bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptation to trichodermin and anisomycin in Physarum polycephalum
Authors: Gorman Jessica A.
Published: February 2005

The effects of the protein synthesis inhibitors trichodermin and anisomycin on the growth of the eucaryotic myxomycete Physarum polycephalum have been examined. When either of these drugs is added to log phase monoxenic cultures of myxamoebae, cell division is immediately arrested, but on continued incubation, growth resumes at a rate only slightly lower than that of drug free cultures. The length of the drug induced growth lag is roughly proportional to drug concentration. When adapted cells are transferred to fresh drug containing medium, growth is not inhibited. However, if the drug concentration is increased, transient inhibition is again exhibited. Measurement of the antibiotic concentration in used media demonstrates no significant external inactivation of either drug during adaptation. The resumption of growth cannot be attributed to the selection of stable drug‐resistant mutants: single amoebal colonies arising on drug plates are found to be as drug‐sensitive as control colonies when retested after subculture. In addition, when adapted cells are transferred to drug free medium, the phenotypic drug‐resistance is completely lost after several generations of growth. As recovery occurs in the continuous presence of drug and is not due to the accumulation of drug‐resistant mutants, this response appears to be an example of drug adaptation. Cross adaptation between anisomycin and trichodermin is also demonstrated, suggesting a common system is involved in adaptation to these structurally dissimilar, but functionally similar, drugs.
Transformation of human cystinotic fibroblasts by SV40: Characteristics of transformed cells with limited and unlimited growth potential
Authors: Oshima Robert G. , Pellett Ocean L. , Robb James A. , Schneider Jerry A.
Published: February 2005

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed “crisis,” characteristics of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postrisis transformed cystinotic cell line was different from that the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.
Repetitive DNA in differntiating chick tissues
Authors: Ayres Kathleen Nelson
Published: February 2005

Embryonic chick DNA from different tissues was examined for diferences in relative content of highly repetitive DNA which might indicate specific DNA amplification in somatic cells. The content of repetitive sequences in DNA isolated from cerebrum, muscle, and neural retina tissues, at the same and at different embryonic stages, was determined by hydroxyapatite fractionation of partially reassociated DNA samples. An unrenatured marker DNA (C14‐labeled E. coli DNA) was added to each chick DNA sample in order to monitor the nonspecific single‐stranded DNA retention by each hydroxyapatite column. When chick DNA samples were sheared to a double‐stranded length of 1,300 nucleotide pairs, an average of 20.2% ± 2.2% of the DNA was found to reassociate at a Cot value of 10. The quantity of the fast reassociating sequences was found to constitute the same fraction of the DNA in all the tissues studied. In addition, all the reassociated DNA samples exhibited the same CsCl density classes. The studies also indicated that most chick DNA repetitive sequences are interspersed with nonrepetitive sequences.
Interrelationships between water and cellular metabolism in Artemia cysts VII. Free amino acids
Authors: Clegg James S. , Lovallo Jeffrey
Published: February 2005

The concentration of total ninhydrin‐positive material (NPM) soluble in 5% trichloroacetic acid was measured in cysts of the brine shrimp, Artemia salina, as a function of hydration level. No net change in NPM was observed until the cysts had achieved a water content of about 0.65 g H2O/g of initially dry cysts. Above this hydration threshold the NPM content increased markedly. Examination of the free amino acid composition of cysts incubated at selected hydration levels revealed that almost all of the amino acids underwent net change above the hydration threshold. However, just below this threshold, the free amino acid composition was essentially the same as in fully dried cysts. The activity generating net changes in the concentration of free amino acids above the hydration threshold was shown to be metabolic in nature and restricted to the cellular component of the cyst.
Effects of multiplication stimulating activity (MSA) on AIB transport into myoblast and myotube cultures
Authors: Merrill Gary F. , Florini James R. , Dulak Norman C.
Published: February 2005

The effects of a somatomedin analog, Temin's multiplication stimulating activity (MSA), on amino acid transport into muscle cells have been characterized in a series of experiments on myoblasts and myotubes in culture. Addition of MSA to serum‐starved L6 myoblasts increased the rate of aminoisobutyrate (AIB) uptake 50‐150% within five hours. This early effect on transport was followed by increases in cell number, protein content and 3H‐thymidine incorporation. Kinetic analyses indicated that MSA increased the maximal velocity of AIB uptake but had no effect on the KM for AIB. When myoblasts were allowed to fuse (and dividing cells eliminated by addition of 10−4 M cytosine arabinoside) the AIB transport system(s) remained similarly responsive to MSA. In myoblasts and in myotubes, both the basal and MSA‐stimulated rate of AIB uptake were sodium‐dependent processes; little stimulation occurred if sodium was absent from the labeling medium. Further suggesting the involvment of cations in response to hormone, MSA stimulated uptake of the potassium analog, 86Rb+, and increased net intracellular potassium in both myoblasts and myotubes. MSA was active at concentrations equivalent to in vivo levels of somatomedins; neither insulin nor growth hormone had any effect at or near physiological concentrations.
The presence of cyclic AMP, and its effects on oral regeneration and in situ ciliary regeneration in stentor coeruleus
Authors: Maloney Michael S. , Burchill Brower R.
Published: February 2005

We have found cyclic AMP in the large, heterotrichous ciliate Stentor coeruleus in amounts per milligram protein similar to those found in another ciliate, Tetrahymena pyriformis. The possible function of cyclic AMP in Stentor was first examined by determining its effects on oral regeneration, the process by which Stentor can replace a missing oral apparatus in eight to ten hours. Once begun (by brief exposure to a 15% sucrose solution, causing shedding of the oral apparatus) regeneration follows eight specific morphological stages visible with the dissecting microscope. Continuous exposure of regenerating cells to either N6, 2′‐0‐dibutyryl adenosine cyclic 3′:5′‐monophosphate (DBC) or theophylline begun at the onset of oral regeneration (stage 0) caused delays in the completion of regeneration. The delays induced by DBC occurred in the early stages prior to stage 5. Regenerating cells exposed to DBC or theophylline at various stages of development were delayed, even at stages 5 and 6. Both DBC and theophylline reversibly bleached the cortical pigment of the cells. Guanosine 3′:5′‐cyclic monophosphate (cyclic GMP), AMP, GMP, and sodium butyrate neither delayed oral regeneration nor bleached the cortical pigment. Excess extracellular calcium ions alone had no effect on oral regeneration, but 10 mM calcium and DBC caused more delay than DBC alone. Thus, the delay of oral regeneration in Stentor caused by cyclic AMP may involve calcium ions.

To determine if cyclic AMP can retard in situ ciliary regeneration by Stentor, as it does in Tetrahymena, a new technique, more accurate than past methods, was developed to monitor ciliary regrowth. Using this procedure we found that both DBC and theophylline significantly delayed the in situ ciliary regeneration by Stentor.
Reversible regulation by magnesium of chick embryo fibroblast proliferation
Authors: Rubin A. H. , Chu Berbie
Published: February 2005

The rates of 3H‐thymidine incorporation and of cell proliferation in chick embryo fibroblast cultures are reduced coordinately when the [Mg2+] of the external medium is reduced below the physiological concentration of about 0.8 mM. These effects of moderately reduced [Mg2+] and the accompanying change in appearance of the cells, resemble the effects produced by lowering the [serum] of the medium. Cells subjected to severe Mg2+ deprivation, especially at low [Ca2+], die and detach from the culture dish. Cells kept at a reduced rate of proliferation for three days by moderate Mg2+ deprivation are quickly restored to rapid proliferation upon restoration of the normal [Mg2+] of the medium. The rate of proliferation of the chick embryo cells is reduced markedly by lowering [Ca2+] about 100‐fold, but unlike the case of Mg2+‐deprivation this can occur without significant effect on the rate of 3H‐thymidine incorporation. More severe Ca2+ deprivation, which does lower the rate of 3H‐thymidine incorporation, produces retraction of cells from one another and from the dish, and results in a distinctly abnormal, rounded appearance. The results lend weight to the thesis that free [Mg2+] plays a central role within the cell in the coordinate control of metabolism and growth. They also suggest that the effects produced by varying [Ca2+] in the medium are caused by changes at the external surface of the cell.
Arginase activity and other cellular events associated with epidermal hyperplasia
Authors: Redmond Audrey F. , Rothberg Simon
Published: February 2005

The unknown biochemical role of arginase in epidermal metabolism was probed by examining the association of elevated arginase activty with epidermal hyperplasia and hyperkeratinization. Epidermal hyperplasia as induced experimentally by topical application of 1‐decanol to the right side of male hairless mice while the contralateral side served as control. Arginase activity, incorporation of 3 H‐thymidine into DNA, DNA and protein content were measured in the separated control and experimental epidermis six hours and on days 1 through 5 and 7 after 1‐decanol application. After six hours, the epidermis appears damaged histologically, and DNA synthesis is inhibited. By day 1, incoporation of 3 H‐thymidine into DNA is elevated and a new hyperplastic epidermis has formed beneath the original epidermis. Epidermal arginase is elevated two through seven days after 1‐decanol application and always is associated with continuing epidermal hyperplasia. The stimulation of DNA synthesis, which parallels the induction of epidermal hyperplasia by 1‐decanol, precedes the induction of epidermal arginase activity. An attempt to relate these results with polyamine synthesis and other metabolic events is made.
Interrelationships between water and cellular metabolism in Artemia cysts. VIII. Sorption isotherms and derived thermodynamic quantities
Authors: Clegg James S.
Published: February 2005

An analysis of water vapor sorption by cysts of the brine shrimp, Artemia salina, has shown that at environmental water activities (aw) of 0.95 or less, the cysts equilibrate with the aw of their environment. Above this aw the metabolic activity of the cysts participates directly in their water content, and equilibration does not occur. In contrast, dried cysts killed by heat treatment or exposure to ammonia fumes equilibrated with all values of aw examined. Analysis of the temperature dependence of sorption isotherms revealed that below cyst hydrations of about 0.3 g H2O/g dried weight the temperature coefficient for water sorption was negative, but became positive at hydrations appreciably in excess of this value. Estimates for the differential and integral net enthalpy and entropy changes accompanying the sorption of water have been calculated from isotherms. These results have been interpreted and integrated with those from previous work on the hydration‐dependence of metabolic activity. All of the examined hydration properties of the cysts have been shown to be due chiefly to the cellular component, and not the acellular shell. Analysis of the data by the Bradley equation has shown that the hydration behavior of the shell obeys this relationship, whereas that of the cellular component does not.
The selection of a stable rat hepatoma variant with concomitant increase in ploidy and permeability to glycerol
Authors: Li C. C. , Lin E. C. C. , Karnovsky M. J. , Lin P. S.
Published: February 2005

By repeated selection for longer survival in an isotonic solution of glycerol, a stable subline of Novikoff rat hepatoma cells has been isolated. The cells exhibit markedly increased resistances to osmotic lysis in isotonic solutions of glycerl. They are twice as large and have twice as many chromosomes as cells of the parental line. It is suggested that the osmotic stress procedure can be extended for the selection of numerous kinds of mutants and can be used as a method of analysis of membrane properties.
Early transport changes during erythroid differentiation of friend leukemic cells
Authors: Mager Dixie , Bernstein Alan
Published: February 2005

Early transport changes occurring during Friend erythroleukemic cell differentiation are reported. A decrease in the rate of 86Rb transport was observed beginning approximately five hours after stimulation with 1.5% dimethylsulfoxide (DMSO), a potent inducer of Friend cell differentiation. By 12 to 14 hours after DMSO addition, the transport rate had stabilized at close to 60% of control level. This decrease in the rate of 86Rb transport preceded a previously reported decrease in cell volume. Other chemical inducers of Friend cells, such as hypoxanthine and ouabain, also caused early decreases in 86Rb influx. In contrast, xanthine, which does not induce Friend cell differentiation, also did not affect 86Rb influx.

The transport of two amino acid analogues, αaminoisobutyric acid and 2‐aminobicyclo [2,2,1]‐heptane‐2‐carboxylic acid, which differ in their mode of uptake, was also measured following induction by DMSO. The transport rates of both compounds decreased after a 12‐hour exposure to DMSO. In contrast, the uptake of 3H‐colchicine, a drug which diffuses passively across the cell membrane, was not significantly affected.

Studies with several variant cell lines which do not synthesize hemoglobin in response to DMSO indicate that these non‐inducible cells can be divided into two classes—those that demonstrate early changes in transport very similar to the changes observed in inducible cell lines and those which exhibit only small changes in transport. Results obtained using a revertant clone have helped to distinguish between those transport changes which are associated with the induction of hemoglobin synthesis and those which are not. In addition, these early transport changes may be useful in defining the stage in the differentiation process at which a particular variant line is blocked.
Effect of hydrocortisone on cell morphology in C6 cells
Authors: Berliner Judith A. , Bennett Kimberly , De Vellis Jean
Published: February 2005

Hydrocortisone has been found to induce cell spreading in rat glial C6 cells by 24 hours after its addition. This spreading phenomenon is correlated with an increase in the fraction of the peripheral cytoplasm occupied by microfilaments. Cytochalasin B causes disorganization of microfilaments in the peripheral cytoplasm of the cells. Additionally, it also prevents cell spreading in response to hormonal stimulation. High levels of calcium prevent recovery of normal microfilament organization and cell spreading following removal of cytochalasin B, but have no effect on normal microfilament organization alone. Additionally both the hydrocortisone induced spreading of C6 cells and increases in peripheral microfilaments are shown to be dependent on RNA and protein synthesis. The levels of protein co‐electrophoresing with actin are not effected by hydrocortisone.
Growth dependence of phosphoglyceric acid dehydrogenase activity in cultured rat liver cells
Authors: Isom Harriet C. , Pizer Lewis I.
Published: February 2005

Rat liver epithelial cells in culture (WIRL‐3C) have the enzymes that synthesize serine from 3‐phosphoglyceric acid. Both phosphoglyceric acid dehydrogenase (PGAD) and serine‐phosphate (serine‐P) forming activities fluctuate with time after subculture and are higher in growing than confluent cells. This activity pattern was not common for other dehydrogenases in WIRL‐3C cells, nor was it common for PGAD activity in other cultured cells. At time of sub‐culture, cells are removed from spent medium, treated with trypsin, and fed fresh medium. None of these parameters causes the rise in activity; in contrast, reduction in cell density and the accompanying stimulation of growth do. PGAD activity decreases when growth is slowed either as the cells progress to the end of the culture cycle, when cells are treated with dexamethasone‐phosphate (Dx‐P) or dibutyryl cyclic AMP (cAMP) and theophylline or when the serum concentration of the medium is reduced to 0.2%. Under these conditions, decreased PGAD activity is paralleled by a decline in growth and DNA accumulation. PGAD activity in WIRL‐3C cells is regulated in a manner closely resembling what has been observed previously in rat liver from the whole animal. The possible use of this system in studying regulation of gene expression in mammalian cells is discussed.
Early changes in the membrane of HeLa cells adsorbing sendai virus under conditions of fusion
Authors: Fuchs P. , Spiegelstein M. , Haimsohn M. , Gitelman J. , Kohn A.
Published: February 2005

Adsorption of Sendai virus at high multiplicity (500‐1,000 HAU/106 cells) to HeLa cells grown in monolayers causes immediate changes in the ion barrier of the cell membrane, as well as changes in the morphology of the virus‐treated cells. Within minutes of adsorption the cells begin to lose potassium and an extensive influx of ions into the cells occurs. Concomitantly with these changes, the cell membrane becomes depolarized, and the resting potential across its membrane decreases. Twenty to sixty minutes post adsorption the damage to the cell membrane is repaired, and both the potassium uptake and the resting potential return to their pre‐exposure values. Scanning electron‐micrographs of Sendai infected cells incubated at 37°C show formation of bridging microvilli in a zipper‐like fashion within two to five minutes post‐adsorption; 30 to 60 minutes thereafter the majority of cells in the monolayer are fused. Biochemical changes induced by virus adsorption and the role of Ca++ ions in the observed effects are discussed.
Polyamine biosynthesis and DNA synthesis in cultured mammary gland explants from virgin mice
Authors: Sakai Tadashi , Lundgren David W. , Oka Takami
Published: February 2005

The mammary cells in virgin mice are essentially non‐proliferative, but they can be induced to undergo DNA synthesis in vitro in the presence of insulin. Time course studies on polyamine biosynthesis and DNA synthesis showed that insulin elicits sequential stimulation of the activity of the polyamine biosynthetic enzymes, ornithine decarboxylase, S‐adenosyl‐L‐methionine decarboxylase (SAMDC) and spermidine synthase, and an increase in the concentration of spermidine prior to the augmentation of DNA synthesis. At 48 to 72 hours of culture when DNA synthesis is maximal, the concentration of spermidine increased 2− to 3‐fold, whereas the level of spermine remained unchanged. Addition of methyl glyoxal bis(guanylhydrazone) (5—10 μM), a potent inhibitor of SAMDC, to the medium at the onset of culture resulted in inhibition of spermidine formation and DNA synthesis, but when added at 24 hours or 48 hours of culture, the inhibitory effect on DNA synthesis was greatly reduced. The drug, however, produced little inhibition of RNA and protein synthesis. Inhibition of DNA synthesis by the drug can be reversed by addition of spermidine or other polyamines such as putrescine, cadaverine and spermine to the culture. Spermidine is, however, the only polyamine that is effective at physiological concentrations (100∼150 pmoles/mg tissue). These results suggest a possibility that spermidine may play a key role in the regulation of mammary cell proliferation.
Induction of erythroid differentiation in friend leukemia cells by bromodeoxyuridine
Authors: Adesnik Milton , Snitkin Harriet
Published: February 2005

Treatment of Friend leukemia cells with BrdU, the thymidine analog which interferes with DMSO induced differentiation in these cells as well as the expression of differentiated character in many other cell systems, is capable of inducing erythroid differentiation. Globin mRNA, as assayed by hybridization to globin cDNA, increases 2.5− to 30−fold after appropriate treatment with BrdU. This effect was observed with several different subclones of three independent Friend tumor cell lines. After BrdU treatment, globin mRNA content may reach up to 10—20% of the levels in DMSO induced cultures. The induction of erythroid differentiation is also apparent when accumulated heme content or the appearance of benzidine positive cells is monitored

One Friend cell line (745) we examined was not induced by BrdU although it incorporated an amount of BrdU into its DNA comparable to that incorporated by the other cell lines. In addition, BrdU did interfere with DMSO induction in this cell line

These results suggest that two different mechanisms may be operative in regulating erythroid differentiation in Friend leukemia cells. While BrdU interferes with the mechanism activated by DMSO treatment, this analog could independently activate an alternative mechanism.
Alterations in the protein synthetic apparatus of friend erythroleukemia cells during their erythroid differentiation
Authors: Nishioka Yutaka , Silverstein Saul
Published: February 2005

The changes in rate of protein synthesis and cell division and the distribution of polyribosomes and globin mRNA on the polyribosomes of Friend erythroleukemia (FL) cells exposed to 2% DMSO and maintained at low cell density, were examined at different times after exposure to DMSO. The rate of protein synthesis and the capacity of cells to divide declined in concert to 50% of the level found in untreated cell cultures at 24 hours after exposure. Thereafter these rates recovered to 70% of the rate found in untreated control cultures until 96 hours post‐exposure and then irreversibly declined as the cells lost the capacity to divide. The proportion of ribosomes present as polyribosomes in cells exposed to DMSO paralleled the capacity of these cells to synthesize protein. The distribution of polyribosomes analyzed by sedimentation in sucrose gradients demonstrated that a discrete, abundant class of polyribosomes composed of pentamers to heptamers appeared as early as 48 hours after exposure to DMSO. The appearance of an abundant class of polyribosomes was correlated with globin synthesis be demonstrating that a discrete class of polyribosomes arises in cells treated with the inducers hexamethylene bisacetamide and hemin.
Deoxyglucose and 3‐O‐methylglucose transport in untreated and ATP‐depleted novikoff rat hepatoma cells. Analysis by a rapid kinetic technique, relationship to phosphorylation and effects of inhibitors
Authors: Graff Jon C. , Wohlhueter Robert M. , Plagemann Peter G. W.
Published: February 2005

Detailed time courses of uptake of labeled 3‐O‐methyl‐D‐glucose and 2‐deoxy‐D‐glucose by untreated and ATP‐depleted Novikoff rat hepatoma cells were determined as function of concentration (0.2–10 mM) by a rapid mixing/sampling technique which allows uptake measurements in time intervals as short as 1.5 seconds. Intracellular accumulation of 3‐O‐methylglucose in untreated and ATP‐depleted cells and of deoxyglucose in ATP‐depleted cells to equilibrium followed pseudo‐first order kinetics and initial velocities were computed from the overall time courses of substrate accumulation. Initial velocity was a Michaelis‐Menten function of exogenous substrate concentration. The estimated kinetic constants for zero‐trans transport of 3‐O‐methylglucose were about the same for untreated and ATP‐depleted cells (K = 1.73 ± 0.24 mM; V = 28.8 ± 3.6 pmoles/μl cell H2O. sec) and were similar to those for deoxyglucose transport in ATP‐depleted cells (K = 0.65 ± 0.1 mM; V = 19.6 ± 1.6 pmoles/μl cell H2O. sec). Similar kinetic parameters were obtained for the transport of D‐glucose and D‐galactose in ATP‐depleted cells. The transport of 3‐O‐methylglucose and deoxyglucose were inhibited by each other in a simple competitive manner with apparent Ki's similar to their transport Km's.

In untreated cells, in which deoxyglucose was phosphorylated, intracellular steady‐state levels of free deoxyglucose accumulated within 10 to 20 seconds of incubation regardless of its concentration in the medium. Thereafter, the rate of deoxyglucose incorporation into total cell material reflected the rate of phosphorylation rather than the transport rate. The rate of deoxyglucose transport exceeded the initial rate of its phosphorylation by 20–40%. The intracellular steady‐state levels observed during the first 2 minutes of incubation decreased from about 40% of equilibrium level at 0.2 mM deoxyglucose to about 8% at 10 mM. Computer fits of a kinetic equation describing transport and phosphorylation as independent processes operating in tandem to these data are consistent with the observed kinetic constants for hexose transport and hexokinase activity with deoxyglucose as substrate. Upon longer incubation (2–10 minutes) the rate of deoxyglucose uptake by the phosphorylating cells decreased progressively, concomitant with a decrease in intracellular ATP and an increase in intracellular deoxyglucose to equilibrium levels. It is demonstrated that the rate of deoxyglucose uptake, measured at two or more minutes, seriously underestimates the hexose transport rate and yields misleading conclusions regarding the extent and type of inhibition by transport inhibitors, such as Persantin or cytochalasin B. Persantin inhibited hexose transport in a simple non‐competitive manner (Ki = 20 μM) indicating that the drug affects the function of the hexose carrier.
Studies of cAMP metabolism in cultured hepatoma cells: Presence of functional adenylate cyclase despite low camp content and lack of hormonal responsiveness
Authors: Leichtling Ben H. , Su Y.‐F. , Wimalasena Jayantha , Harden T. Kendall , Wolfe Barry B. , Wicks Wesley D.
Published: February 2005

The ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in a line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined.

The concentration of cAMP in BRL cells (∼ 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (∼ 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. Binding studies with [125I]‐iodohydroxybenzylpindolol, a specific β‐adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 β‐adrenergic receptors whereas BRL cells contain 2–5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phoshodiesterase activities.
On the stimulation of rat thymocyte 3‐0‐methyl‐glucose transport by mitogenic stimuli
Authors: Hume David A. , Weidemann Maurice J.
Published: February 2005

Pretreatment of rat thymus lymphocytes with N‐ethyl‐maleimide prevents the stimulation of 3‐0‐methyl‐glucose transport by concanavalin A or ionophore A23187 but does not affect the ability of concanavalin A to induce a rapid increase in cellular Ca2+ uptake. N‐ethyl‐maleimide added after concanavalin A amplifies rather than prevents the subsequent stimulation of 3‐0‐methyl‐glucose transport. Incubation of thymocytes with concanavalin A produces a decrease of 43% in the apparent Ki for phloretin, a competitive inhibitor of 3‐0‐methyl‐glucose transport, without affecting the apparent Km for the substrate. Similarly, very low concentrations of cytochalasin B inhibit concanavalin A‐stimulated glucose transport preferentially, without markedly affecting the unstimulated transport rates. The similarity between concanavalin A‐stimulated 3‐0‐methyl‐glucose transport in thymocytes and insulin‐stimulated glucose transport in adipose tissue, with particular emphasis on the effects of the modifying agents described in this paper, is discussed.
Morphological and biochemical correlates of skeletal muscle contractility in the cat. I. Histochemical and electron microscopic studies
Authors: Van Winkle W. B. , Schwartz A.
Published: February 2005

Three cat hind limb muscles have been examined, histochemically and ultrastructurally, in a multiparameter correlative study of structure and function in skeletal muscle contractility. The soleus, a histochemically pure, slow‐twitch muscle possesses ultrastructural features which are, in many cases, significantly different from those of almost pure fast twitch caudofemoralis muscle. Although stereological analysis of fiber types indicates a correlation between speed of relaxation and volume of sarcoplasmic reticulum, morphological features such as fenestrated collars and triad morphology are identical in all fiber types. The fast twitch‐oxidative‐glycolytic fiber possesses features common to both slow twitch fibers (high mitochondrial content) as well as fast twitch fibers (high sarcoplasmic reticulum content) in addition to Z band width which falls in between these two fiber types.

Sarcoplasmic microtubules have been described in all three fiber types in all muscles examined. They occur in predictable orientation and their possible function (s) is described.
Morphological and biochemical correlates of skeletal muscle contractility in the cat. II. Physiological and biochemical studies
Authors: Van Winkle W. B. , Entman M. L. , Bornet E. P. , Schwartz A.
Published: February 2005

Isometric twitch characteristics and biochemical parameters of isolated myosin and sarcoplasmic reticulum have been compared in three cat hind limb muscles. The fast twitch caudofemoralis and the slow twitch soleus are almost pure muscles as judged from histochemical studies. Isolated myosin from the caudofemoralis is not only 2‐ to 3‐fold higher in its ATPase activities than that of the soleus, but also in non‐dissociated forms has greater electrophoretic mobility than the soleus myosin. Purified myosins from fast muscles as well as soleus exhibited three light chains upon electrophoresis. However, the intact non‐solubilized myosins differed in electrophoretic mobilities. The sarcoplasmic reticulum fraction isolated from caudfemoralis exhibits faster rates of Ca++ binding and uptake than soleus, and when fit to a two component model, the caudofemoralis SR exhibits a higher amount of a fast binding site than does soleus SR, features reflected in differences in the relaxation time of the two muscles.

In contrast, the fast twitch tibialis anterior has been shown to be a gradient of fiber types and its isometric twitch may be separated by selective nerve stimulation, into a fast and a slow twitch component. Our findings that myosin fraction, as well as sarcoplasmic reticulum fractions isolated from these two components differ with respect to their biochemical characteristics add support to the possibility of a dual function in this muscle.
Rates of protein synthesis by hepatocytes isolated from rats of various ages
Authors: Ricca George A. , Liu Daniel S. H. , Coniglio John J. , Richardson Arlan
Published: February 2005

The rate of total protein synthesis in isolated hepatocytes was determined. The incorporation of L‐[3H]valine into protein is linear for at least two hours of incubation and is affected by the concentration of amino acids in the medium. Uptake of valine by hepatocytes from 1.5‐ and 18‐month‐old rats was identical and appears to occur by simple passive diffusion. Within five minutes, the specific activities of the intracellular and extracellular valine pools are equivalent. The specific activities of these pools are saturated by 1.6 mM valine and remain constant for 60 minutes of incubation. The rates of protein synthesis by hepatocytes from 1‐ to 2‐month‐old rats is 96.8 pmoles of valine per minute per milligram protein. This is comparable to rates of protein synthesis reported for perfused liver and liver in vivo and is approximately 64% higher than the rate of protein synthesis by hepatocytes from 18‐month‐old rats.
Inhibition of sugar uptake in adenosine‐treated 3T3 cells
Authors: Barnes David W. , Brown Verona T. , Colowick Sidney P.
Published: February 2005

Addition of 5 to 250 micromolar adenosine to the culture medium resulted in a 30–80% inhibition of the rate of uptake of 2‐deoxyglucose or 3–0‐methylglucose by sparse or confluent 3T3 cells within three hours. The inhibition of deoxyglucose uptake could be reversed partially by changing the cells to medium without adenosine for two hours and could be prevented completely by the addition of persantin, an inhibitor of nucleoside uptake. The adenosine effect is not due to inhibition of pyrimidine synthesis, since it is not prevented by uridine. It is not seen in 3T6 cells lacking adenosine kinase. The inhibition could be observed on confluent cells whose deoxyglucose uptake was stimulated by insulin, epidermal growth factor (EGF), calf serum or calcium phosphate. Although the percentage stimulation over control by these factors varied, the percentage inhibition by addition of adenosine of the stimulated rates, as well as the unstimulated rate, was relatively constant. EGF, insulin and calcium phosphate caused little or no stimulation of deoxyglucose uptake by sparse cells, whether adenosine treated or untreated. The results suggest that adenosine acts intracellularly after phosphorylation to regulate sugar uptake through a mechanism which is independent of the regulation by hormones and cell density.
Sequential changes in ribosomal activity during the activation and cessation of growth in lymphocytes stimulated with concanavalin A
Authors: Harms‐Ringdahl Mats , Cooper Herbert L.
Published: February 2005

The ribosomal activity in human peripheral blood lymphocytes stimulated by concanavalin A has been characterized during the passage of cells from the resting (G0) state, through maximum mitotic activity, and into a secondary resting state. The total ribosome content increased progressively after the addition of concanavalin A and after 72 hours reached a maximal 10‐fold increase. Lymphocytes cultured for 120 hours returned to a low metabolic activity, but still contained four times more ribosomes per cell than unstimulated lymphocytes. During the first 24 hours free ribosomes, which were originally 64% of resting cell ribosomes, decreased to 27% of total particles. Thereafter they reaccumulated until, at 120 hours, free ribosomes again comprised 61% of the total ultraviolet absorbance and the total polysome profile resembled that for resting cells, despite the higher ribosome content.

Analysis of the rate of protein synthesis and the corresponding quantity of polysomes at different times in the growth sequence revealed that protein synthesizing efficiency of polysomes was initially low and rose for the first 48 hours of culture. After 48 hours, polysomes with low protein synthesizing efficiency reappeared. Following pulse labeling of rRNA with [3H]uridine, the redistribution of newly synthesized ribosomal particles was followed. A decreased flow of ribosomes through polysomes was evident at 48 hours and thereafter. Diminution in polysome efficiency, reduced flow of particles through polysomes, and accumulation of free ribosomes thus characterize the cessation of growth which occurs as a physiological event in Con A‐stimulated lymphocyte cultures. Nevertheless, these changes begin at or before the peak of RNA, protein and DNA synthetic activity, showing that the biochemical mechanisms related to cessation of cell growth begin to function during the height of cell growth in a system under physiological control.
Requirements of BHK cells for the exit from different quiescent states
Authors: Talavera Antonio , Basilico Claudio
Published: February 2005

We have investigated the kinetics of exit from the resting state of BHK cells which had been arrested by isoleucine deprivation, serum starvation, or high temperature in the case of three ts G1 mutants. In addition, we have studied the effect of imposing a secondary deprivation on cells which had been released from one of the above mentioned blocks. The results obtained show that the quiescent states reached by BHK cells following serum or isoleucine deprivation cannot be differentiated on the basis of the exit kinetics from Smith and Martin's probabilistic A‐state. Nevertheless, the response of cells to secondary deprivation is different, depending on the nature of the primary arresting condition used, reflecting physiological differences between the different resting states. A model is presented which postulates that cycle transition specific genes require the presence of different proliferative agents for their expression.
Lithium transport by fibroblastic mouse cells: Characterization and stimulation by serum and growth factors in quiescent cultures
Authors: Smith Jeffrey B. , Rozengurt Enrique
Published: February 2005

Serum enhances the rate of Li+ entry and exit in quiescent cultures of mouse fibroblasts by 2‐ to 3‐fold. Tertiary cultures of whole mouse embryos as well as established fibroblast lines (3T3, 3T6) show the increase in Li+ permeability when serum is added to cultures whose growth has been arrested by serum deprivation. Growing cells are only slightly more permeable to Li+ in the presence of serum. Purified compounds which initiate DNA synthesis also rapidly increase Li+ entry; mitogenic levels of thrombin and the combination of epidermal growth factor, insulin, and bovine serum albumin were the most effective ones tested. The effect of serum on Li+ uptake occurs within a few minutes, is not affected by inhibitors of macromolecular synthesis, and appears mainly to increase the Vmax of entry. Inhibitors of energy production partially reduce Li+ entry but do not block the activation by serum. One portion of Li+ uptake (−40%), which is inhibited by ouabain, phloretin, or Na+ deprivation, is mediated by the Na+/K+ pump in the plasma membrane. A second mechanism of Li+ entry which is blocked by Na+ or amiloride appears to be a Na+ specific “porter.” The activity of both components is stimulated by serum. The increased activity of the putative Na+ porter would increase Na+ availability to the Na+ pump and may account for its enhancement by serum, which was also noted previously (Rozengurt and Heppel, '75).
Cultured human fibroblasts: Distribution of cell generations and a critical limit
Authors: Harley Calvin B. , Goldstein Samuel
Published: February 2005

In circular outgrowths of human skin fibroblasts we found that mitotic cells at the circumference had consumed more of their replicative lifespan than cells located more centrally. The lifespan remaining in cells at a given radial position could be predicted by determining their generation level based on the rate at which the outgrowth expanded and the cell doubling time. The data also show that outgrowths contain a heterogeneous mixture of cells described by a linear distribution of generations which can account for the variable replicative capacity observed in clones and the exponential increase in the fraction of nondividing cells with serial passage. These results support the concept that a critical limit of cell divisions determines the replicative lifespan.
Selective changes in tRNA methyltransferase activity in confluent monolayers of WI‐38 cells stimulated to proliferate
Authors: Wilkinson C. Ray , Kerr Sylvia J.
Published: February 2005

In quiescent confluent monolayers of WI‐38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzymes show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2μ/ml). The increases in activity were not equivalent for the different base‐specific enzymes. The contribution of the N2‐methylguanine specific enzyme remained relatively constant, while that of the N2,N2‐dimethylguanine specific and 1‐methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1‐methylguanine and the 7‐methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non‐coordinate variations in the expression of the different base‐specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.
Differential effects of inhibition of polyamine biosynthesis on cell cycle traverse and structure of the prematurely condensed chromosomes of normal and transformed cells
Authors: Sunkara Prasad S. , Pargac Mary B. , Nishioka Kenji , Rao Potu N.
Published: February 2005

The objective of this study was to determine the points in the cell cycle at which normal and transformed cells become arrested as a result of polyamine deprivation. Treatment of normal (human fibroblast line PA2 and mouse 3T3) and transformed (CHO, HeLa and SV3T3) cells with methylglyoxal bis‐(guanyl‐hydrazone) resulted in a significant decrease in the levels of spermidine and spermine which was associated with an inhibition of growth. Examination of the prematurely condensed chromosomes (PCC) of the polyaminedepleted cells, revealed that normal fibroblasts were preferentially arrested in early G1 phase while a majority of cells in the transformed lines were blocked in S phase. A close examination of the PCC of the transformed cells indicated a significant decrease in the number of DNA replication sites suggesting that polyamines have an important role in DNA chain initiation.
Characterization of cell lines showing growth control isolated from both the wild type and a leucyl‐tRNA synthetase mutant of chinese hamster ovary cells
Authors: Pollard J. W. , Stanners C. P.
Published: February 2005

The genetic approach to the problem of cellular growth control is limited by the availability of recessive mutations in cell lines which are capable of growth control in vitro. The CHO cell line has yielded many recessive mutations including, for example, tsH1, a temperature sensitive leucyl‐tRNA synthetase mutant, which under non‐permissive conditions rapidly shuts down protein synthesis and generates uncharged tRNA. Both CHO and tsH1 are transformed, however, and do not respond to environmental stimuli with the coordinated regulation of macromolecular processes observed in normal diploid fibroblasts. We describe here the isolation and characterization of growth control revertants obtained from both CHOwt and tsH1. The best of these GRC+L‐73, isolated from tsH1, had 20 chromosomes, one less than tsH1, had normal fibroblastic morphology, would not grow in suspension, required high serum concentrations for growth, grew to relatively low cell densities at saturation in monolayer culture and showed a stationary phase characterized by arrest in a G1‐like state with maintenace of high viability for several weeks. It is expected that this line as well as a ts revertant GRC+LR‐73 will greatly facilitate the genetic investigation of growth control and, in particular, will help to elucidate the role of uncharged tRNA in the regulation of macromolecular synthesis in mammalian cells.
Interactions between purified GM‐CSF, purified erythropoietin and spleen conditioned medium on hempoietic colony formation in vitro
Authors: Metcalf D. , Johnson G. R.
Published: February 2005

Preincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250‐fold excess concentration of purified GM‐CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM‐CSF were added to agar cultures stimulated by pokeweed mitogen‐stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non‐erythroid colonies or the non‐erythroid content of mixed erythroid colonies.

Although neither GM‐CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM‐CSF was also able to support the survival in vitro of a small proportion of erythroid colony‐forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony‐forming cells in similar cultures of adult marrow cells.

The results do not support the hypothesis that GM‐CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM‐CSF and EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM‐CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM‐CSF is unable to stimulate the formation of colonies of these types.
Erythroid and granulocytic colony growth in cultures supplemented with human serum lipoproteins
Authors: Aye M. T. , Seguin J. A. , McBurney J. P.
Published: February 2005

The ability of human serum to support erythroid an dgranulocytic colony formation has been investigated. It was found that normal human serum could replace fetal calf serum in the cultures and was able to support the growth of these hemopoietic colonies. Serum fractions enriched for low density lipoproteins, either by precipitation with Heparin‐Mn++ or by ultra‐centrifugation, was found to contain this growth supporting activity of human serum.