bluuebunny/crossref_metadata_2025 huggingface dataset:
Density‐dependent resistance to puromycin in cell cultures
Authors: Cass Carol E
Published: February 2005

Pig kidney cells exhibit resistance to puromycin when cultured at high cell densities, growing progressively in otherwise lethal concentrations of drug. Comparison of population growth of cultures inoculated at different densities indicates that survival in puromycin is a function of cell density. Uptake of puromycin from culture media is less at higher cell densities resulting in decreased inhibition of protein synthesis by puromycin. Enhanced survival in puromycin at high population densities is attributed to reduced accumulation of puromycin from the culture media.
Genetics of somatic mammalian cells. XIV. Genetic analysis in vitro of auxotrophic mutants
Authors: Kao Fa‐Ten , Fuck Theodore T.
Published: February 2005

Genetic analysis has been carried out on auxotrophic mutants produced by treatment of Chinese hamster ovary and the Chinese hamster lung cells with mutagenic agents in vitro. Thirty‐six different mutants were subjected to complementation analysis and biochemical tests. The different mutations studied result in growth requirements for proline; glycine; glycine or folinic acid; adenine or several of its precursors; inositol; adenine plus thymidine; and glycine plus adenine plus thymidine.

The mutants which require glycine fall into four different complementation classes while those requiring adenine or hypoxanthine form two different complementation classes. The biochemical blocks of the latter two classes both occur somewhere in the steps involved in conversion of 5‐phosphoribosyl‐1‐pyrophosphate to 5‐aminoimidazole 4‐carboxylic acid ribonucleotide.

The auxotrophic mutants described exhibit all‐or‐none responses to their specific nutrilite supplements and are stable with respect to reversion. They involve alterations in ten different genes, and hence form a useful set of mutants for a variety of genetic studies. All the auxotrophies produced in a single exposure to a mutagen are due to single gene mutations, even when multiple nutritional requirements were produced. All the mutations studied are recessive.
Control of macromolecular synthesis in proliferating and resting syrian hamster cells in monolayer culture. III. Electrophoretic patterns of newly synthesized proteins in synchronized proliferating cells and resting cells
Authors: Becker H. , Stanners C. P.
Published: February 2005

Electrophoretic patterns of newly synthesized proteins have been compared for hamster embryo fibroblasts in asynchronous cultures, mitotically synchronized cultures, and stationary phase cultures. Only proteins with molecular weight between 30,000 and 150,000, comprising 60–70% of the total cell proteins and excluding histones and collagen were included in the comparison. Although no significant differences could be detected between such patterns for cells at different stages of the cell cycle, significant differences were detected between patterns for cells in stationary phase and for proliferating asynchronous or synchronous cells in any stage of the cell cycle. These differences amounted to at least 5% of the newly synthesized cellular proteins. Much larger differences were detected between patterns from a nuclear fraction of proliferating and resting cells. These results indicate that normal cells in stationary phase are arrested in a state distinct from any phase of the normal cell cycle and may provide a biochemical marker for resting cells.
Synthesis of nuclear acidic proteins in density‐inhibited fibroblasts stimulated to proliferate
Authors: Tsuboi Atsushi , Baserga Renato
Published: February 2005

Previous work from this laboratory (Rovera and Baserga, 1971) has shown that, when density‐inhibited WI‐38 human diploid fibroblasts are stimulated to proliferate by a change of medium, the synthesis of nuclear acidic proteins increases within 30 minutes after stimulation; several hours before DNA synthesis begins to increase. Similar results have now been obtained with density‐inhibited 3T6 mouse fibroblasts, also stimulated by a change of medium.

Gel electrophoretic analysis of nuclear acidic proteins in both WI‐38 human diploid fibroblasts and 3T6 mouse fibroblasts stimulated to proliferate indicates that the increased synthesis of nuclear acidic proteins is limited to certain classes of proteins while other classes are totally unaffected.

The increase in nuclear acidic proteins synthesis is inhibited when WI‐38 cells or 3T6 cells are stimulated in the presence of 5‐azacytidine (10 μg/ml), a treatment which also inhibits the subsequent stimulation of DNA synthesis.

These results, confirming and extending similar findings previously reported in other models of stimulated DNA synthesis, lend further support to the hypothesis that nuclear acidic proteins may play a critical role in the control of DNA synthesis and cell division in mammalian cells.
Transport changes in synchronously growing CHO and L cells
Authors: Sander Greta , Pardee Arthur B.
Published: February 2005

A reduced rate of transport of 2‐amino‐isobutyric acid (AIB), was found to be associated with M and early G1 periods in synchronized populations of CHO and L cells. A doubling of the transport rate occurred with progress of CHO cells further into G1, and the specific transport activity remained constant during the rest of the cycle, The rate of uridine and thymidine transport also doubled at this point in the cell cycle. There was no change in Km values. The rise in transport rate was blocked by cycloheximide, or when the cells were prevented from leaving metaphase by colcemid.
Opposite effects of dibutyryl adenosine 3′:5′ cyclic monophosphate and serum on growth of Chinese hamster cells
Authors: Rozengurt Enrique , Pardee Arthur B.
Published: February 2005

The effect of dibutyryl adenosine cyclic 3′:5′ monophosphate and testosterone on growth, response to serum and transport properties of Chinese hamster ovary cells was studied. There was a marked depression of growth in the presence of both compounds. A change of medium was sufficient to permit a partially synchronized burst of growth of the treated cells either in the presence or absence of dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone. However, in the presence of these compounds a second round of cell division was prevented. Initiation of the cell cycle by cells exposed to dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone displayed a greater serum requirement than untreated cells. It is concluded that serum and cyclic AMP could have antagonistic interactions in growth regulation.

The treated cells had a reduced ability to accumulate amino‐isobutyrate and glutamine, but no difference was observed with uridine uptake. The data suggest that a functional alteration of the membrane is induced by the exposure to dibutyrl adenosine cyclic 3′:5′ monophosphate plus testosterone.
Separation of mouse spermatogenic cells by velocity sedimentation
Authors: Meistrich Marvin L.
Published: February 2005

Improvements have been made in methods of preparation of single cell suspensions from mouse testes for separation by sedimentation at unit gravity. Mechanical means of preparation specifically damage spermatogonia, young primary spermatocytes, and spermatids at various stages of development, resulting in apparent sedimentation velocities which correspond closely to those of free nuclei. Trypsin has been used to release these cells from the seminiferous tubules allowing measurement of the sedimentation properties of the intact cell. The exclusion of trypan blue from the cells, resistance to trypsin, and the persistence in vitro of the highly differentiated forms of macromolecular synthesis demonstrate the integrity of the cells in the suspensions prepared with trypsin. Differentiation pathways in terms of sedimentation velocities of the cells and nuclei have been constructed and are in agreement with morphological changes.
Requirement for calcium ions in lymphocyte transformation stimulated by phytohemagglutinin
Authors: Whitney Richard B. , Sutherland Robert M.
Published: February 2005

Medium calcium ions were essential to the development of the lymphocyte transformation response induced by PHA. Magnesium ions could not substitute for calcium ions. Calcium exerted its influence during the induction phase of the process before DNA synthesis began since its removal after the response had fully developed did not alter subsequent levels of nucleic acid synthesis. The early development of RNA synthesis was prevented by low calcium levels indicating that calcium either directly influenced the initiation of this process or some prior event(s).
On the selection for the modal chromosome number in Chinese hamster cells
Authors: Terzi M.
Published: February 2005

Chinese hamster cell lines have a strong tendency to maintain as modal chromosome number the euploid one characteristic of the species. By following the evolution of karyotypes in cloned subpopulation it has been possible to show that these pseudodiploid constitutions are not simply established due to a selective advantage but they are actually generated in populations where pseudodiploid classes had been absent. The possible role of the mitotic apparatus in keeping and restoring the euploid number is discussed.
The mechanical triggering of bioluminescence in marine dinoflagellates: Chemical basis
Authors: Hamman J. P. , Seliger H. H.
Published: February 2005

In both photosynthetic (Pyrodinium bahamense, Gonyaulax polyedra, Pyrocystis Iunula, P. noctiluca, P. fusiformis) and nonphotosynthetic (Noctiluca miliaris) bioluminescent dinoflagellates chemical stimulation can by‐pass mechanical stimulation. The effective ions are Ca++, K+, NH4+ and H+. Other chemicals found effective are those implicated in Ca++ transport or binding. There are interspecies differences in degrees of mechanical and chemical stimulability. Photoinhibition of mechanical stimulability is the result of two effects, the first a reduction in total bioluminescence potential and the second a decrease in mechanical stimulability resulting experimentally in a decreased rate of light emission. This latter effect can be reversed with Ca++ ions. Chemicals which bind Ca++ or displace Ca++ can mimic the effects of photoinhibition. The chemical inhibition of mechanical stimulability is also reversed by Ca++ ions. A scheme is proposed which is consistent for all species examined.
Selective inhibition of the cell cycle of cultured human diploid fibroblasts by aminonucleoside of puromycin
Authors: Studzinski George P. , Gierthy John F.
Published: February 2005

Aminonucleoside (AMS) inhibited the cell cycle of human lung fibroblasts at a point in G1 phase and at another point in G2 phase. Even when this inhibition was fully established, DNA synthesis and mitosis which were in progress proceeded normally. Inhibition of RNA synthesis in the cultures preceded the effects on DNA synthesis and mitosis, but inhibition of protein synthesis could not be detected.

These points of potential inhibition do not exist in the cell cycle of HeLa cell, or are not affected by aminonucleoside. Here inhibition of cell proliferation by AMS was less marked, and when inhibition eventually occurred it was not specific for any point of the cell cycle. The rate of entry of the inhibitor was similar in both types of cell.
Tetrodotoxin‐resistant electric activity in chick skeletal muscle cells differentiated in vitro
Authors: Kano Masaakira , Shimada Yutaka
Published: February 2005

The embryonic chick skeletal muscle cells differentiated in cell culture from trypsin‐dissociated myoblasts produce a spike response which is tetrodotoxin‐sensitive. It has been found that many cells also produce a plateau response which is resistant to tetrodotoxin. The plateau response frequently occurs even in the muscle cells which do not normally exhibit the spike response. During the plateau response membrane resistance is greatly reduced below its resting value. The current‐voltage relation in muscle cells with the plateau response is always S‐shaped. It is suggested that the plateau arises from a voltage‐dependent increase in permeability to external cations whose influx produce the maintained depolarization, and from low level of repolarizing potassium outflux. The plateau response is sensitive to manganese ions. This finding, together with resistibility to tetrodotoxin, suggests that calcium ions are the dominant carriers for the depolarizing current.
A partially purified polypeptide fraction from rat liver cell conditioned medium with multiplication‐stimulating activity for embryo fibroblasts
Authors: Dulak Norman C. , Temin Howard M.
Published: February 2005

A polypeptide fraction with multiplication‐stimulating activity for chicken and rat embryo fibroblasts was partially purified from serum‐free medium conditioned by the growth of a line of rat liver cells. The specific multiplication‐stimulating activity of this fraction was 27,000 times that of serum. The rat liver cell multiplication‐stimulating activity had a molecular weight of approximately 10,000 daltons and was inactivated by mercaptoethanol and dithiothreitol. It had sulfation factor and non‐suppressible insulin‐like activities, but did not have anti‐trypsin activity. The rat liver cell multiplication‐stimulating activity resembled both multiplication‐stimulating activity from calf serum and somatomedin.
Isolation, purification, and chemical characterization of the serum mitogen for diploid human fibroblasts
Authors: Houck J. C. , Cheng R. F.
Published: February 2005

We have found that diploid human fibroblasts, but not heteroploid human fibroblasts, stringently required serum for their multiplication. Using Diaflo ultrafiltration membrane units, isoelectric focusing and preparative gel electrophoresis, we have isolated and purified this mitogenic activity from mammalian sera. This electrophoretically homogeneous sialoprotein is 120,000 daltons in size, with an iso‐electric point of pH 5.2–5.4; it is made up of two electrophoretically identical dimers weighing 57,000 each; it is thermostable and is inactivated by both trypsin and neuraminidase.
Purine excretion by mammalian cells deficient in adenosine kinase
Authors: Chan Teh‐Sheng , Ishii Kazuhiro , Long Cedric , Green Howard
Published: February 2005

An adenosine kinaseless (AK−) mutant of the mouse fibroblast line 3T6 has been obtained in cell culture by evolution of resistance to 6‐thio‐methylpurine ribonucleoside and tubercidin. The mutant excretes purines (xanthine and hypoxanthine) into the culture medium. Human or mouse cells lacking hypoxanthine‐guanine phosphoribosyl transferase (HPT−) excrete increased amounts of purines, but a human cell mutant lacking both HPT and AK excretes considerably more hypoxanthine. The difference in hypoxanthine excretion between the HPT− mutant and the HPT− AK− mutant originates from the adenosine normally reutilized through the activity of adenosine kinase. The activity of adenosine kinase is essential to retard the adenosine cycle and to prevent cellular loss of purines.
Enhancement by purine nucleosides and nucleotides of serum‐induced DNA synthesis in quiescent 3T3 cells
Authors: Schor Seth , Rozengurt Enrique
Published: February 2005

Purine nucleotides and nucleosides were found to enhance the DNA synthesis in quiescent 3T3 cells induced either by fresh medium containing calf serum or by the addition of the β‐globulin serum fraction to depleted medium. A two to threefold enhancement has been obtained under these conditions. The adenine and hypoxanthine based compounds were more effective than the guanine ones in the concentration range tested (10−5−−10−3 M), while the pyrimidine compounds were without effect. The degree of enhancement obtained with the purine compounds depended on the type and amount of serum used to stimulate DNA synthesis. A maximal degree of enhancement was obtained when the purine compounds were present for only the first three hours after the addition of serum and thus 18–20 hours before the actual onset of DNA synthesis.
Stimulation of DNA synthesis and 2‐deoxy‐D‐glucose transport in chick embryo cultures by excessive metal concentrations and by a carcinogenic hydrocarbon
Authors: Rubin H. , Koide T.
Published: February 2005

Chick embryo cultures deprived of serum synthesize DNA at a reduced rate. DNA synthesis in serum‐deprived cultures is stimulated as much as ten‐fold by the addition of Zn++, Mn++ or Cd++ in concentrations just below the toxic level. These metals, in the same concentration range, also stimulate the uptake of 3H‐2‐deoxy‐D‐glucose (2‐DOG). The increase in uptake of 2‐DOG precedes the increase in synthesis of DNA, and is probably an indicator of a more general membrane perturbation. The metals also stimulate DNA synthesis in serum‐containing, density‐inhibited cultures. The carcinogenic hydrocarbon 9,10‐dimethyl‐1,2‐benzanthracene stimulates DNA synthesis and 2‐DOG uptake in serum‐deprived cultures at those concentrations which also cause morphological changes in the culture. Other carcinogenic hydrocarbons, which produce no morphological changes in the culture do not stimulate DNA synthesis. In contrast to these non‐specific effects, DNA synthesis which is inhibited by low concentrations of either ethylene diamine tetraacetate (EDTA) or diethylene triamine pentaacetate (DTPA) is stimulated specifically by Zn++. These findings are interpreted to mean that certain metals and carcinogens, like a variety of other agents, interact non‐specifically with the plasma membrane to initiate a chain of events leading to DNA synthesis, and that one of these events is the liberation of Zn++ for enzyme reactions leading to DNA synthesis.
Electrical properties of normal and dysgenib mouse skeletal muscle in culture
Authors: Powell Jeanne A. , Fambrough Douglas M.
Published: February 2005

Electrical properties of normal and dysgenic mouse skeletal muscle were studied by intracellular recording from embryonic cells developing in vitro. Passive membrane constants were determined from records of transmembrane potential responses to hyperpolarizing pulses of current using two types of analyses, assuming the tubes to be finite cylinders: the off transient and steady state analyses. The following properties of normal and dysgenic fibers were also studied. (a) membrane potentials (b) acetylcholine sensitivity (c) α‐Bungarotoxin binding and (d) maximum rate of rise, overshoot and one‐half fall time of the action potential. Rare electrotonic coupling between fibroblasts and myotubes was noted. An anomalous type of rectification Was observed in some fibers in which the transmembrane potential responses possessed under and overshoots. These responses may have affected the values of membrane constants as derived by the off transient analysis. In all parameters studied, including membrane constants derived by the steady state analysis, the cultured mouse cells resembled adult denervated mammalian muscle rather than innervated muscle. There were no differences between normal and dysgenic fibers with respect to any of the parameters studied. Dysgenic fibers did not contract although they displayed passive and active membrane properties like those in normal, non‐dysgenic fibers.
Trichodermin, a possible inhibitor of the termination process of protein synthesis
Authors: Stafford Mary E. , McLaughlin Calvin S.
Published: February 2005

The mode of action of the antibiotic, trichodermin, on yeast cells has been investigated. Trichodermin specifically inhibits protein synthesis and, during the in vivo inhibition of protein synthesis, ribosomes remain in polyribosomes rather than shifting to monoribosomes. This observation suggests that trichodermin inhibits either an elongation step or a termination step of protein biosynthesis. These two possibilities were distinguished by comparing the action of trichodermin with that of cycloheximide, a known elongation inhibitor, upon the reformation of polyribosomes during recovery from a block in polypeptide chain initiation. Cycloheximide slows the recovery of polyribosomes from monoribosomes following a block in polypeptide chain initiation whereas trichodermin enhances the recovery of polyribosomes. This observation is interpreted to mean that trichodermin primarily inhibits the termination step of protein biosynthesis.
Attempted hybridizations with senescent human fibroblasts
Authors: Littlefield John W.
Published: February 2005

If the aging of diploid human fibroblasts reflects stable genetic or epigenetic changes which are few in number and different in different cells, then complementation could occur in hybrids between individual senescent cells. However neither pairs of aged fibroblasts nor even pairs of young and senescent fibroblasts produce viable hybrids under conditions known to promote cell fusion.
Adrenergic antagonists, and a possible link between the increase in cyclic adenosine 3′,5′‐monophosphate and DNA synthesis during liver regeneration
Authors: Macmanus John P. , Braceland Brian M. , Youdale Tony , Whitfield James F.
Published: February 2005

The cyclic‐AMP concentration in the liver remnant after 70% hepatectomy increases in a biphasic manner with peak values at 3 and 12 hours, and DNA synthesis begins at 18 hours. Propranolol (dl) injected at 30 minutes after surgery stopped the first wave of cyclic‐AMP accumulation, but did not affect the second accumulation or the initiation of DNA synthesis. However, dl, propranolol injected at eight hours equally delayed (by 6 to 8 hours) the second wave of cyclic‐AMP accumulation and the initiation of DNA synthesis. Propranolol (dl) did not affect DNA replication per se, since it was totally ineffective after the second wave of cyclic‐AMP accumulation had passed and DNA synthesis had been initiated. Propranolol (dl) action was not due to a blockade of β‐adrenergic receptors, since its d or l isomers were separately without effect, as were unrelated β‐adrenergic blockers (Kü 1313 and M&B 17‐803A).

On the other hand, an activation of α‐adrenergic receptors may be involved in the induction of hepatocyte proliferation, since α‐adrenergic antagonists, such as phenoxybenzamine and phentolamine, both delayed and considerably reduced the second wave of cyclic‐AMP accumulation and the subsequent initiation of DNA synthesis. It is concluded that the second wave of cyclic‐AMP accumulation is somehow associated with the initiation of DNA synthesis.
Inhibition of marrow granulocytic colony formation by phytohemagglutinin
Authors: Burns Maria V. , Lozzio Bismarck B.
Published: February 2005

The effect of phytohemagglutinin (PHA) on the growth and number of granulocytic colonies (GC) developing on agar from bone marrow and spleen cells of normal and erythroleukemic mice inoculated with Rauscher leukemogenic virus was studied. Equal number of marrow cells from erythroleukemic mice produced twice as many colonies as those from normal mice. The number of GC developing from either normal and leukemic spleen cells was only 20% to 25% of that arising from marrow cells. The number of cells within each colony was significantly larger in GC formed by myelogenous leukemic cells than those arising from normal cells even though they had similar morphologic features. The addition of 100 μg of PHA per 105 cells reduced the number of GC arising from normal and leukemic cells by 35% and 50%, respectively. Treatment with periodate which mainly inhibits its mitogenic activity, abolished the inhibitory effects of PHA on proliferation of granulocytic cells.
The effect of ploidy on chemical mutagenesis in cultured chinese hamster cells
Authors: Chasin Lawrence A.
Published: February 2005

The frequency of mutations induced by ethyl methane sulfonate was compared in a pseudodiploid Chinese hamster cell strain and in a tetraploid substrain derived from it. The frequency of reverse mutations from glycine auxotrophy to glycine independence was similar in the two strains, as expected for a dominant phenotype. Forward mutation to 6‐thioguanine‐resistance was 25 fold lower in the tetraploid as compared to the diploid strain. The resistant mutants lack hypoxanthine phosphoribosyl transferase activity and their resistant phenotype is recessive in somatic cell hybrids. A combination of chromosomal segregation and mutation could account for the frequency of these recessive drug‐resistant mutants in the tetraploid population.
A kinetic analysis of purine nucleotide synthesis and interconversion in intact Ehrlich ascites tumor cells
Authors: Snyder Floyd F. , Henderson J. Frank
Published: February 2005

A pseudo‐first order kinetic analysis has described the reactions of purine ribonucleotide synthesis and interconversion in Ehrlich ascites tumor cells in vitro. Rate constants were determined for 13 reactions at non‐saturating precursor concentration (50 μM); nucleotide formation from adenine‐14C and hypoxanthine‐14C was rate limiting for ATP synthesis and nucleotide formation from guanine‐14C and hypoxanthine‐11C was rate limiting for GTP synthesis. The initial rites of nucleotide synthesis from 1.0 mM adenine‐14C and hypoxanthine‐14C were equivalent to the rates of nucleotide synthesis at non‐saturating base concentration, but the rate of nucleotide synthesis at saturating base concentration after 30 minutes incubation was 20‐25% that of non‐saturating precursor concentration.
The mechanism of regulation of fibroblastic cell replication II. Participation of the nucleoli
Authors: Mironescu Stefan , Ellem K. A. O. , Love R.
Published: February 2005

Cultures of human diploid fibroblasts (HDFs) exhibiting density dependent inhibition of replication (DDIR) resumed their progression through the cell cycle following medium replacement and, after a lag period of two hours, showed a dramatic increase in the incidence of isonucleolinar 4 cells and in the levels of uptake of 3H‐uridine into the nucleoli. Between five and ten hours after refeeding these nucleolar changes were maximal, leveling off at the highest values, in periods corresponding to late G1 and early S. Concomitantly, a parallel increase in the number of nucleolini per cell occurred. As cells progressed through S and G2 phases the nucleolini decreased in number and reverted to the aniso‐nucleolinar type. The intensity of nucleolar labeling by 3H‐uridine and its correlate, the frequency of cells with labeled nucleoli, also decreased during these cell cycle stages. Both pre‐ and postreplicative periods of mitotic quiescence were characterized by high levels of anisonucleolinosis (60–80% of the cells) and by very low levels of nucleolar 3H‐uridine incorporation.

The magnitude of these nucleolar changes occurring during G1 stage was found to be strongly dependent on: (1) the length of time of contact between the cells and the fresh medium, at least eight hours of contact being necessary for a maximal response; (2) the amount of serum in the medium, the optimal serum concentration being between 10 and 50%, and (3) the pH of the medium. The nucleolar response was completely abolished at pH values below 7.0. These nucleolar changes were very sensitive to the presence of cycloheximide (10 μg/ml) and actinomycin D (0.003 μg/ml). The behavior of the nucleoli in response to these parameters was similar to the activation response of the cells to initiate DNA synthesis.

During the time period of maximal nucleolar (activation) the onset of DNA synthesis as well as the morphological and autoradiographic manifestations of the nucleolar activation were completely inhibited by very low levels of actinomycin D (Ellem and Mironescu, '72), a selective inhibitor of nucleolar RNA synthesis (Perry, '65). This suggested a possible role of nucleolar metabolism, in normal diploid cells, in the initiation of DNA synthesis. Our results, however, seem to indicate that the nucleolar changes are necessary but not sufficient for the subsequent initiation of DNA synthesis, since with graded serum concentrations or medium volumes, smaller levels of a stimulus were needed to produce maximal isonucleolinosis than to effect a maximum replicative response in the cells.
Studies on Down's syndrome in tissue culture. I. Growth rates protein contents of fibroblast cultures
Authors: Segal David J. , McCoy Ernest E.
Published: February 2005

Skin fibroblast cultures from six patients with Down's syndrome (Trisomy 21) were compared with four in vitro age‐matched normal fibroblast cultures. Growth rates were calculated from increases in cell number and total protein during exponential growth, early in culture lifetime (less than 20 doublings). The Down's syndrome (D.S.) cultures had an average population doubling time of 35.6 ± 1.1 hours and average mass doubling time of 38.6 ± 3.2 hours, significantly lower (p<0.005) than the corresponding normal culture values of 23.0 ± 0.7 hours, and 23.3 ± 1.9 hours. D. S. Cells also contained 4.46 ± 0.19 ± 10−4 μg protein/cell as compared to 3.06 ± 0.13 × 10−4 μg/cell (p<0.001) for normal fibroblasts.

Similar in vitro observations of increased doubling time and protein content have been reported in normal fibroblasts from older donors, and from individuals with premature aging syndromes, as well as in normal fibroblasts near the end of their in vitro lifetime. The present results, obtained from cultures young in vitro, may therefore suggest that D.S. fibroblast cultures age prematurely. This hypothesis is consistent with clinical manifestations of premature aging in D.S. patients and points to a defect in growth regulation, both in vivo and in vitro, resulting from an extra copy of chromosome 21.
Effect of growth conditions on the activity of ornithine decarboxylase in cultured hepatoma cells. I. Effect of amino acid supply
Authors: Hogan Brigid L. M. , Murden Susan
Published: February 2005

Ornithine decarboxylase activity in high density, stationary phase rat hepatoma (HTC) cells in suspension culture has an extremely short half‐life of between 5 and 15 minutes, as measured after inhibiting protein synthesis. Following dilution of these cells into fresh medium there is a large increase in ornithine decarboxylase activity, reaching a peak often several hundred times the initial level at about four hours. At least part of this stimulation is due to an increase in the apparent half‐life of the enzyme, to between 30 and 90 minutes. Evidence is presented that the supply of amino acids can control the turnover of ODC under some conditions. For example supplementing high density cells with glutamine, asparagine, serine, glycine and proline, either singly or together, increases ODC activity and decreases its apparent turnover. The stimulation by amino acids is enhanced by serum.
Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar
Authors: Lin Hsiu‐San , Stewart Carleton C.
Published: February 2005

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony‐forming cells from the peritoneal exudate are similar to bone marrow colony‐forming cells in vitro in that they both require a substance(s) present in conditioned medium from L‐cells or mouse embryo fibroblasts or the serum from endotoxin‐treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony‐forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L‐cell conditioned medium than bone marrow colony‐forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.
Evidence of a common site of action for the antitumor agents, hydroxyurea and guanazole
Authors: Wright J. A. , Lewis W. H.
Published: February 2005

Hydroxyurea and guanazole were used as selective agents in tissue culture to obtain independent Chinese hamster ovary cell lines resistant to the cytotoxic effects of hydroxyurea or guanazole. In all cases tested a cell line selected for resistance to one of the antitumor agents exhibited resistance to both drugs. This result supports the view that these two drugs act at a common site.
Effect of PCMBS on water transfer across biological membranes
Authors: Naccache P. , Sha'afi R. I.
Published: February 2005

P‐chloromercuriphenylsulfonate, PCMBS, and 5, 5′ dithiobis‐(2‐nitrobenzoic acid), DTNB at a concentration of 1 mM are found to inhibit the rate of water transport across human red cell membrane. In addition PCMBS inhibits the rates of transport of small hydrophilic but not hydrophobic nonelectrolytes. Other sulfhydryl reagents such as N‐ethylmaleimide and iodoacetamide have no significant effect on the rate of water transfer in these cells. The results suggest that there are at least two populations of membrane bound SH‐groups which differ in their topical location which participate in the control of water transfer. One is located closer to the outer surface of the membrane, and thus is readily accessible to PCMBS while the other component is probably located in the membrane interior. These two populations can be dissociated by pH. The effect of PCMBS on water transfer can be greatly influenced by pH and temperature. The main effect of temperature and pH is on the permeability of the membrane to the drug. The same concentration of PCMBS is also found to inhibit to a lesser degree water transfer across other biological membranes.
Erythropoietic progenitors capable of colony formation in culture: Response of normal and genetically anemic W/Wv mice to manipulations of the erythron
Authors: Gregory C. J. , Tepperman A. D. , McCulloch E. A. , Till J. E.
Published: February 2005

Changes in the number and erythropoietin sensitivity of erythropoietic progenitor cells (CFU‐E or E) capable of erythropoietin‐dependent colony formation in culture have been measured in mice subjected to a variety of manipulations known to affect erythropoiesis. Injection of erythropoietin into mice whose E levels had been reduced by transfusion‐induced plethora stimulated a striking increase in this population within 24 hours in both spleen and marrow. Following irradiation and marrow transplantation E were found to regenerate in the spleen with a population doubling time of 6 hours. Evidence of erythropoietin‐dependent stimulation of E in vivo under conditions of hemopoietic regeneration was also obtained, although substantial numbers of E were detected in both regenerating spleen and marrow of plethoric mice even without erythropoietin administration, In the same experiments comparable effects of erythropoietin on pluripotent stem cells or the progenitors of granulopoietic colonies in culture were not observed. Detailed studies of the dependence of colony formation on erythropoietin concentration in culture showed variations in erythropoietin sensitivity of E from normal, regenerating and plethoric, erythropoietin‐stim‐ulated spleen and marrow. This finding provides the basis for an extremely fine mechanism regulating the flow of erythropoietic differentiation at the level of the production of E.

The number of E present in the marrow of untreated W/WV mice was normal, but their sensitivity to erythropoietin in culture was decreased to a similar extent as E from normal mice exposed to a heightened erythropoietic demand. Plethora had a more marked effect in reducing E numbers in W/WV mice than in +/ + controls and stimulation of E following subsequent erythropoietin administration was highly defective. The results of these studies provide further evidence that cells identified as E represent a stage of differentiation along the erythropoietic pathway that is several steps removed from pluripotent stem cells and support the view that erythropoiesis, granulopoiesis and stem cell self‐renewal are regulated to a major degree by independent mechanisms.
Effect of endotoxin on the production of colony‐stimulating factor by human monocytes and macrophages
Authors: Cline Martin J. , Rothman Belina , Golde David W.
Published: February 2005

Colony‐stimulating factor (CSF) is necessary for the clonal growth of human bone marrow in vitro. Human blood monocytes and macrophages produce CSF. Endotoxin was found to increase the level of CSF generated by macrophages, but had no stimulatory effect on monocytes. Several other substances known to influence the pinocytic or phagocytic activity of mononuclear phagocytes failed to enhance cellular CSF generation.
Contact inhibition, polyribosomes, and cell surface membranes in cultured mammalian cells
Authors: Levine Elliot M. , Jeng Dai‐Yun , Chang Yvonne
Published: February 2005

An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell‐cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.
Concanavalin A and the initiation of thymic lymphoblast DNA synthesis and proliferation by a calcium‐dependent increase in cyclic GMP level
Authors: Whitfield J. F. , Macmanus J. P. , Boynton A. L. , Gillan D. J. , Isaacs R. J.
Published: February 2005

Exposure of a thymic lymphocyte population (suspended in serum‐free synthetic medium) to the phytomitogen concanavalin A (Con A) causes brief (within the first 8 to 12 minutes) rises in the cellular contents of cyclic AMP and cyclic GMP. However, the rise in the cyclic GMP level is calcium (extracellular)‐dependent, but the cyclic AMP rise is not. These changes are followed during the next hour by the initiation of DNA synthesis by a large fraction of the lymphoblast subpopulation which, like the preceding cyclic GMP rise, is calcium‐dependent. The stimulated lymphoblasts eventually progress into mitosis. Additional observations indicate that Con A operates by sensitizing lymphoblasts to calcium ions which, in turn, cause the initiation of DNA synthesis by a process mediated by cyclic GMP, but not cyclic AMP.
Agglutination by concanavalin A of normal chick embryo fibroblasts treated by 5‐bromodeoxyuridine (BrdU)
Authors: Biquard Jean‐Michel
Published: February 2005

When chick embryo fibroblasts are grown for two days in the presence of low doses (18 μg/ml) of 5‐bromodeoxyuridine (BrdU), their agglu‐tinability by Concanavalin A is increased to about the same degree as following viral transformation of the cells by Rous sarcoma virus. Simultaneous addition of a large excess, but not of a low dose of thymidine prevents this effect of BrdU.

Treatment with BrdU also enhances the agglutinability of fibroblasts infected with a conditional It mutant of Rcus sarcoma virus at the temperature of 41° which restricts morphological transformation.
Uridine uptake in a unicellular eukaryote during the interdivision period and after growth arrest
Authors: Wolfe Jason
Published: February 2005

The rate of uridine uptake was measured in Tetrahymena after shiftdown to non‐nutrient physiological salt solution. Uptake follows Michaelis‐Menten kinetics and an apparent Km of transport of 2 × 10−6 M has been estimated. This value is in good agreement with those reported for tissue‐derived cells in culture. Incorporation of uridine into RNA follows similar kinetics suggesting that uptake is rate limiting for incorporation. Within three hours after shiftdown the rate of uptake is decreased by an order of magnitude. Also at three hours after shiftdown pairing occurs between cells of complementary mating types. It seems likely that the change in uptake is a reflection of a surface change associated with differentiation. The rate of uptake was also measured during the interdivision period using cells synchronized by a physical selection procedure. A change in rate occurs at the time the cells begin replication of DNA and is essentially stable thereafter. These results indicate that there exists in Tetrahymena a relationship between surface properties as assayed by uridine uptake and properties of growth and differentiation.
Cyclic AMP, membrane transport and cell division. I. Effects of various chemicals on cyclic amp levels and rate of transport of nucleosides, hypoxanthine and deoxyglucose in several lines of cultured cells
Authors: Sheppard J. R. , Plagemann Peter G. W.
Published: February 2005

Nutrient transport rates and cyclic AMP levels have been implicated in the regulation of cell proliferation. In the present study, however, changes in intracellular cyclic AMP level induced in several lines of cultured cells (normal 3T3 and SV40 and polyomavirus‐transformed 3T3 cells; 3T6, C6 glioma, mouse L, and Novikoff rat hepatoma cells) by treatment with papaverine, prostagladine E, or isoproterenol did not correlate with the inhibition of the uridine, hypoxanthine or deoxyglucose transport rates by these chemicals. Transport inhibitions by above chemicals or Persantin or Cytochalasin B occurred in most cell lines in the absence of any measurable change in intracellular cyclic AMP concentration. Furthermore, treatment of several cell lines with 1 mM dibutyryl cyclic AMP had no immediate effect on the transport of uridine, thymidine or deoxyglucose, although the transport capacity of the cells for uridine and thymidine, but not that for deoxyglucose, decreased progressively with time of treatment. We also observed that the uridine transport system of all cell lines derived from 3T3 cells and the hypoxanthine transport system of L cells exhibited high degrees of resistance to inhibition by the various chemicals. On the other hand, deoxyglucose transport was inhibited to about the same extent by these chemicals in all the cell lines investigated.
Reversible alterations in the mitotic cycle of chick embryo cells in various states of growth regulation
Authors: Rubin H. , Steiner R.
Published: February 2005

Chick embryo cells which have been kept overnight at pH 6.8 in the absence of serum multiply very slowly. Only a small fraction of cells is in the S period at any given time, and the rate of uptake of 2‐deoxy‐D‐glucose is very low. Upon raising the pH to 7.4 and adding serum (“turn‐on”) the uptake of 2‐deoxy‐D‐glucose increases immediately; the rate of DNA synthesis increases after a lag of about 4 hours, and represents an increase in the fraction of cells synthesizing DNA. The uptake of 2‐deoxy‐D‐glucose is rapidly returned to its original low rate at any time by again lowering the pH and removing serum (“turn‐off”). The synthesis of DNA in the culture remains constant or continues to rise at a markedly reduced rate following the same treatment. Lowering pH or removing serum independently of each other is less efficient at inhibiting the increase in DNA synthesis than the combined treatment but each accomplishes a similar result. Cultures which have been “turnedoff” during the early stages of the rapid increase in DNA synthesis, resume their prior rate of increase immediately if “turned‐on” again within 2.5 hours. If the cultures have been “turned‐off” for 5.5 hours before restoring the “turn‐on,” there is a 2 hour delay before they resume an increased rate of DNA synthesis. The results indicate that chick embryo cells do not become committed to the initiation of DNA synthesis until shortly before, or at the time of the onset of the S period.

Up to 96% of the cells in post‐confluent cultures growing in conventional medium become labeled upon continuous, prolonged exposure to 3H‐thymidine. Seventy‐eight percent of the cells in serum‐deprived cultures growing at a very low rate become labeled. These and other considerations suggest that the inhibition of cell multiplication by high population density or serum deprivation is caused by a lengthening of the time cells remain in the prereplicative G1 period rather than by shifting cells into a qualitatively distinct G0 period. There may, however, be a period common to all cells regardless of growth rate, in which cells are not progressing toward the S period. The length of this variable period would then determine the growth rate of a population of cells.
Heterogeneity of in vitro colony‐ and cluster‐forming cells in the mouse marrow: Segregation by velocity sedimentation
Authors: Metcalf D. , Macdonald H. R.
Published: February 2005

C57BL bone marrow cells were separated on the basis of their sedimentation velocity at unit gravity and cell fractions cultured in agar using three types of colony stimulating factor (CSF). Colony‐forming cells separated as a single peak (s = 4.4 mm/hr) in cultures stimulated by mouse lung conditioned medium (CSFMLCM) or endotoxin serum (CSFES). Clusterforming cells were separable into two peaks and the majority were larger than colony‐forming cells (s = 5.7 mm/hr). Partial segregation of colony‐forming cells was observed according to the morphological types of colonies generated, large cells tending to generate macrophage colonies and small cells, granulocytic colonies. Large colony‐forming cells were more responsive to stimulation by CSF than small cells. Human urine (CSFHU) appeared unable to stimulate the proliferation of most small colony‐forming cells.

Colony‐forming cells appear to be a highly heterogeneous population with intrinsic differences in responsiveness to CSF and with differing capacities to generate colonies whose cells differentiate to granulocytes of macrophages.
Interaction of two mechanisms regulating alkali cations in hela cells
Authors: Cook John S. , Vaughan Gerald L. , Proctor William R. , Brake Emily Tate
Published: February 2005

The alkali cation content of HeLa cells is independent of culture density and of whether the cells are grown in suspension or attached to the culture vessel. With a cell doubling time of 28 hours, the cell K content turns over approximately once per hour. Following partial blockade of the alkali‐cation transport system with ouabain, two distinct but interrelated mechanisms operate in the cellular response: (a) an increase in intracellular Na stimulates the pump so that the short‐term alteration in electrolyte composition is less than would be expected from the fraction of pump sites inhibited, and (b) there is a cyclo‐heximide‐sensitive recovery in transport capacity reflecting a restoration of functional transport sites to their normal density on the cell surface. Experimental manipulations that mimic the effect of ouabain lead to a stimulation of transport, but they do not result in an increase in the number of ouabain‐binding sites on the surface. The data are consistent with a four‐to‐six‐hour turnover of transport sites at the surface, but there is no evidence for a specific induction of the transport system within this short‐term recovery period.