells distal to cyclic AMP formation in the ACTH-stimulated steroidogenic pathway The four cell lines described are potentially useful in elucidating the mechanism of action of ACTH in adrenal cells as well as in determining the factors required for maintaining differentiated function in cultured cells

Discrepancies between precursor uptake and DNA synthesis in mammalian cells.

Abstract: The incorporation of tritiated thymidine and deoxycytidine into DNA of x-irradiated mammalian cells was studied. Both inhibition and stimulation were found due to pool changes rather than to effects on DNA synthesis, indicating that precursor uptake can be a misleading method to measure DNA synthesis rate.

A comparative study of the role of mitochondria and the sarcoplasmic reticulum in the uptake and release of Ca++ by the rat diaphragm

Abstract: The respective importance of mitochondria and of sarcoplasmic reticulum in the uptake and maintenance of Ca++ by the isolated rat diaphragm has been compared. Diaphragms were incubated at 30° in conditions optimal for Ca++ uptake either by isolated mitochondria or by sarcoplasmic reticulum: more Ca++ was taken up from the “mitochondrial” medium. For maximal uptake, Pi and Mg++ were necessary; substitution of NaCl and KC1 with sucrose had no effect on the uptake. The uptake was markedly inhibited by uncouplers of oxidative phosphorylation, by respiratory inhibitors, and by lowering the temperature of the incubation medium to 0°; it was not affected by oligomycin, aurovertin, DCCD, nor by inhibitors of Ca++ transport in the isolated sarcoplasmic reticulum (ergotamine, ergobasinine, caffeine). The lack of effect of caffeine was not due to lack of penetration into the muscle. Permeability barriers for ergotamine and ergobasinine could not be excluded. The maintenance of Ca++ by the diaphragm was optimal in a medium contaming Pi and Mg++. Uncoupling agents and respiratory inhibitors accelerated the rate and extent of release of Ca++ by the diaphragm. Lowering the temperature of the incubation medium to 0°, or addition of oligomycin, aurovertin, DCCD, had no effect on the release. The release of Ca++ was also unaffected by ergotamine, ergobasinine, caffeine. The results suggest a role for mitochondria in the uptake and maintenance of Ca++ by the isolated diaphragm.

Purification and ultrastructural properties of the calcium accumulating membranes in isolated sarcoplasmic reticulum preparations from skeletal muscle.

Abstract: Sarcoplasmic reticulum fragments, prepared from skeletal muscle homogenates, were found to consist of two major types when examined after negative staining. One type possessed 90 A subunits and was thought to be of mitochondrial origin. The other had 35 A subunits and ws presumably derived from the sarcoplasmic reticulum. Only the latter type accumulated visible calcium oxalate deposits inside the vesicle when they were exposed to a medium containing ATP, MgCl2, K2C2O4 and CaCl2. The calcium oxalate loaded vesicles were strikingly angular in shape and did not have tails. The calcium oxalate loaded vesicles had identical membrane subunit arrangement to inactive companion membranes and nonincubated controls; this suggested that the membrane subunits were not the critical structural requirement for calcium transport. A method was described whereby the calcium accumulating membranes could be purified 3- to 4-fold on the basis of the best previously used preparation procedures.

Migration and proliferation of diploid human fibroblasts following "wounding" of confluent monolayers.

Abstract: Six diploid human fibroblast strains were grown in confluent monolayers. Holes were scraped in these monolayers and the number of cells proliferating into these “wounds” with time were determined. The migration and mitotic aspects of the proliferation of fibroblasts into these wounds were analyzed separately. Small amounts of undialysed or dialysed serum were essential for cell division but not migration. Saline extracts of skin could not substitute for serum in the medium. Neither zinc nor cupric ion at tolerable concentrations (10−5M) increased the rate of cell proliferation. Normal human fibroblasts did not immediately start to divide from confluency into the “wound” space. Their generation time was about 32–39 hours. Fibroblasts from patients with cystic fibrosis began to divide almost immediately into the “wounded” area. Their generation time was about 48 to 56 hours.

Effect of exogenous ATP on the volume of TA3 ascites tumor cells.

Abstract: When exogenous ATP is added to suspensions of TA3 ascites tumor cells suspended in Ca++ and Mg++ free media, a significant increase in cell volume can be measured. This increase is reversible upon addition of Ca++ and/or Mg++ back to the media. The enlargement of these cells is temperature sensitive and specific for ATP; no other nucleotides, EDTA or ouabain were effective. The evidence suggest that this phenomena may be due to an alteration in membrane permeability and that the regulation of membrane permeability is an energy dependent process.

The effect of exogenous ATP on the electrolyte content of TA3 ascites tumor cells.

Abstract: In the presence of 1 mM ATP in the external medium, TA3 mouse ascites tumor cells showed a dramatic loss of potassium and gain of sodium down their respective concentration gradients. The volume changes detected by size discrimination with the Coulter counter have been adequately confirmed by densimetric techniques. Further, some experiments were so designed that net losses of both ions occurred and the cell shrank in response to ATP, a response which was predictable if the volume change was produced by a loss of cell water. We believe that ATP produces a major change in the passive permeability of the membrane to these ions and the effect may be due to a response of a contractile protein in the membrane to ATP.

The effect of serum components on sterol biosynthesis in L cells

Abstract: L cells were cultivated in test medium which contained 14C-sodium acetate, and the amount of labeled digitonin-precipitable sterol was assayed in medium and cells. Increasing concentrations of whole serum in the medium had two effects: depressed cellular synthesis and enhanced release of synthesized sterol from the cells. In experiments with delipidized serum containing unesterified cholesterol, cellular sterol synthesis decreased as free cholesterol concentration in the medium increased. In other experiments using medium containing increasing lecithin concentration and no exogenous sterol, the concentration of lecithin markedly influenced the distribution of synthesized sterol between the cells and the medium which then directly influenced the amount of sterol synthesized. These experiments indicate that cell sterol synthesis is regulated by internal levels of free sterol. This, in turn, is a function of cellular sterol flux which is regulated by the concentration and composition of serum lipoprotein in the medium.