bluuebunny/crossref_metadata_2025 huggingface dataset:
Immature megakaryocytes in the mouse: In vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes
Authors: Long Michael W. , Williams Neil , McDonald Ted P.
Published: February 2005

An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5–7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell‐conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell‐conditioned medium, lung‐conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5–6 mm hr‐1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony‐stimulating factor devoid of detectable potentiator activity present in WEHl‐3‐conditioned medium. In contrast, serum‐free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony‐stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony‐stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase‐containing cells can undergo cellular division.
Regulation of sugar transport in cultured diploid human skin fibroblasts
Authors: Germinario Ralph J. , Rockman Howard , Oliveira Maureen , Manuel Susannia , Taylor Melarie
Published: February 2005

The regulation of hexose transport under glucose‐starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2‐DG and 3‐O‐methyl‐D‐glucose (3‐OMG) but not of L‐glucose. Glucose‐starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation‐induced change in 2‐DG transport was due to an increase in the Vmax of both the high and low affinity transport sites (2.8‐ and 2.4‐fold, respectively) with no effect on their Kms. The presence of 5.55 mM galactose, fructose, or L‐glucose in the medium resulted in transport increases similar to those seen in glucose‐starved cells, while the presence of 5.55 mM glucose, mannose, or 3‐OMG repressed 2‐DG transport. Glucose‐starvation enhancement of 2‐DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α‐amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose‐starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3‐OMG to glucose‐starved cells had little effect on reversing the transport increases, glucose plus 3‐OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose‐fed or glucose‐starved cells exhibited rapid decreases in 2‐DG transport when exposed to glucose‐containing serum‐free medium. These decreases were prevented by employing glucose‐free, serum‐free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.
Regulation of hexose transport in BALB/c 3T3 preadipose cells: Effects of glucose concentration and 12‐O‐tetradeconoylphorbol‐13‐acetate
Authors: O'Brien Thomas G. , Saladik Douglas
Published: February 2005

Like many cell types in culture, both undifferentiated and differentiated BALB/c 3T3 preadipose cells respond to glucose deprivation with an increased uptake of 2‐deoxy‐D‐glucose (deoxyglucose) and 3‐O‐methyl‐D‐glucose (methylglucose). Glucose readdition to glucose‐deprived cultures resulted in a prompt fall in uptake activity; in undifferentiated cells, a half‐maximally effective concentration of glucose was approximately 0.5 mM, while 0.1 mM was ineffective. Several hexoses differed in their efficacy of “deactivating” methylglucose transport in glucose‐deprived cells; it appeared that a particular hexose must be metabolized beyond the 6‐phosphate from to deactivate the transport system. Previous studies have shown that the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) stimulates hexose transport in undifferentiated and differentiated BALB/c 3T3 cells. In this study, it was found that TPA (and insulin in differentiated cells) prevented the glucose‐induced deactivation of transport activity. Glucose‐induced deactivation of transport activity was also prevented by cycloheximide or actinomycin D addition concomitantly with glucose. In glucose‐starved cells, agents such as TPA and insulin appear to override a cellular control mechanism sensitive to the external concentration of glucose, so that elevated levels of transport activity are maintained under environmental conditions (i.e., a return to physiological glucose concentrations) that normally induce a fall in transport activity.
Regulation of Na+, K+ pump activity by nerve growth factor in chick embryo dorsal root ganglion cells
Authors: Boonstra Johannes , Skaper Stephen D. , Varon Silvio
Published: February 2005

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+, K+‐pump‐mediated) K+ influx over a period of several hours. Addition of NGF to NGF‐deprived cells caused (1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by (2) a very rapid, but transient overstimulation of the Na+, K+‐pump‐mediated K+ influx. Restoration of normal Na+, K+‐pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1–100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF‐deprived cells and could not be elicited in NGF‐supported steady‐state cells or in cells having already received delayed NGF once. This transient Na+, K+‐pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+, K+‐pump was specific for the NGF molecule and could not be mimicked by other proteins.
Temperature‐dependent alteration of cellular morphology by cholera toxin in rat liver epithelial cells which are TS for maintenance of transformed properties
Authors: Niles Richard , Loewy Barbara , Krah David
Published: February 2005

Cholera toxin via its ability to increase intracellular cyclic AMP levels can induce drastic changes in cell morphology. This report describes a temperature sensitive mutant of chemically transformed rat liver epithelial cells which only display cell shape alterations in response to cholera toxin at the permissive temperature. Shift up‐shift down experiments indicate that the change in the response occurs fairly rapidly, i.e., within 2 hours at the new temperature. The behavior of the temperature sensitive cells at the nonpermissive temperature mimics that of the untransformed rat liver epithelial cells (i.e., no morphological change in response to cholera toxin) while at the permissive temperature the positive cell shape change is identical to that exhibited by chemically transformed rat liver epithelial cells. The temperature sensitive response to cholera toxin is not a function of cyclic AMP production, since the amount of cyclic AMP found as a function of either time or concentration of cholera toxin is quite similar in cells treated at either temperature.
Induced release and metabolism of arachidonic acid from myeloid cells by purified colony‐stimulating factor
Authors: Ziboh Vincent A. , Miller Alan M. , Wu Ming‐Chi , Yunis Adel A. , Jimenez Joaquin , Wong Glen
Published: February 2005

The in vitro incubation of cells from turpentine‐induced rat myeloid hyperplastic marrow and peritoneal monocyte/macrophage with 14C‐arachidonic acid resulted in the incorporation of the radiolabel into the particulate phospholipids. Challenge of the radiolabeled cells with a highly purified type I CSF (CSF I) from human pancreatic carcinoma cells in continuous culture resulted in the hydrolysis and release of the 14C‐arachidonic acid from the cellular phospholipids. The simultaneous challenge of the prelabeled cells with CSF‐I and its specific antibody (anti‐CSF‐I antibody) inhibited the CSF‐I induced hydrolysis of 14C‐arachidonic acid from the cells. These results confer a specificity on the CSF‐I induced release of arachidonic acid from the cellular phospholipids. Our data also demonstrated that the 14C‐arachidonic acid released from the cellular phospholipids was further transformed into products of the cyclooxygenation and lipoxygenation pathways by cellular enzyme systems in both populations of cells. Interestingly, our data also indicate that the challenge of the granulocytic hyperplastic marrow cells and the monocyte/macrophage cells with purified CSF‐I resulted in a higher generation of lipoxygenase products in the predominantly granulocytic cell population than in the population rich in monocyte/macrophage cells. The biological significance of this observation remains to be further explored. Thus, the CSF‐I‐induced release of cellular arachidonic acid explains, at least in part, the presence of prostaglandins and other metabolites of arachidonic acid that are found in the media of hemopoietic cells incubated with a variety of CSF preparations.
Nuclear ligation of RNA polynucleotide kinase products
Authors: Winicov Ilga
Published: February 2005

RNA polynucleotide kinase has been shown to transfer [γ32P] from ATP to 5‐OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′‐OH kinase products are polyadenylated. A sizeable fraction of the [γ32P] label from ATP is also found in internal phosphodiester bonds after 30‐minute nuclear incubation in vitro. The possibility of substantial [32P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α32P] UTP, (3) different nearest‐neighbor distribution for RNA molecules > 12S and 12‐3S, (4) relative insensitivity of the [γ32P] incorporation to α‐amanitin as compared with total RNA synthesis, (5) internal [32P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [32P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ32P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′‐OH RNA kinase in a ligation reaction.
The prolongation of mitotic stages in SV40‐transformed vs nontransformed human fibroblast cells
Authors: Sisken Jesse E. , Bonner Susan V. , Grasch Sally D.
Published: February 2005

Early studies on the duration of mitotic stages and on metaphase‐to‐prophase ratios in a number of normal and neoplastic cells indicated that the process of mitosis becomes altered during the course of oncogenesis. However, the nature of these changes and their effects on each of the mitotic stages are still unclear. With the use of time lapse cinemicrography, we have compared the durations of mitotic stages of SV40/Wl‐38 and SV40/Wl‐26 cells to those of their normal counterparts and to other nontransformed human fibroblasts. We also examined the relative frequencies of the individual mitotic stages in fixed preparations of Wl‐38 and SV40/Wl‐38 cells. The data show that metaphase durations are increased in the transformed cells and as much as 3‐4.7‐fold in SV40/Wl‐38 cells compared to Wl‐38 and other nontransformed cells. Other stages are also prolonged though to lesser degrees. These findings suggest that increased metaphase/prophase ratios observed in many tumors are due to increases in duration of metaphase rather than to shorter prophases, and that increased mitotic indices commonly observed in malignant tumors and sometimes used as an index of growth rate are at least in part due to the prolongation of mitotic stages.
Glucose metabolism, insulin effects, and developmental age of cultured neonatal rat heart cells
Authors: Schroedl Nancy A. , Hartzell Charles R. , Ross Philip D. , McCarl Richard L.
Published: February 2005

Cultured heart cells from 2–3 day old and 5–6 day old neonatal rats have been used as a model system for the characterization of carbohydrate metabolism in developing cardiac tissue. The rate of depletion of glucose from the growth medium was dependent on (1) the age of the animals from which the cultured cells were obtained, and (2) the presence and absence of serum and/or insulin in the growth medium. The glucose depletion rate in insulin and serumcontaining medium was 9.63 ± 0.96 nmol/min/mg protein for heart cell cultures from, 2 day old rats and 3.51 ± 0.68 nmol/min/mg protein in heart cell cultures from 5 day old rats. Appearance of lactate in the medium during these experiments occurred at the rates of 18.6 ± 7.9 nmol/min/mg and 6.4 ± 1.2 nmol/min/mg, respectively. In the absence of serum and insulin, the medium glucose depletion rates were 5.7 ± 1.6 and 2.2 ± 0.5 nmol/min/mg for cells derived from 2‐day‐old and 5‐day‐old rats, respectively. It is apparent from these data that immature cardiac cells depend upon glucose as a primary source of energy for muscle contraction and cellular growth, and that less‐efficient energy‐yielding metabolic pathways are used to obtain ATP.
The competitive ability of stem cells from mice with Hertwig's anemia
Authors: Barker Jane E. , McFarland Eleanor C. , Bernstein Seldon E.
Published: February 2005

Mice homozygous for the gene, an, have a macrocytic, normochromic anemia. In this report, attempts have been made to cure Hertwig's anemia (an/an) by injecting genetically normal (+/+) stem cells. The anemia of unirradiated an/an mice was alleviated but not completely cured by injection of as many as 3 × 107 +/+ bone marrow cells. Lethal irradiation of the an/an recipients was necessary before injections of 107 +/+ marrow cells were effective in normalizing the blood parameters. The inability to achieve normal blood values without first destroying the host's own stem cells suggested that the indigenous an/an cells compete effectively with injected +/+ cells. This hypothesis was tested by injecting varying numbers of stem cells from C57BL/6J‐ +/+ mice, together with stem cells from either WBB6F1‐an/an or, as controls, from their WBB6F1‐ +/+ littermates, into lethally irradiated hosts. The C57BL/6J and WBB6F1mice have electrophoretically distinguishable hemoglobins. The an/an cells are able to compete in the repopulation of the host hematopoietic tissue as shown by the presence of WBB6F1 hemoglobin in the recipients. The cells from mice with Hertwig's anemia, however, do not compete as effectively as do the same number of cells from the +/+ littermates. These results indicate that the pluripotent hematopoietic stem cells of an/an mice are reduced in number, seeding capacity, or proliferative potential.
Low‐frequency voltage noise in a mammalian bone cell clone
Authors: Ferrier Jack , Dixon Jeff , Illeman Alan , Dillon Eva , Smith Inka
Published: February 2005

Measurements were made of plasma membrane voltage noise in cells of a bone cell clone. The measurements were made under conditions intended to approximate in vivo conditions more closely than in previous electrical measurements on small mammalian cells. Mononuclucell of normal size, imbedded in a collagen matrix, were used. The electrical state of the cell membrane under normal conditions was characterized by low‐frequency random fluctuations (noise) of high magnitude. Hyperpolarizing spikes were observed in some cells. Power spectrum analysis revealed that the random fluctuations were actually a sum of incoherent spike patterns, with spikes of the same time width as those seen in the clearly spiking patterns. This analysis, combined with similar measurements in a high [K+], low [Na+] medium, showed that the fluctuation/spiking phenomenon resulted from modulation of K+ and Na+ transport by a control process at a level higher that that of the individual channels. This process persisted when the membrane potential was depolarized. These results indicate that the membrane potential is not part of the feedback loop producing the fluctuation/spiking phenomenon.
Lens epithelial cell adhesion to lens capsule: A model system for cell‐basement membrane interaction
Authors: Cammarata Patrick R. , Spiro Robert G.
Published: February 2005

Adhesion of calf lens epithelial cells to lens capsule, their natural basement membrane was found to be considerably more rapid than either to plastic or to type I or type IV collagen coated surfaces. No polarity of the basement membrane was observed as the cells were able to attach to either side of the anterior or posterior lens capsule; a prerequisite for adhesion to the lenticular side of the anterior capsule was the prior removal of its epithelial cell layer. The attachment was energy‐dependent and required calcium and magnesium ions, but was not enhanced by the presence of serum. Neither exogenous fibronectin nor laminin was able to stimulate attachment or spreading of lens cells to the capsule even when the cells had been treated with cycloheximide. Since rapid adhesion and spreading takes place in this lens cell‐lens capsule system without requirement of exogenous macromolecules, it provides a favorable model for investigating the determinants in epithelial cell‐basement membrane interactions.
Selective utilization of ω 6 and ω 3 polyunsaturated fatty acids by human skin fibroblasts
Authors: Rosenthal Miriam D. , Whitehurst M. Candace
Published: February 2005

Incorporation of exogenous [14C] arachidonate by human skin fibroblasts was found to be significantly greater than that of either [14C] linoleate or α‐[14C] linolenate. Arachidonate was preferentially esterified in the PI + PS and PE classes of phospholipids. Over 40% of the incorporated [14C] arachidonate was chain elongated in 24 hours. Cells were also grown in lipid‐free medium to enhance PUFA desaturation and elongation and the utilization of various ω 6 and ω 3 metabolites examined. Whereas [14C] linoleate partitioned approximately 50:50 between PL and TAG, eicosatrienoate (20:3 ω 6) was selectively sequestered in TAG. Arachidonate and docosatetraenoate (22:4 ω 6) were preferentially incorporated into phospholipids; the PI + PS fraction was most highly enriched with arachidonate. Modification of α‐[14C] linolenate was more extensive than that of [14C] linoleate. Docosapentaenoate (22:5 ω 3) was the major ω 3 [14C] PUFA of PI + PS and PE. Eicosapentaeonate was not selectively incorporated into phospholipids; within phospholipids the 20:5 ω 3 was primarily in PC. These results indicate that human skin fibroblasts exhibit acyl specificity in the esterification of polyunsaturated fatty acids, including preferential utilization of arachidonate rather than other prostaglandin precursors in the PI + PS fraction.
Antigen‐specific stimulation of amino acid transport in bovine lymphocytes
Authors: Tate Emily H.
Published: February 2005

Treatment of bovine lymphocytes isolated from animals which were either infected with Mycobacterium bovis or sensitized to a purified protein derivative (PPD‐B) from this organism induced an increase in the transport of α‐aminoisobutyric acid (AIB) and α‐methylaminoisobutyric acid (MeAIB). PPD‐B did not stimulate these transport activities in lymphocytes from nonsensitized animals. The transport stimulation was first measurable after about 7 hours of treatment, reached about a two‐fold enhancement after 20 hours, and continued to increase to 30‐ to 40‐fold after 6 days. The stimulation of AIB transport was inhibited by both ouabain and cycloheximide. Experiments to determine transport system specificities in nonstimulated lymphocytes showed that MeAIB transport was primarily by the Na+‐dependent, A‐system, and leucine transport was mostly by Na+‐independent system(s). In contrast, AIB transport was about 25% by the A‐system, 25% by at least one Na+‐dependent, non‐A‐system, and 50% by one or more Na+‐independent system(s). Analysis of the three components of AIB transport after treatment with PPD‐B showed that: (1) transport by both the A‐system and the Na+‐independent system(s) was stimulated; (2) A‐system transport was stimulated to a larger extent than Na+‐independent transport; and (3) Na+‐dependent, non‐A‐system transport was not stimulated significantly.
Enhancement of altered‐cell foci in baby mouse skin cultures by antitubulin treatment: Nuclear mechanisms
Authors: Cooper Peter D. , Marshall Susan A. , Masinello Gillian R.
Published: February 2005

When primary baby mouse skin (BMS) cultures were subcultured for 48 hours into media containing 10−6 to 10−7 M colchicine or demecolcine, the number of altered cell foci appearing after 3–4 weeks' maintenance at 36°C was substantially enhanced over drug‐free controls. This applied whether or not the primary cultures had been irradiated with white fluorescent light. The additional presence of cytochalasin D and 12‐O‐tetradecanoyl‐phorbol‐l3‐acetate (TPA) sometimes improved and sometimes partly suppressed the enhancing effect of the antitubulin drugs, and these drugs were omitted for reproducible focus enhancement. The enhancement depended on passage through DNA synthesis in presence of colchicine, which did not prevent concurrent or subsequent DNA synthesis but induced a substantial proportion (>33%) to replicate in the tetraploid (4n to 8n) chromosome configuration. Another effect was to induce widespread asymmetric nuclear division, allowing the potential for chromosome loss. All these effects occurred within the first one or few cell cycles after removal of the antitubulin drugs. The results suggest that the generation of tetraploidy perhaps followed by chromosome loss may be an important factor in the rapid induction of altered cell foci. Pre‐existing DNA damage is another important factor.
Mg2+‐sensitive alterations in Ca2+ regulation associated with cell transformation
Authors: Vidair Charles , Rubin Harry
Published: February 2005

The intracellular Ca2+ content of nontransformed Balb/c3T3 cells is two to three times higher than that of a spontaneously transformed derivative. Depriving either cell type of extracellular Mg2+ causes a 2‐ to 3‐fold increase in their Ca2+ content over a 24‐hr period. Restoring Mg2+ to the medium decreases the Ca2+ content of the cells to their original values in about the same time. The increase in Ca2+ content is not blocked by cycloheximide suggesting that normal rates of protein synthesis are not required to produce this effect. Mg2+ deprivation also decreases the initial rate of Ca2+ efflux from the transformed cells and increases the size of the slowly exchanging fraction of Ca2+ to the levels found in the nontransformed cells. Since Mg2+ deprivation normalizes the appearance and growth behavior of the transformed cells, the possible intermediary role of Ca2+ in this normalization was studied. Large changes in extracellular Ca2+ produced large changes in the Ca2+ content of the transformed cells with little change in appearance or thymidine incorporation rate. Ca2+ deprivation did inhibit thymidine incorporation in early passage nontransformed cells; however with repeated passage, this effect decreased, as did the Ca2+ content of these cells. The possible role of Mg2+ in regulating cellular Ca2+ content and distribution is discussed, as is the relation of Ca2+ content and distribution to the development of the transformed state.
Membrane potential and sodium flux in neuroblastoma X glioma hybrid cells: Effects of amiloride and serum
Authors: O'Donnell Martha E. , Villereal Mitchel L.
Published: February 2005

The Na+ uptake into neuroblastoma x glioma hybrid cells was measured in Hepes‐buffered EMEM containing 10% calf serum and 5 mM ouabain in the presence and absence of amiloride (1.0 mM). Amiloride was found to markedly inhibit net Na+ influx (by approximately 50%). Examination of the effect of amiloride on net Na+ influx in the absence of calf serum revealed that a significant amiloride‐sensitive Na+ influx remains even under serum‐deprived conditions, although the degree of amiloride inhibition (35%) is substantially lower than that found in the presence of serum. The amiloride‐insensitive portion of Na+ influx was found to be independent of serum effects. Estimation of resting membrane potential was made by measurement of the steady state distribution of the lipophilic cation, TPP+, in the presence and absence of amiloride. A large, immediate increase in TPP+ uptake, indicative of a membrane hyperpolarization, was seen upon addition of amiloride. Determination of the effect of amiloride on resting membrane potential of serum‐deprived cells showed that cells are hyperpolarized to a greater extent in the presence than in the absense of amiloride, and that serum exerts a depolarizing effect on the cells. Thus, serum‐stimulation of Na+ influx results in a depolarization of resting membrane potential, while amiloride inhibition of Na+ influx causes a hyperpolarization. These data strongly suggest that NG108‐15 cells possess an electrogenic Na+ influx pathway that is sensitive to amiloride inhibition and enhanced by serum.
Hereditary orotic aciduria, Lesch‐Nyhan syndrome, and xeroderma pigmentosum probed by herpes simplex virus: 125I‐Iododeoxycytidine incorporation as an assay for viral growth
Authors: Campisi Judith , Hafner Julianne , Boorstein Robert , Pardee Arthur B.
Published: February 2005

125I‐Iododeoxycytidine (125IdC) incorporation into acid‐insoluble material was a sensitive, rapid, and quantitative assay for the growth of herpes simplex virus type 1 (HSV‐1) in human fibroblasts. Cellular utilization of the isotope was 10 to 25% of the incorporation by infected cells and could be 80% inhibited by tetrahydrouridine (THU). Viral utilization was inhibited by acycloguanosine, thioguanine (TG), and cytosine arabinoside. Isotope was incorporated equally well by growing or quiescent infected cells. HSV‐1 was used to probe the metabolic capabilities of three mutant human fibroblast strains. 125IdC incorporation quantitatively measured the ability of the virus to grow in these cells. Viral 125IdC incorporation was sensitive to TG in normal fibroblasts but showed a 8‐ to 10‐fold greater resistance to TG in fibroblasts derived from patients with Lesch‐Nyhan syndrome (LN). Similarly, the growth of ultraviolet irradiated HSV‐1 in normal fibroblasts, as judged by 125IdC incorporation, was 5‐fold greater than in fibroblasts derived from patients with xeroderma pigmentosum. In fibroblasts derived from patients with hereditary orotic aciduria, viral 125IdC incorporation was sensitive to adenosine (AD) at concentrations which were slightly stimulatory in normal fibroblasts. This was a 2‐fold difference in AD sensitivity, which the radioassay reliably and quantitatively documented. Previous attempts to quantify and characterize this difference by immunofluorescence were unsuccessful. HSV‐1 infected cells could be individually identified by their incorporated 125IdC; such cells had blackened nuclei in autoradiograms prepared 12 hr after infection. Normal cells infected in the presence of TG had many fewer labeled nuclei than LN cells similarly infected in the presence of the drug. Thus, this viral assay can be a rapid and accurate probe of cellular function and has potential for the identification of mutant cells in amniocentesis samples or tumor speciments. Towards this end, we determined that the AD and TC dose responses of infected amniotic fluid cells closely paralleled that of the normal human fibroblasts.
DNA synthesis and cell division related to adipose differentiation of 3T3 cells
Authors: Kuri‐Harcuch Walid , Marsch‐Moreno Meytha
Published: February 2005

Adipose differentiation of 3T3‐F442A cells in surface cultures depends on adipogenic factor present in the culture medium. We found that after stimulation with adipogenic serum, 3T3T442A cell underwent a burst of DNA synthesis before adipose conversion was manifested by an augmented li‐pogenic enzyme activity. In differentiating cells, DNA synthesis, judged by a 100‐fold higher rate of [3H]thymidine incorporation into TCA‐insoluble material, was followed by a 100‐fold increase in the activity of glycero‐phosphate acyltransferase. Cytosine arabinoside, added to the cultures at a concentration of 3 μg/ml, exerted 95% inhibition of [3H]thymidine incorporation and also inhibited adipose conversion. The burst of DNA synthesis correlated with a 2.5‐fold increase in the amount of DNA and in the number of cells in the culture. The DNA content was the same in differentiated and nondifferentiated cells. We conclude that after the interaction with the adipogenic factor, the cells go through DNA synthesis and cell division essential for adipose conversion.
Tunicamycin inhibits the expression of surface Na+ channels in cultured muscle cells
Authors: Bar‐Sagi Dafna , Prives Joav
Published: February 2005

We have investigated the effect of tunicamycin (TM), an inhibitor of protein glycosylation, on surface Na+ channels in cultured chick skeletal muscle cells. The expression of Na+ channels, estimated by the measurement of batrachotoxin (BTX)‐activated 22Na+ uptake, was found to be significantly diminished after exposure of muscle cells to TM. This effect is partially reversed by the protease inhibitor leupeptin and is associated with a markedly enhanced rate of disappearance of Na+ channels from the surface of TM‐treated cells. Our findings suggest that protein glycosylation contributes to the metabolic stability of voltage‐sensitive Na+ channels.
Analysis of myogenesis by somatic cell hybridization. II. Retention of myogenic competence and suppression of transformed properties in hybrids between differentiation competent and incompetent rat L6 myoblasts
Authors: Lawrence Jeanne Bentley , Coleman John R.
Published: February 2005

This study describes the characteristics of hybrids between two closely related rat myoblast lines, which differ both in the ability to express their program of differentiation and in the expression of neoplastic properties. Myogenic, nonneoplastic L6J1‐S cells were hybridized with nonmyogenic, neoplastic L6J1‐N1 cells. Six hybrid clones were isolated and expanded for analysis of myogenic competence, and four of these clones were also evaluated for parameters of transformation, including tumorigenicity, ability to clone in agar, and surface fibronectin. In addition to our analysis of isolated clones, we also assessed myogenic differentiation in colonies representing 226 early hybrid clones. Results of all these analyses demonstrate that the myogenic phenotype is retained and that the tumorigenic/transformed phenotype is suppressed in the hybrids. Furthermore, our results indicate that when the programs for myogenesis and neoplastic transformation are confronted within a single cell, they are expressed as mutually exclusive alternatives. In contrast to these results on myogenic × nonmyogenic L6 hybrids, it has been reported that isolated clones of A9 × L6 exhibited extinction of myogenic competence and retention of transformed properties. We have evaluated myotube formation in over 300 early hybrid clones between A9 and either diploid or subtetraploid L8 rat myoblasts. Our results demonstrate that all of these hybrid clones exhibit extinction regardless of the ploidy of the myoblast parent, and they further indicate that extinction is not a consequence of chromosome loss. These results support the conclusion that in A9 × L6 hybrids, the nonmyogenic, transformed phenotype is dominant.
RNA turnover in cultured hamster embryo cells: Identification of modified nucleoside end products
Authors: Lothrop Clinton D. , Uziel Mayo
Published: February 2005

Fourteen methylated nucleosides (N‐2‐dimethylGuo, N‐2‐methylGuo, N‐1‐methylGuo, N‐5‐methylUrd, N‐3‐methylUrd, N‐1‐methylAdo, N‐3‐methylCyd, N‐5‐methylCyd, N‐1‐methyllno, 2′‐0methyl‐Cyd, 2′‐0‐methylUrd, 2′‐0‐methylGuo, 2′‐0‐methyllno, and thymidine) and one methylated base (m7Gua) have been identified as normal excretion products of cultured hamster embryo cells. The methylated nucleosides are excreted in the culture media subsequent to RNA turnover. The excretion pattern of the base‐methylated nucleosides was determined by continuous labeling of serum‐stimulated quiescent hamster embryo cells with [3H‐CH3]methionine and measurement of radioactivity in the excreted nucleosides between 23 and 811/2 hours after the label was added. These nucleosides accumulate exponentially until a maximum level is reached after 60 hours. These maximum levels were maintained for at least an additional 20 hours.
Cultured endothelial cells increase their capacity to synthesize prostacyclin following the formation of a contact inhibited cell monolayer
Authors: Eldor Amiram , Vlodavsky Israel , Hy‐Am Esther , Atzmon Ruth , Weksler Babette B. , Raz Amiram , Fuks Zvi
Published: February 2005

The synthesis of the prostaglandins (PG), prostacyclin (PGI2), PGE2, and thromboxane A2 (TXA2), has been investigated in actively growing and contact‐inhibited bovine aortic endothelial cell cultures. Cells were stimulated to synthesize prostaglandins by exposure to exogenous arachidonic acid or to the endoperoxide PGH2 and by the liberation of endogenous arachidonic acid from cellular lipids with melittin or ionophore A23187. Increased capacity of the cells to synthesize PGI2 and PGE2 was observed as a function of time in culture, regardless of the type of stimulation. TXA2 production increased with time only upon stimulation of the cells with ionophore A23187. This increased PG synthetic capacity was independent of cell density since it was mainly observed in confluent, nondividing endothelial cell cultures. The fact that increased PGI2 production in confluent cells was also observed with PGH2, a direct stimulator of PGI2 synthetase, implies that this process is independent of the arachidonate concentration within the cells or in the culture medium. This increased capacity is likely to reflect an increased activity of the PG synthetase system associated with the formation of a contact inhibited endothelial cell monolayer. A similar time‐dependent increase in the PGI2 production capacity was also observed during growth of cultured bovine corneal endothelial cells.
Treatment of human tumor cells with ADP or ATP yields arrest of growth in the S phase of the cell cycle
Authors: Rapaport Eliezer
Published: February 2005

Treatment of a variety of cultured human tumor cells with low levels (40–80 μM) of adenosine 5′‐dephosphate (ADP) or adenosine 5′ ‐triphosphate (ATP) has produced arrest of these cells in the S phase of their cycle followed by cellular death. ADP and ATP are demonstrated to be incorporated into the cellular acid‐soluble nucleotide pools, presumably by permeation through the plasma membrane of these cells. Since AMP, adenosine, 3′,5′ cycle AMP, or a variety of diadenosine polyhosphates do not produce similar cytostatic effects or similar alterations of the cellular acid‐soluble nucleotide pools, we suggest that the effects of ADP and ATP are not due to their prior breakdown or modification. Cultured animal cells have been widely acknowledged not to incroporte acid‐soluble adenine nucleotides although incorporation of exogenous intact nucleoside monophosphate into cellular pools has been demonstrated in a few cases. A 48‐hour treatment of logarithmically growing human tumor cells with 40mUM of ADP or ATP produced substantial arrest of cell populations in the S phase of their cycle and a dramatic reduction in total cellular uridine 5′ ‐triphosphate (UTP) pools. AMP, adenosine, 3′,5′ cyclic AMP, Ap3A, Ap4A or Ap5A produced small and dissimilar effects. The experiments reported here suggest that the plasma membranes of several lines of human tumor cells are premeable to low levels of intact ADP and ATP. The suggestion of en bloc incorporation of low ADP and ATP levels into human tumor cells is supported by the almost identical effects of ADP and ATP on total cellular acid‐soluble nucleotide pools and cellular growth. The rapid conversion of intracellular ADP pools into ATP in tumor cells has been reported.
Analysis of angiotensin‐stimulated sodium transport in cultured smooth muscle cells from rat aorta
Authors: Smith Jeffrey Bingham , Brock Tommy A.
Published: February 2005

Angiotensin peptides (AI, AII, AIII) increased the rate of Na+ accumulation by smooth muscle cells (SMC) cultured from rat aorta. The stimulatory effect of All on Na+ uptake was observed which Na+ exodus via the Na+/K+ pump was blocked either by ouabain or by the removal of extracellular K+. All was at least ten times more potent than AIII and about 100 times more potent than AI in stimulating Na+ uptake. Saralasin had little effect on Na+ uptake by itself but almost completely blocked the increase caused by All. The stimulation of net Na+ entry by AI, but not AII, was prevented by protease inhibitors. The stimulation of Na+ uptake was almost completely blocked by amiloride. Tetrodotoxin, which prevented veratridine from increasing Na+ uptake, had no effect on the response to AII. Angiotensin increased the rate of ouabain‐sensitive 86Rb+ uptake (Na+/K+ pump activity) but had no effect on ouabain‐sensitive ATPase activity in frozen‐thawed SMC or in microsomal membranes isolated from cultured SMC. The stimulation of ouabain‐sensitive 86Rb+ uptake by All was blocked by saralasin. Omitting Na+ from the external medium prevented All from increasing 86Rb+ uptake. All had no effect on cell volume or cyclic AMP levels in the cultured SMC. These results suggest that angiotensin peptides activate an amiloride‐sensitive Na+ transporter which supplies the Na+ /K+ pump with more Na+, its rate‐limiting substrate.
Differential calcium dependence of contractile responses and 86Rb efflux from the rabbit aorta induced by vasoactive stimuli
Authors: Martin W. , Gordon J. L.
Published: February 2005

86Rb was used to monitor potassium movements in strips of rabbit aorta simultaneously with measurements of tension. Histamine, noradrenaline, the prostaglandin endoperoxide analogue U46619, angiotensin II, and 144 mM K+ each induced an increase in 86Rb efflux concomitantly with contraction. For the first four agonists there was a rank‐order correlation between the contractile response and 86Rb efflux, but 144 mM K+ induced a massive increase in 86Rb efflux although it was the weakest contractile stimulus. Contraction and increase in 86Rb efflux‐induced K+ were both reduced by verapamil, which blocks voltage‐sensitive calcium channels, implying that both effects of K+ were mediated mainly by a depolarisation‐induced influx of calcium. Noradrenaline increased both tension and 86Rb efflux through an action on alpha‐adrenoceptors, but its effect on efflux, unlike its effect on tension, was apparently totally dependent on the presence of extracellular calcium. Experiments performed in the presence of lanthanum, which blocks calcium influx, showed that the intracellular store of calcium released by noradrenaline apparently played no role in inducing 86Rb efflux, although it could trigger contraction. Lanthanum also blocked contraction induced by K+ but less effect on the increase in 86Rb efflux induced by K+. Thus, agonist‐induced vascular contraction and 86Rb efflux can be dissociated, but under normal conditions all the contractile stimuli tested induced 86Rb efflux.
Variables controlling somatomedin production by cultured human fibroblasts
Authors: Clemmons David R. , Shaw Debra S.
Published: February 2005

Somatomedin is secreted by multiple types of cultured cells including human fibroblasts. Since the control of somatomedin (Sm) production by fibroblasts may be an important regulatory step in cell division, we undertook studies to define the variables in tissue culture experimental design that may have significant effects on the secretion of Sm. Cell density was an important parameter in determining basal Sm production rates. Cultures plated at 2.5 × 104 cells/well produced 0.38 ± 0.06 U/ml/105 cells whereas an increase in culture density to 6.2 × 104 cells/well was associated with a decrease in Sm production per cell to 0.23 ± 0.04 U/ml/105 cells (P < .01). Cultures stimulated by platelet‐derived growth factor (PDGF) showed a similar reduction in Sm production with increasing cell density. Epidermal growth factor was stimulatory (5.4‐fold increase) in low‐density cultures but had no effect when high‐density cultures were used. If the experiments were initiated between 2 and 4 days after the last media change there were no significant differences in basal or PDGF‐stimulated Sm concentrations. Between days 5 and 9 however, there was a progressive increase in the basal Sm production rate. The duration of incubation was an important variable since Sm production increased during the first 12 h in noncycling cells and showed an accelerated increase between 4 and 8 h in cycling cells. Cells between the eighth and 12th passage had similar basal Sm production (0.22 ± 0.04 U/ml/105 cells) rates; cells between the 19th and 20th passages had significantly higher basal Sm production rates (0.41 ± 0.05 U/ml/105 cells) (P < .01). These results suggest that several variables, particularly cell density and passage number, are critical variables when quantitating the effect of hormones and growth factors on Sm production by cultured fibroblasts.
Calcium effects on epidermal growth factor receptor‐mediated endocytosis in normal and SV40‐transformed human fibroblasts
Authors: Tupper Joseph T. , Bodine Peter V.
Published: February 2005

Lowering of extracellular Ca2+ levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus40(SV40)‐transformed WI38 cells do not exhibit this Ca2+‐dependent arrest. One possibility for this difference in Ca2+ requirement is that extracellular or surface membrane‐bound Ca2+ may be required for growth factor receptor‐mediated endocytosis and this Ca2+ requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca2+ in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV40 WI38 cell. The binding of [125I]EGF to the cell surface is not significantly altered by lowering of Ca2+ to 10−5‐M levels in either the normal or transformed cell. At this Ca2+ level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca2+ deprivation. Degradation of the EGF‐receptor complex is also sensitive to Ca2+. A twofold reduction in the rate of release of acid‐soluble 125I occurs in the normal but not the transformed cell under conditions of lowered medium Ca2+. In contrast, 2‐chloro‐10‐3‐aminopropyl phenothiazine (CP), an inhibitor of the Ca2+‐dependent regulator protein calmodulin, causes an inhibition of [125I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [125I]EGF binding to the cell surface receptor of the transformed cell but not the normal cell.
Analysis of morphology and receptor metabolism in clonal variant A431 cells with differing growth responses to epidermal growth factor
Authors: Lifshitz Aliza , Lazar Cheri S. , Buss Janice E. , Gill Gordon N.
Published: February 2005

Human epidermoid carcinoma A431 cell clones have been obtained whose growth is inhibited, stimulated, or unaffected by epidermal growth factor (EGF). In clones exhibiting each type of growth response, EGF induced similar morphologic changes consisting of aggregation of cells into dense clusters with baring of large areas of the culture dish. The similarity of the clones' morphologic responses, despite their differing growth responses, indicates that the effects of EGF on morphology are distinct from effects on growth. Cells whose growth was inhibited by EGF contained high numbers of EGF receptors, whereas the concentration of EGF receptors was reduced in cells whose growth was stimulated or unaffected by EGF. There were, however, no consistent differences in EGF receptor concentrations between stimulated or null clones. Cells that exhibited each type of growth response displayed similar rates of EGF binding to receptors, rates of internalization of EGF, and rates and extent of EGF‐induced receptor down‐regulation. Changes in EGF‐stimulated tyrosine‐specific protein kinase activity paralleled changes in EGF receptors, both between clones and upon down‐regulation. These studies indicate that a reduction in the concentration of EGF receptors in A431 cells allows escape from the growth inhibitory effects of EGF, but suggest that the pattern of growth response depends on biochemical events subsequent to EGF‐receptor metabolism and activation of tyrosine‐specific protein kinase.
Inhibition of chemotactic factor‐induced neutrophil responsiveness by arachidonic acid
Authors: Naccache P. H. , Molski T. F. P. , Volpi M. , Mackin W. M. , Becker E. L. , Sha'Afi R. I.
Published: February 2005

Arachidonic acid when added simultaneously with the chemotactic peptide formyl‐methionyl‐leucyl‐phenylalanine (f‐Met‐Leu‐Phe) inhibits the ability of the latter to initiate several but not all of its effects on rabbit peritoneal neutrophils. Stimulated neutrophil aggregation, calcium uptake, and increases in the steady state level of exchangeable calcium are all inhibited by 1–10 μM arachidonic acid. The binding of f‐Met‐Leu‐Phe and the parameters of intracellular calcium redistribution (calcium efflux and changes in the steady state level of exchangeable calcium in the absence of extracellular calcium) and of stimulated sodium uptake are, on the other hand, unaffected by the same concentrations of arachidonic acid. Arachidic acid, the saturated analog of arachidonic acid, was found not to inhibit f‐Met‐Leu‐Phe‐stimulated aggregation and calcium uptake. Arachidonic acid, therefore, in addition to its well‐described agonist properties, also possesses antagonist activities toward rabbit neutrophils. These results add a new level of complexity to the study of the role of arachidonic acid in cell activation.
A voltage‐clamp study of the effects of colchicine on the squid giant axon
Authors: Chang D. C.
Published: February 2005

The effects of colchicine applied inside a squid giant axon were studied using voltage‐clamp and internal perfusion techniques. It was found that colchicine selectively and reversibly suppresses the sodium conductance during excitation. The possible involvement of the microtubular structure in the functioning of the excitable channel is discussed.