bluuebunny/crossref_metadata_2025 huggingface dataset:
Studies on the regulation of the three enzymes of the leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line
Authors: Stern Elizabeth Sell , Krooth Robert S.
Published: February 2005

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, α‐D‐galactose‐1‐phosphate:UDP glucose uridylyl transferase and UDP galactose 4‐epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose‐1‐phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue‐specific functions of liver.

The failure of galactose to stimulate increased cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.
Induction of hyaluronic acid synthetase activity in rat fibroblasts by medium change of confluent cultures
Authors: Tomida Mikio , Koyama Hideki , Ono Tetsuo
Published: February 2005

Hyaluronic acid synthesis in cultured cells usually occurs during the growth phase. The relation between hyaluronic acid synthetase activity and cell proliferation is studied. The synthetase activity in rat fibroblasts is high during the growth phase, but low in the stationary phase. When the old medium of stationary cultures is renewed with fresh medium containing 20% calf serum, DNA synthesis occurs synchronously between 12 and 20 hours, followed by cell division. Under these conditions, the hyaluronic acid synthetase activity is significantly induced within two hours, reaching a maximum level at 5–8 hours, and then decreases gradually. This induction of the synthetase, which shows a high turnover rate, requires continued synthesis of both RNA and protein. Furthermore, the induction of both DNA and hyaluronic acid synthesis is found to be caused by calf serum added in the medium. However, dialysis and ultrafiltration of the serum permit us to concentrate an active fraction with a high molecular weight, which induces the synthetase activity, but not DNA synthesis.
A reduction in energy‐dependent amino acid transport by microtubular inhibitors in ehrlich ascites tumor cells
Authors: Fyfe Mary Jo , Loftfield Sharon , Goldman I. David
Published: February 2005

Vincristine, other periwinkle alkaloids, and colchicine partially inhibit the energy dependent transport of α‐aminoisobutyric acid in Ehrlich ascites tumor cells. The properties of this phenomenon were characterized in detail for vincristine. Maximum depression of the steady‐state intracellular α‐aminoisobutyric acid level was achieved with a vincristine concentration of > 0.5 m̈M. The inhibitory effect of vincristine increases as the extracellular α‐aminoisobutyric acid concentration is increased reaching a maximum, however, of only ∼25% at a level of 5 mM, leaving a large gradient for α‐aminoisobutyric acid across the cell membrane. Vincristine produced an asymmetrical effect on the bidirectional fluxes decreasing the initial uptake rate, while increasing the efflux of α‐aminoisobutyric acid. Inhibition of net α‐aminoisobutyric acid transport by vincristine was partially reversible (∼40%). Colchicine (50 m̈M) reduced the steady‐state α‐aminoisobutyric acid level by 30%, an effect that was not reversible. Inhibition by vinleurosine and vinrosidine was comparable to that of vincristine. Addition of glucose to the medium resulted in a small, but significant, decrease in the inhibitory effects of both vincristine and colchicine.

The data indicate that these agents inhibit a small component of the uphill transport of α‐aminoisobutyric acid in Ehrlich ascites tumor cells. The inhibitory effect of vincristine cannot be attributed to an increase in the passive permeability of the cell membrane to this agent. Rather, the data along with other studies from this laboratory suggest that vincristine reduces the energy‐dependent transport of α‐aminoisobutyric acid by either inhibiting cellular energy metabolism or by inhibiting the coupling of energy‐metabolism to the transport of this amino acid and raises the possibility that cellular microtubules play a role in these processes.
Cell Attachment to cllagen: The requirement for energy
Authors: Klebe Robert J.
Published: February 2005

It has previously been demonstrated that cell attachment to collagen depends on the presence of a serum‐derived cell attachment factor. Two steps in the cell attachment process have been defined. First, the cell attachment factor can bind to collagen in the absence of divalent cations. Secondly, either Ca++ or Mg++ are required for cells to attach to the collagen‐cell attachment factor complex. A third requirement for cell attachment to collagen is demonstrated here; namely, cellular metabolic energy.

A simple method for the assay of ATP in tissue culture cells is presented.
Effect of pharmacological agents on human keratinocyte mitosis in vitro. II. Inhibition by catecholamines
Authors: Harper Robert A. , Flaxman B. Allen
Published: February 2005

Catecholamines produce mitotic inhibition in primary cell cultures of human keratinocytes probably via a block in the G2 part of the cell cycle. Epinephrine produced significant mitotic inhibition (49%) at a concentration as low as 4.5 × 10−10 M, while its analog, isoproterenol, produced 47% inhibition at 1 × 10−10 M. Norepinephrine elicited a 49% inhibitory response at 1 × 10−8 M. One other catecholamine, dopamine, caused a 53% decrease in mitosis at 1 × 10−6 M. Other structurally related amines to exhibit mitotic inhibition were phenylephrine, 58% at 1 × 10−7 M; octopamine, 47% at 1 × 10−5 M; and tyramine, 52% at 1 × 10−4 M. Serotonin showed no mitotic inhibition at 1 × 10−4 M.

Various alpha and beta adrenergic blocking agents were added to the cell system. The alpha blocking agent, phentolamine, had no effect on mitosis. When added in conjunction with epinephrine or norepinephrine, no reduction of the catecholamine‐induced mitotic inhibition was observed. The beta blocking agent, propranolol, by itself showed slight mitotic inhibition at 1 × 10−6 M. When added along with epinephrine or norepinephrine, propranolol reduced the catecholamine‐induced mitotic inhibition approximately 65%. In addition, propranolol blocked mitotic inhibition caused by phenylephrine, an alpha adrenergic agent. However, another beta blocking agent, dichloroisoproterenol, showed strong mitotic inhibition (53%) when added alone to the cultures at a concentration of 1 × 10−8 M. The effect was reduced to zero in the presence of propranolol. These data suggest that while beta receptors may be involved in the catecholamine‐induced mitotic inhibition of human keratinocytes in vitro, the nature of the receptor‐molecule interaction may be complex.
The effects of a glucocorticoid on the cell surface of RLC‐GAI cells
Authors: Berliner Judith A. , Gerschenson Lazaro E.
Published: February 2005

The purpose of these studies was to examine the effects of a synthetic glucocorticoid, dexamethasone sodium phosphate, on the cell surface of an epithelial cell line, RLCGAI, and to investigate the mechanism of these effects. Within hours after addition of dexamethasone sodium phosphate to the cultures, the cells begin to spread going from a more bipolar to a more epithelioid form; this change is maximal by 24 hours. In the spread cells the density of surface microvilli, as visualized in the scanning electron microscope, is considerably reduced. The changes in cell surface and shape elicited by the glucocorticoid are blocked by actinomycin D but not by hydroxyurea. Cell spreading is probably related to the configuration of cell microfilaments since these are increased in numbers in the presence of the hormone and spreading is inhibited by cytochalasin B. An important role of microfilaments in the general mechanism of action of glucocorticoids is suggested.
The effect of Lanthanum on electrophoretic mobility and passive cation movements of the ehrlich ascites tumor cell
Authors: Smith Thomas C.
Published: February 2005

We have investigated the effects of La+3 binding to the surface of Ehrlich ascites tumor cells on cell electrophoretic mobility and passive movements of Na+ and K+. Incubation of tumor cells in La+3‐containing media results in a La+3 concentration‐dependent decrease in net surface charge negativity. At [La+3] greater than 0.5 mM, the net surface charge becomes positive with maximum positivity occurring at [La+3] = 0.9 mM. The effects of La+3 binding on passive Na+ and K+ movements were investigated by following 22Na and K+ losses from ouabain‐inhibited cells. Neither low (0.02) nor high (1.0 mM) [La+3] had any effect on the K+ efflux rate coefficient. 22Na losses from control and La+3‐treated cells were consistent with washouts from two cellular compartments. Low [La+3] (0.02 mM) was without effect on Na+ loss from the cells. However, higher [La+3] (1.0 mM) resulted in a 48% inhibition of Na+ loss from the more slowly exchanging compartment. These results are not consistent with simple electrostatic interactions exerting a major influence on the passive movements of Na+ and K+. It is suggested that La+3 interacts with sites specific for Na+, perhaps involved in a carrier‐mediated exchange system.
Rapid breakdown of diphosphoinositide and triphosphoinositide in erythrocyte membranes
Authors: Garrett N. E. , Burriss Garrett R. J. , Talwalkar R. T. , Lester Robert L.
Published: February 2005

Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37°C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0° thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di‐ and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75°C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10‐second intervals. This method was more convenient than standard procedures and gave yields of di‐ and triphosphoinositide equivalent to that obtained by the method of Folch.
Selection of chinese hamster somatic cell mutants after irradiation of BUdR‐Labelled cells
Authors: Thirion Jean‐Paul , Labrecque Romain , Vu Thuy‐Phung
Published: February 2005

The selection of somatic cell mutants after irradiation of BUdR‐labelled cells is improved with the use of a mercury lamp combined with a plastic sheet which filters the unwanted wave lengths below 303 nm. New somatic cell mutants defective for galactose metabolism or auxotrophe for fetal calf serum were thus isolated.
Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization, cellular ATP and associated membrane changes in D+ and D− cells
Authors: Vlodavsky Israel , Fibach Eitan , Sachs Leo
Published: February 2005

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D+ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D− myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D+ than in D− cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D− than in D+ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D+ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D+ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D− cells, were found before most of the D+ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D− cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D+ cells, were associated with differences in the organization of the cell surface membrane.
Polynucleotide aggregates enhance the transport of protein at the surface of cultured mammalian cells
Authors: Ryser H. J.‐P. , Termini T. E. , Barnes P. R.
Published: February 2005

It was known that polycationic polymers enhance the entry of macromolecules into cells. We now show that polynucleotides may have similar effects, when used as large aggregates. Poly(1‐vinylcytosine):polyinosinic acid, an inducer of interferon production in human cells, can cause at 40 μg/ml a 75‐fold enhancement of albumin uptake by sarcoma cells in culture. Most of this activity (85%) is related to the presence of aggregates retained by 0.65 μ millipore membranes. The prior finding that enhancers of albumin transport have increasing effects with increasing molecular sizes may thus extend to complexes of supramolecular sizes.
The interaction of chloride with the sulfate transport system of ehrlich ascites tumor cells
Authors: Levinson Charles
Published: February 2005

The effect of Cl− on SO4−2 efflux was studied in both Cl−‐containing and Cl−‐free ascites tumor cells loaded with 35SO4−2 to test the hypothesis that Cl−‐SO4−2 exchange is mediated by the same mechanism responsible for SO4−2‐self exchange.

The addition of Cl−‐free, 35SO4−2 loaded cells to a SO4−2‐free, Cl− medium results in: (1) SO4−2 efflux that is dependent on the extracellular Cl− concentration (Km = 4.85 mM; ke = 0.048 min−1 at 50 mM Cl−) and (2) net Cl−‐uptake that exceeds SO4−2 loss. Both SITS (4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulfonate) and ANS (1‐anilino‐8‐napthalene sulfonate) inhibit SO4−2 efflux but are without effect on Cl− uptake.

The addition of Cl−‐containing, 35SO4−2 loaded cells to a SO4−2‐free, C1− medium results in: (1) a slight gain in cellular Cl− and (2) k efor SO4−2 efflux identical to that for Cl−‐free cells.

The results are compatible with the suggestion that: (1) Cl− interacts with a membrane component responsible for transmembrane SO4−2 movement; (2) Cl− interaction stimulates the rate of unidirectional SO4−2 efflux from cells initially free of Cl− as well as the rate of SO4−2 turnover in cells maintained in the steady state with respect to Cl− and SO4−2; and (3) in the case of cells initially free of Cl−, the Cl−‐SO4−2 pathway represents only a small fraction of the total unidirectional Cl−‐influx the remainder being compatible with the electroneutral accumulation of NaCl and KCl.
Nucleotidase activity in regenerating liver and in clonal strains of hepatoma and pituitary cells in culture
Authors: Tjernshaugen Hans , Gautvik Kaare M.
Published: February 2005

A nucleotidase of the combined 3′‐ and 5′‐type (nucleotide phosphohydrolase, E.C.3.1.3.31) was present in the cytosol of regenerating rat liver cells, and of rat hepatoma and pituitary cells in culture. The enzyme activity per milligram of cell protein was very similar in regenerating liver and in three of the different cell types. The hepatoma cell strain which showed the slowest growth rate had a three‐fold higher basal enzyme activity. After the first days of regenerative growth in rat liver and during early plateau phase of cell growth, there was a 50–120% increase in specific enzyme activity. In the hepatoma cells, the enzyme activities were also compared to the cellular content and synthesis of RNA and DNA. The increase in enzyme activity occurred concomitantly with a reduced incorporation of 3H‐thymidine into DNA. The possible physiological role of this nucleotidase in nucleic acid and nucleotide metabolism is discussed.
Cell surface control in Blepharisma intermedium (Protozoa ciliata). Blocking of gamone II‐induced homotypic pairing by different membrane interacting ligands
Authors: Ricci Nicola , Esposito Fulvio , Nobili Renzo , Revoltella Roberto
Published: February 2005

PHA, Con‐A, or anti‐tubulin antibodies inhibit homotypic pair formation, in B. intermedium mating type‐I cells in the presence of suboptimal concentrations of gamone II. The inhibition is dependent on the dose of gamone added; the structural conformation and the relative concentration of the inhibitor; and the time of addition of the inhibitor. The block can be selectively prevented by competitive inhibitors of each ligand. The receptors for the inhibitors are distinctive and there is no cross‐reaction between the ligands. It is concluded that ligand binding and subsequent receptor‐ligand aggregation must induce a change within the cell‐surface membrane, which distorts the distribution and/or affects an optimal conformational aspect of a specific membrane‐receptor system for the gamone, a prerequisite for cell pair formation.
Selective isolation of reversible cold sensitive variants from chinese hamster ovary cell cultures
Authors: Ohlsson‐Wilhelm B. M. , Freed J. J. , Perry R. P.
Published: February 2005

Variants of the Chinese hamster ovary cell line CHO‐K1 (ATCC CCL 61) which grow almost normally at 38.5°C but very poorly or not at all at 30°C were obtained after treatment with mutagens and application of an indirect selection procedure. Two kinds of variants were recovered. In the first of these, the cold sensitive phenotype is expressed completely only at low cell densities. At higher cell density, growth continues at the nonpermissive temperature, but at a reduced rate. In the second class, the cold sensitive phenotype is independent of cell density. Two members of the latter class were studied in detail. In both lines, after shift to the nonpermissive temperature, the rate of 3H‐thymidine incorporation declines markedly; the rates of 3H‐uridine and 3H‐phenylalanine uptake are less drastically reduced. Autoradiographs indicate that the decline in thymidine uptake at the nonpermissive temperature is due to an elongation of part of the cell cycle, so that a smaller proportion of the cells lie in the synthetic (S) phase of the cell cycle with a consequent reduction in the fraction of labeled cells. The uridine labeling patterns of the mutants appear to rule out a ribosomal lesion. Low temperature growth inhibition of both cell strains was reversible. In one of the cell lines, an apparent stretching of the cells at the low temperature produces substantial alterations in cell shape.
The partial replacement of the serum growth factor requirement of SV3T3 cells by lactalbumin hydrolyzate
Authors: Young Delano V.
Published: February 2005

Lactalbumin hydrolyzate (LH), a commercially prepared, enzymatic digest of a milk protein fraction, can partially replace the serum requirement of SV3T3 cells. In a low, but obligatory, background of calf serum (0.15% v/v), LH causes a large increase in the final cell density (5–10 x over the control) while modestly stimulating the actual growth rate. Lactalbumin hydrolyzate does not contain survival activity for SV3T3 cells and does not affect the growth of 3T3 cells.

Since even prolonged exposure to pH 4 or 2 results in complete abolishment of the growth‐rate stimulatory capability without affecting the capacity to increase the final cell density, it is possible that the LH growth activity may consist of two dissociable components. All of LH growth activity is water soluble, autoclavable, and resists proteolytic treatment. On Sephadex G15 growth activity appears as a single peak at the void volume but on G25 it is retained beyond the void volume as a broad, skewed peak. The relevant molecular weight range lies between 1,500 and 4,000. The putative low pH resistant material is strongly adsorbed to Dowex 1 × 2 and can be displaced from the column by a reduction in the pH.

A comparison of the properties of the LH factors with those of known growth promoting agents isolated from serum and various enzymatic digests indicates that these new factors do not correspond to any of these agents.
Stimulation by N6,O2′‐dibutyryl adenosine 3′,5′‐cyclic monophosphate of RNA and DNA synthesis and of cell proliferation of rat hepatocytes in primary tissue culture
Authors: Armato Ubaldo , Draghi Enrica , Andreis Paola G. , Meneghelli Virgilio
Published: February 2005

The effects of DBcAMP in doses from 1.5 × 10−8 to 1.5 × 10−3 M on the compartmental apparent surface area (ASA) and (5‐3H)uridine radioactivity concentration (URC), (methyl‐3H)thymidine labelling index per 1 hour ([Me‐3H]Tdr LI/h) and per cent mitotic index (MI%) and colchicine metaphase index (CMI%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographic methods. In these cells DBcAMP was found to elicit: (1) progressive increments in the ASA of nucleoli, karyoplasm and cytoplasm; (2) peak increases in nucleolar URC at 1.5 × 10−8 and 10−5 M, but a slight decrease at 1.5 × 10−3 M; (3) significant increments in karyoplasmic and total nuclear URC at all doses, except at 1.5 × 10−6 and 10−4 M, when such parameters remained at control levels; (4) steady and progressive increases in cytoplasmic and total cell URC values; (5) marked increments in (Me‐3H)Tdr LI/h, MI% and CMI% up to the dose of 1.5 × 10−4 M, but at 1.5 × 10−3 M these parameters were found to be either much less enhanced or to approach closely to control values.

cAMP in doses from 1.5 × 10−8 to 10−4 M also markedly incremented the in vitro hepatocyte CMI%, while having a lesser stimulatory effect at 1.5 × 10−3 M.

Finally of the various possible metabolites of DBcAMP administered at 1.5 × 10−8 M to liver cultures, N6‐ and O2′‐MBcAMP and, again, cAMP significantly increased the CMI% of cultured hepatocytes, whereas 5′‐AMP, adenosine and allantoin had no significant effect and Na‐butyrate slightly decreased it.

The present observations strengthen the hypothesis that cAMP and its butyrated derivatives, by possibly amplifying the template activity of the liver chromatin, accelerate the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.
Variation in melanosome numbers in cultured B‐16 melanoma cells
Authors: Doezema Philip , Slesinski Ronald S.
Published: February 2005

Melanosomes from B‐16 mouse melanoma cells in culture were isolated by treatment of pigmented cells with 2% SDS, sonication, and heating at 100°C. The total number of melanosomes in cultures of B‐16 mouse melanoma cells increased exponentially during the rapid phase of sigmoid growth. The numbers of melanosomes per cell decreased during rapid phase of growth, and repigmentation was observed only when the cultures attained the stationary growth phase. BUdr at a minimum concentration of 0.5 μg/ml decreased both cell growth and numbers of melanosomes per cell, and completely inhibited repigmentation following a period of active growth. Cells cultured in 0.1 μg/ml BUdr grew at the same rate as untreated cells but contained fewer melanosomes/cell and lower total numbers of melanosomes during the late stages of the growth cycle.
Stimulation of WI‐38 cell cycle transit: Effect of serum concentration and cell density
Authors: Bartholomew James C. , Neff Nicola T. , Ross Priscilla A.
Published: February 2005

Flow microfluorometry has been used to characterize the effects of serum concentration and cell density on the initiation of cell cycle transit of stationary phase (G0) human diploid fibroblasts (strain WI‐38). The concentration of serum used to stimulate these cultures had no effect on the time cells began appearing in S (the DNA synthetic period), nor on the synchrony with which they moved around the cell cycle. However, as the serum concentration increased, the fraction of the stationary phase population released from G0 increased. Cell density modulated the ability of serum to stimulate cell cycle traverse. For example, at a cell density of 1.81 × 104 cells/cm2, 78% of the population was sensitive to serum stimulation; whereas, when the density was increased to 7.25 × 104 cells/cm2, only 27% of the population could be stimulated. This effect of cell density on the serum response is not simply the result of changing the ratio of serum concentration to cell density, but appears to reflect a true modulation of the population's sensitivity to serum stimulation. These results are consistent with the interpretation that the primary action of serum is to determine the transition of cells from a non‐cycling G0 state to a cycling state and that cell density determines the proportion of the population capable of undergoing this transition.
Stimulation of DNA synthesis, cell multiplication, and ornithine decarboxylase in 3T3 cells by multiplication stimulating activity (MSA)
Authors: Nissley S. Peter , Passamani John , Short Patricia
Published: February 2005

Multiplication stimulating activity (MSA) has been purified from the conditioned media of rat liver cells in culture by a modification of the procedure of Dulak and Temin. Purified MSA stimulates [3H] thymidine incorporation into DNA in subconfluent, serum starved 3T3 cells. Cell cycle analysis by the flow microfluorometer shows that the [3H] thymidine incorporation data reflects DNA synthesis. MSA also stimulates the multiplication of serum starved subconfluent 3T3 cells. MSA is approximately 10‐fold less active in 3T3 cells than in chick embryo fibroblasts in stimulating [3H] thymidine incorporation into DNA.

MSA causes a 2–10‐fold increase in ornithine decarboxylase (ODC) activity in 3T3 cells and the dose response curve parallels the dose response curve for [3H] thymidine incorporation into DNA. The Km of ODC for ornithine is 0.12 mM. There is a 30% decrease in the activity of ornithine transaminase (OTA) during the time period in which MSA causes an increase in ODC activity. Insulin also stimulates [3H] thymidine incorporation into DNA, cell multiplication and ODC activity over the same concentration range as shown for MSA, however, the extent of stimulation by insulin is less than that observed following MSA addition.
Clonal lines of teratocarcinoma cells in vitro: Differentiation and cytogenetic characteristics
Authors: McBurney Michael W.
Published: February 2005

Clonal lines of embryonal carcinoma cells have been established in culture from four independently‐derived transplantable teratocarcinomas of mice: three from strain C3H and one from strain 129/Sv. Cells from all lines retain the capacity to differentiate into a variety of tissue types both in tumors formed following the injection of cells into syngeneic animals and in vitro under appropriate culture conditions. Analysis of their G‐banded chromosomes indicated that the four lines have near‐diploid but not absolutely normal karyo‐types. The same chromosomal abnormalities were often present in more than one line.

Tetraploid embryonal carcinoma cells made by Colcemid or cytochalasin B treatment were also pluripotential in spite of chromosomal instability. Hybrid cells were readily obtained between diploid or tetraploid embryonal carcinoma cells and mouse 3T3 fibroblasts. Hybrid cells failed to differentiate and were contact inhibited like the 3T3 parent.
Effect of uncoupler on “Downhill” substrate efflux of Escherichia coli is dependent on (Mg2+,Ca2+)· adenosine triphosphatase
Authors: Rotman Boris
Published: February 2005

Previous studies have shown that mutations in the unc gene of Escherichia coli K12 cause defects in energy transduction as well as in a membrane‐bound (Mg2+,Ca2+)· adenosine triphosphatase.

We studied the effect of this mutation on the “downhill” efflux of methyl‐β‐D‐galactopyranoside, a substrate of the mgl transport system. While unc+ and unc− isogenic strains of E. coli K12 did not show significant differences in substrate influx or efflux, a differential effect of an uncoupler, 2,4‐dinitrophenol was demonstrated. In contrast to the unc+, dinitrophenol failed to inhibit significantly the rate coefficient of efflux in the unc− strain.

Analyses of spontaneous unc+ revertants of the unc− mutant provided additional evidence that a functional unc gene is necessary for dinitrophenol inhibition of efflux.

Other uncouplers tested in the unc+ strain showed different effects on efflux. While arsenate, azide and carbonyl cyanide p‐trifluoromethoxyphenylhydrazone caused little or no effect, 2,4‐dibromophenol and pentachlorophenol increased efflux by a considerable factor.
Transport of nutrients by a thermophilic bacterium—reconstitution of vesicles from crystalline ATPase or solubilized alanine carrier
Authors: Kagawa Yasuo
Published: February 2005

A strain of aerobic thermophilic bacteria was selected in order to purify highly stable membrane proteins and to reconstitute proteoliposomes capable of transporting nutrients from them. These proteins responsible for the transport could be divided into (1) proteins which supply energy to the transporting system, and (2) specific nutrient carriers driven by the energy. The former included a stable ATPase (TF1) and a lipoprotein (TF0) which rendered TF1 sensitive to energy transfer inhibitors. The complex of TF0 and TF1 could transport protons into reconstituted proteoliposomes upon hydrolysis of ATP. And one of the latter reported in this paper was alanine carrier protein which was driven by proton movement.

TF1 was the first crystallized ATPase in biomembranes, and was reconstituted from its five different polypeptides, two of which were necessary for ATPase activity and four of which, for proton translocation. Purification of alanine carrier and reconstitution of proteoliposomes capable of alanine accumulation were also demonstrated.
The application of rapid kinetic techniques to the transport of thymidine and 3‐O‐methylglucose into mammalian cells in suspension culture
Authors: Wohlhueter Robert M. , Marz Richard , Graff Jon C. , Plagemann Peter G. W.
Published: February 2005

Techniques are described by which the transport of nutrients into mammalian cells in suspension can be measured at intervals of 1.5 seconds. By application of these techniques, the existence of a saturable (Km = 85 μM), non‐concentrative, transport system for thymidine was demonstrated in Novikoff rat hepatoma cells depleted of ATP. At concentrations of thymidine less than the Km, this system operated at velocities sufficient to nearly completely equilibrate intra‐ and extra‐cellular thymidine pools within 8 seconds. In phosphorylating cells, the transport system operated with similar rapidity, so that intracellular phosphorylation was rate‐limiting for the incorporation of thymidine into nucleotides. Uptake of 3‐O‐methylglucose occurred at comparable velocities, attaining 90% of equilibrium between internal and external pools within 25 seconds. Uptake of cytosine by simple diffusion was 100 times slower.
Glucose transport and metabolism in cultured cells of nervous tissue
Authors: Passonneau Janet V.
Published: February 2005

The effect of the concentration of glucose in the medium on the intracellular concentrations of metabolites in C‐6 astrocytoma cells and C‐1300 neuroblastoma cells in culture has been investigated. The intracellular concentrations of glucose, glycogen, glucose 6‐P and UDP‐glucose were measured at intervals after feeding the cells. A rapid increase in glucose and glucose 6‐P levels occurred when fresh medium containing 5.5 mM glucose was applied to the cells, followed by slower increases in UDP‐glucose and glycogen. When the medium glucose was increased ten‐fold, the intracellular concentration of glucose was increased, but the levels of glucose 6‐P, UDP‐glucose and glycogen were not altered, nor were the rates of accumulation. The addition of insulin to the medium resulted in an increase of intracellular glucose, glucose 6‐P and glycogen. The transport of glucose into the cells is not the rate‐limiting step of the regulation of metabolite levels in the cells.
Transport changes associated with growth control and malignant transformation
Authors: Weber Michael J. , Hale Arthur H. , Yau Tom M. , Buckman Trent , Johnson Marcia , Brady Terrance M. , Larossa Denise D.
Published: February 2005

We can distinguish two classes of membrane transport changes in cultured cells: (a) growth‐rate contingent changes are those which occur in coordination with the onset of density‐dependent inhibition of growth; (b) transformation‐specific changes are those which occur when cells become transformed, and which can be detected even when normal and transformed cells are growing at the same rate. Growth‐rate contingent changes include the density‐dependent changes in phosphate, nucleoside, glucose, amino acid, and potassium transport. Only one transformation‐specific transport change has been found in Rous‐transformed chicken embryo fibroblasts: an increased rate of hexose transport. The variations in potassium transport are associated with variations in the number of ouabain binding sites in the membrane. The molecular basis for changes in the rate of hexose transport is unknown, although gross changes in membrane bilayer composition and “fluidity” seem not to be involved. In analyzing the regulation of hexose transport activity, we find that decreased cAMP may play a role in the transformation‐specific increase in hexose transport, but that fibrinolytic activity is not necessary.
Genetic alteration in the (Na+ + K+ ATPase transport system expressed in human lymphoblasts and their isolated plasma membranes
Authors: Lever Julia E.
Published: February 2005

A series of ouabain‐resistant human lymphoblastoid lines were shown to be genetically altered in the (Na+ + K+) ATPase transport system. The sensitivity to ouabain of Rb+ uptake in intact cells and (Na+ + K+)‐dependent ATP hydrolysis in purified plasma membrane vesicles was compared with measurement of ouabain binding to intact cells. Preliminary evidence for at least two categories of ouabain resistance was obtained.
The function and activity of certain membrane enzymes when localized on – and off – the membrane
Authors: Hochstadt Joy , Quinlan Dennis
Published: February 2005

A group of enzymes known to be involved in group translocation‐type transport mechanisms for the uptake of a variety of nucleotide precursors are enzymatically active both in their natural membrane milieu and in aqueous solution. The activity in aqueous solution markedly differ, however, from the enzymatic activity when the enzyme is membrane localized. The adenine phosphoribosyltransferase (PRT) of E. coli (Hochstadt‐Ozer and Stadtman, 1971 a) is capable of carrying out an exchange reaction between the base moieties of adenine and AMP without requiring P‐ribose‐PP as an intermediate; the enzyme in aqueous solution requires P‐ribose‐PP, indicating a different reaction mechanism in the two environments. Like the adenine PRT of E. coli, the hypo‐xanthine PRT of Salmonella typhimurium (Jackman and Hochstadt, 1976) also carried out an exchange reaction on the membrane only and also is more sensitive to a number of inhibitors in aqueous solution relative to the sensitivity when embedded in the membrane. In addition, however, the hypoxanthine PRT, while restricted to hypoxanthine as a substrate in the membrane, also accepts guanine as substrate in its soluble form. The membrane capacities reflect the in situ capacities of the enzyme and the gain of guanine specificity was determined in a guanine PRT deletion strain (Jackman and Hochstadt, 1976). Finally, in mammalian cell lines purine nucleoside phosphorylase, which translocates the ribose moiety of inosine across the plasma membrane of mouse fibroblasts undergoes a 30‐fold increase in substrate turnover number upon liberation from the membrane. These data raise two important caveats with respect to study of membrane enzymes and transport. Firstly, an enzyme once solubilized and found to differ kinetically from substrate transport in situ cannot be excluded from participating in translocations in the membrane on the basis of its activity in aqueous solution. Secondly, an enzyme which “appears” largely soluble upon cell rupture cannot be assumed to be a cycloplasmic enzyme because the majority of the solubilized activity may represent only a small fraction of the enzyme molecules highly activated concomitant to their solubilization. In this latter case the ability to activate enzyme still residing on the membrane (e.g., with detergents) would be necessary in order to estimate total membrane associated activity after cell rupture.
G2 + M arrest of cultured mammalian cells after incorporation of tritium‐labeled nucleosides
Authors: Marz R. , Zylka J. M. , Plagemann P. G. W. , Erbe J. , Howard R. , Sheppard J. R.
Published: February 2005

Novikoff rat hepatoma cells were propagated in suspension cultures containing 0.5 to 10 μC of 3H‐methyl‐thymidine, 3H‐5‐uridine, 3H‐G‐adenosine or 3H‐8‐adenine. The presence of the 3H‐labeled precursors caused an inhibition of cell replication which was due to a delay or arrest of the cells in G2 and M. The degree of inhibition was proportional to the amount of radioactivity incorporated into nucleic acids. Almost immediate and complete inhibition resulted from incubation with 10 μC 3H‐thymidine/ml. The presence of 0.5 μC 3H‐thymidine/ml caused a significant increase in the relative proportion of cells in G2 + M, even though the population doubling time of the culture appeared to be unaltered.
Phosphohydrolases on the cell surface of BHK cells: Loss during long term culture
Authors: Deppert W. , Walter G.
Published: February 2005

Low passage BHK 21/13 cells contain two cell surface enzymes, a nucleotide pyrophosphatase and a monophosphoester hydrolase, which together hydrolyze exogenous UDP‐galactose to free galactose. During serial passage, BHK cells successively lose both enzymes. Concomitant with the loss of these enzymatic activities, changes in cell morphology, as well as in the serum requirement for the initiation of DNA synthesis, were observed. Clonal sublines of BHK cells were isolated, which differed qualitatively in their ability to hydrolyze UDP‐galactose. Clonal BHK sublines, which exhibited both enzymatic activities on their cell surface, resembled low passage BHK cells in morphology and serum requirement for the initiation of DNA synthesis. Sublines not containing these enzymes resembled BHK cells of high passage cultures. The ability of intact BHK cells to hydrolyze exogenous nucleotide sugars may serve as an indicator for the progression of BHK cells from a normal to a more transformed state.
Chromosome replication in somatic hybrids of mouse and temperature sensitive Chinese hamster cells
Authors: Marin G. , Labella T.
Published: February 2005

Hybrid clones were obtained between a mouse cell line (3TP) and a temperature‐sensitive Chinese hamster cell line (K12) unable to grow at 40° because of a ts defect apparently located at the G1/S transition. The great majority of hybrid clones grew at 40°, showing the ts defect to be “recessive.” Chromosome DNA replication was analyzed in some detail in three hybrid clones with balanced complements. Although the S period of these hybrids was longer than that of K12, DNA replication in mouse and hamster chromosomes started and ended synchronously. Upon prolonged culture, mouse chromosomes were lost as they are in hybrids involving a non ts Chinese hamster partner, in which case asynchronous chromosome replication appears to be the rule. It seems therefore that asynchronous replication is not the determining factor in chromosome loss from cell hybrids.
Production of 5‐hydroxyindoleacetic acid from serotonin by cultured endothelial cells
Authors: Small Roy , Macarak Edward , Fisher Aron B.
Published: February 2005

Endothelial cells from bovine aorta and human umbilical vein and fibroblasts from human foreskin were cultured and subsequently evaluated for ability to metabolize serotonin (5‐HT) to 5‐hydroxyindoleacetic acid (5‐HIAA). Cells were incubated for three hours with 4 × 10−6 M [14C] 5‐HT creatinine sulfate. [14C] 5‐HIAA was separated from labeled 5‐HT by column chromatography and measured by scintillation counting. Production of 5‐HIAA by bovine aorta cells was 39.0 ± 7.5 (S.E.M., n = 6) nmoles per 109 cells per hour. Production of 5‐HIAA was markedly inhibited by the presence of 10−4 M iproniazid (an inhibitor of monoamine oxidase) or 10−4 M imipramine (an inhibitor of amine transport). 5‐HIAA was the only product of 5‐HT metabolism detected by thin layer chromatography. Production of 5‐HIAA by human umbilical vein endothelial cells was 5.4 ± 2.0 nmoles per 109 cells per hour (n = 5) and by human foreskin fibroblasts was 3.9 ± 1.4 nmoles per 109 cells per hour (n = 5). The results obtained during incubation in the presence and absence of inhibitors indicate that bovine aorta endothelial cells maintained in tissue culture are able to transport serotonin with subsequent production of 5‐HIAA. By contrast, human umbilical vein endothelial cells and fibroblasts exhibited relatively low rates of 5‐HT uptake and metabolism.
Development of spike potentials in skeletal muscle cells differentiated in vitro from chick embryo
Authors: Kano Masaakira , Yamamoto Mitsue
Published: February 2005

The development of spike potential mechanisms during cell differentiation was studied in chick myotubes formed in vitro from trypsin‐dissociated myoblasts. The spike potential and its rate of rise were measured in myotubes from 4–14 day old cultures. A depolarizing current pulse was delivered to evoke the spike potential after the steady membrane potential had been adjusted to a standard level of −80 mV in all cases. This gives the greatest maximum rate of rise of the spike potential and eliminates variation due to differences in the resting membrane potential of the myotubes. The size and maximum rate of rise of the spike potential increased significantly during the period examined. The spike potential was blocked by tetrodotoxin in almost all myotubes. These results suggest that during differentiation myotubes develop the ability to generate a spike potential due to an inward current carried by sodium ions.
Essential factors in the kinetic analysis of RNA synthesis in HeLa cells
Authors: Puckett Larry , Darnell James E.
Published: February 2005

Further evidence of mRNA in HeLa cells with a half‐life two hours or less is given. A kinetic model of RNA synthesis in HeLa cells is described in which equilibration of label occurs first into the acid soluble pool (evidence is given that this pool feeds RNA synthesis) and thence in nuclear and cytoplasmic molecules. The measured accumulation of label in nuclear and cytoplasmic poly(A) is examined with the model and parameters were found which are consistent with the quantitative transfer of nuclear poly(A) to the cytoplasm. The strengths and limitations of the model are discussed.
Cell cycle changes in transformed cells growing under serum‐free conditions
Authors: Bush H. , Shodell M.
Published: February 2005

Polyoma transformed hamster cells (PyBHK) and SV40 transformed mouse cells (SV3T3) were transferred in culture using crystalline trypsin followed by neutralisation with soybean trypsin inhibitor. Such cells were able to proliferate freely in defined medium without any serum supplement and without any intervening period of adaptation. However, growth rates were reduced under serum‐free conditions. Re‐establishment of rapid growth rates could be achieved by addition of serum, with the rate attained being proportional to the serum concentration. Irrespective of the prevailing rates of growth, percentages of cells synthesising DNA were the same. However, the rate at which DNA was being synthesised was found to change proportionately with the changes in overall growth rate.
Pentose utilizing variants of Novikoff hepatoma cells: Modification of growth and morphological properties
Authors: Hoffee Patricia , Jargiello Patricia , Zaner Linda , Martin Judith
Published: February 2005

A series of variant lines that utilize multiple pentoses for growth in place of glucose have been isolated from an 8‐azaguanine resistant line of Novikoff hepatoma cells (N1S167). These variants utilize for growth ribose, xylose, arabinose, and/or deoxyribose. The variants growing on pentose containing medium (a) exhibit a density dependent cessation of growth, (b) have a morphology change to a more flattened cell type, (c) become binucleated in the presence of cytochalasin B, and (d) show an altered sensitivity to trypsin treatment.
Expression of liver and placental alkaline phosphatases in Chang liver cells
Authors: LudueñA Marilyn A. , Iverson G. Michael , Sussman Howard H.
Published: February 2005

This paper presents evidence that a protein characteristic of differentiated liver cells, liver alkaline phosphatase, is synthesized by the Chang liver cell line. Liver alkaline phosphatase was demonstrated by immumochemical assay, 32P‐labeling and polyacrylamide gel electrophoresis, immunofluorescence microscopy, and the fluorescence‐activated cell sorter. The synthesis of the liver enzyme by the Chang liver cells is interpreted to indicate fidelity of the Chang cells to their origin from human liver tissue. Chang liver cells also synthesize a phosphatase which is similar if not indentical to the placental alkaline phosphatase. Since a placental‐type alkaline phosphatase has been observed in a number of non‐trophoblastic cell lines and also in some neoplasms, it does not seem reliable as an index of the origins of the cell line. Because of the claims that Chang liver cells are actually HeLa cells, HeLa cells were studied in tandem with the Chang cells. The results showed that the HeLa cells do not make the liver type phosphatase. The data are discussed in relation to the question of HeLa cell contamination of the Chang cell line and the validity of criteria normally used to identify cell lines.
Effects of hormones and serum on glycogen metabolism in adult rat liver parenchymal cell primary cultures
Authors: Walker P. Roy , Grindle Marie J.
Published: February 2005

Parenchymal cells from adult rat liver, isolated by a collagenase perfusion technique, have been maintained in primary culture and a detailed study on carbohydrate metabolism carried out over the initial 48‐hour culture period. The glucose concentration of the medium exerts a major influence on glycogen accumulation by the cells. Insulin, particularly at high glucose concentrations, stimulates glycogen biosynthesis, whereas glucagon prevents glycogen accumulation. Dexamethasone was without effect on glycogen metabolism. Glucose appears to stimulate glycogen accumulation by activation of glycogen synthetase enzyme. However, there is a gradual loss of synthetase activity throughout the culture period. Similar decreases in activity were noted for pyruvate kinase, aldolase and hexokinase. Glucose, insulin and dexamethasone were unable to prevent these decreases in enzyme activity. Foetal bovine serum contains fructose and this hexose appears to be the factor in serum which is responsible for the activation of glycogen accumulation in the presence of physiological glucose concentrations. The lactic acid content of the serum may also stimulate glycogen accumulation. In general, there is a gradual loss of the pattern of carbohydrate metabolism typical of differentiated hepatocytes during the culture period.