bluuebunny/crossref_metadata_2025 huggingface dataset:
Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides
Authors: Hightower Lawrence E.
Published: February 2005

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P110, P88, P71,72, and P23 had unique peptide maps. Evidence of a 27,000 dalton precursor to P23 was obtained. The analogue‐stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose‐regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment.

Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue‐stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine‐mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS‐polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P88 and P71,72 increased from basal to new plateau levels in about 1.5 hours, while P23 required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the metabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue‐mediated response fulfilled several of the criteria for inducible eucaryotic gene expression.

The amino acid analogue p‐fluorophenylalanine and the chain‐terminating analogue of amino acyl‐tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.
The stimulation of the initiation of DNA synthesis by fibroblast growth factor in swiss 3T3 cells: Interactions with hormones during the pre‐replicative phase
Authors: Richmond K. M. Veronica , Kubler Anne‐Marie , Martin Francisco , De Asua Luis Jimenez
Published: February 2005

Fibroblast Growth Factor (FGF) stimulates quiescent Swiss 3T3 cells to initiate DNA synthesis and divide. Cells begin to enter the S‐phase after a lag of 13–15 hr, and the rate of initiation of DNA synthesis in the population can be quantified by a first order rate constant, k. A subsaturating concentration of FGF may establish the lag phase, while the value of k is dependent on the FGF concentration present during the second half of the lag phase. Insulin and hydrocortisone enhance the effect of FGF by increasing k without changing the lag phase, and they can act when added at any time after FGF. Prostaglandin E1 (PGE1) causes a decrease in k and a lengthening of the lag phase, and acts only when added during the first 8 hr. None of these agents stimulate DNA synthesis in the absence of FGF.

These results show that the stimulation of growth by FGF follows the same basic pattern as was previously shown with Prostaglandin F2α (PGF2α). However, since hydrocortisone inhibits stimulation by PGF2α when added during the first 4 hr of the lag phase, there are clearly differences in some events stimulated by the two growth factors.
Changes in RNA polymerase II in a cell cycle‐specific temperature‐sensitive mutant of hamster cells
Authors: Rossini Mara , Baserga Susan , Huang C. H. , Ingles C. James , Baserga Renato
Published: February 2005

tsAF8 cells are a temperature‐sensitive mutant of BHK cells that arrest at the nonpermissive temperature in the G1 phase of the cell cycle. The activity of solubilized RNA polymerase II and its ability to bind [3H]‐γ‐amanitin decrease in tsAF8 cells at 40.6°, with a half‐life of ∼ 10 hr. No appreciable changes occur in these two parameters in tsAF8 cells at 34° or in BHK cells at either 34° or 40.6°. Protein synthesis is not appreciably affected for at least 24 hr after tsAF8 cells are shifted to 40.6°. These results indicate that in tsAF8 cells at the nonpermissive temperature, there is a defect in either the synthesis, the assembly, or the stability of RNA polymerase II, and that the loss of RNA polymerase II molecules is not due to widespread cellular damage.
Relationships between polyamine metabolism and RNA synthesis in post‐ischemic liver cell repair
Authors: Ferioli Maria E. , Schiaffonati Luisa , Scalabrino Giuseppe , Cairo Gaetano , Bernelli‐Zazzera Aldo
Published: February 2005

In liver cells recovering from reversible ischemia the increase in RNA synthesis by isolated nuclei is preceded by activation of ornithine decarboxylase, leading in turn to an increase in putrescine concentration. Treatment of the animals with 1,3‐diaminopropane and putrescine prevents ornithine decarboxylase activation but does not hinder the enhancement of RNA synthesis in post‐ischemic liver nuclei; therefore, ornithine decarboxylase activation does not seem to be a necessary prerequisite for the increase in RNA synthesis. Hypophysectomy does not prevent the post‐ischemic increases of ornithine decarboxylase and RNA synthesis; but pre‐treatment of the animals with cycloheximide—which has a dual effect on the activity of ornithine decarboxylase—abolishes the post‐ischemic enhancement of RNA synthesis. In contrast with regenerating liver, changes in ornithine decarboxylase activity and putrescine concentrations in reversible ischemia are not associated to changes in S‐adenosylmethionine decarboxylase activity and in spermine and spermidine concentrations that seem to be characteristic of tissues where increases in RNA synthesis are followed by DNA synthesis and cell multiplication.
The stimulation by epidermal growth factor (Urogastrone) of the growth of neonatal rat hepatocytes in primary tissue culture and its modulation by serum and associated pancreatic hormones
Authors: Draghi Enrica , Armato Ubaldo , Andreis Paola G. , Mengato Loreta
Published: February 2005

Epidermal growth factor (EGF) added in a single dose (between 10–16 and 1.7 ± 10–9M) to neonatal rat hepatocytes in primary culture with subsequent incubation for 12 and 24 hours in Eagle's MEM fortified with 10% (v/v) FBS stimulated their entry into S and M phases, as shown by (3H)thymidine labeling and autoradiography and by a 1‐hour exposure to colchicine (0.1 mM). Growth stimulation by EGF was detectable after 4 hours, peaking between 12 and 16 hours, and thereafter declining in intensity. Rat hepatocytes exposed for 72 hours (between the fourth and the seventh day in vitro) to no serum or to 10% fresh FBS possessed similar growth rates and absolute numbers in the cultures. A 24‐hour exposure to 20 to 50% FBS stimulated hepatocytic DNA synthesis and mitotic activity and resulted (except for the 50% FBS treatment) in increased hepatocytes' numbers, which were relatively greater than the concurrent increases in connective tissue cell numbers. In serum‐devoid medium EGF (10–11M) enhanced hepatocytic mitotic, but not DNA‐synthetic activity. To be fully effective EGF required a 10% FBS addition to the medium, then eliciting within 24 hours a marked increase in hepatocytes' number with respect to cultures incubated with 10% serum only. When associated with 20 to 30% FBS, EGF stimulated parenchymal cell growth at rates slightly higher, but not significantly different, than those elicited by the same serum concentrations alone. However, when used in conjunction with 10 to 30% FBS, EGF preferentially increased the number of hepatocytes rather than that of non‐parenchymal cells. Moreover, comparative proliferation kinetic studies showed that in the presence of 10% FBS, an equimolar (10–14M) mixture of EGF, insulin, and glucagon promoted an early and marked increase in the DNA‐synthetic and mitotic activities of hepatocytes, which peaked after 8 hours. Within a 24‐hour time lag this growth stimulation was as effective in increasing the final hepatocytes' number as was a 1000‐fold higher EGF concentration, and was twice as active as either an equimolar (10–14M) mixture of the two pancreatic hormones or EGF by itself at 10–14M.

These results show that the growth‐promoting effect of EGF on primary neonatal rat hepatocytes is modulated by serum factor(s) and can be additively amplified by the simultaneous administration of subphysiological doses of glucagon and insulin.
Maintenance of glucokinase activity in primary hepatocyte cultures
Authors: Spence Joseph T. , Pitot Henry C.
Published: February 2005

When primary cultures of hepatocytes are maintained for 2 weeks from the time of perfusion, the activity of the enzyme glucokinase decreases rapidly, so that the activity can no longer be detected after the fourth day in culture. Concomitantly, there occurs an increase in the activity of hexokinases, the low‐KM isozymes, which predominate in fetal liver. We have made several modifications of the culture medium in an attempt to prevent the decrease in glucokinase activity. When the medium was supplemented with a mixture of insulin, thyroxine, glucagon, dexamethasone, testosterone, and estradiol, the activity of the enzyme in the hepatocytes was present at approximately 15% of in vivo levels after 2 weeks in culture. When this hormone mixture was present during the first 4 hrs of culture and when the hepatocytes were allowed to attach to the collagen support and were maintained thereafter in medium supplemented with fetal bovine serum, insulin, and dexamethasone, the activity of glucokinase increased after an initial decrease for 3 days and was maintained thereafter at levels comparable to those observed in vivo. This effect of the hormone mixture was found to be the result of the presence of glucagon in the mixture, since the presence of glucagon with no other hormones added, except insulin, during the attachment period produced the same pattern of increased glucokinase activity. Immunoprecipitation of glucokinase from the hepatocytes, using monospecific antibody, indicated that the increase in enzyme activity was the result of increased glucokinase enzyme protein and not an increased synthesis of the other hexokinase isozymes. These studies demonstrate the specific hormonal requirements for the maintenance of glucokinase levels in primary hepatocyte culture at those seen in vivo and lends support to the hypothesis that fetal gene expression in primary hepatocyte cultures is selectively regulated rather than being a general effect with a common regulatory mechanism.
Serum stimulation of potassium fluxes, ouabain binding, and sodium fluxes in quiescent chicken embryo fibroblasts
Authors: Johnson Marcia A. , Weber Michael J.
Published: February 2005

Growth‐contingent alterations in potassium and sodium fluxes, ouabain binding, and potassium ion content were examined following serum stimulation of quiescent, density‐inhibited chicken embryo fibroblasts. Serum stimulation resulted in very rapid 1.5‐ to 1.8‐fold increases in ouabain‐sensitive potassium influx and lesser 1.4‐ to 1.5‐fold increases in potassium efflux and sodium influx. Potassium influx stimulation was maximal after addition of 5–20% calf serum and was unaffected by cycloheximide inhibition of protein synthesis. Reflecting the slightly greater stimulation of potassium influx versus potassium efflux, potassium ion levels were 10–15% higher in serum‐stimulated compared to unstimulated cells. Specific ouabain binding levels in stimulated and unstimulated control cells were initially similar, however, by four hours after stimulation a 40–50% increase in specific ouabain binding was observed. Incubation with ouabain was found also to inhibit later serum‐stimulated hexose uptake and thymidine incorporation; this blockage may be a consequence of subnormal potassium levels rather than ouabain inhibition of the serum‐stimulated potassium influx.
Differences between rat liver epithelial and fibroblast cells in metabolism of purines
Authors: Berman Jules J. , Tong Charles , Williams Gary M.
Published: February 2005

Epithelial and fibroblast cells from adult rat liver were found to differ markedly in their metabolism of the purine hypoxanthine. Both cell types took up hypoxanthine and possessed hypoxanthine‐guanine phosphoribosyl transferase for phosphoribosylating the purine. However, in the transferase assay, lysates from epithelial cells converted hypoxanthine predominantly to inosine monophosphate, with small amounts of the nucleoside inosine as product, whereas fibroblast cell lysates converted hypoxanthine predominantly to inosine. The inosine appeared not to be produced by direct ribosylation of the base, since fibroblast cell lysates had less purine nucleoside phosphorylase activity than epithelial cell lysates. Rather, the inosine produced by fibroblast lysates appeared to be derived from inosine monophosphate through catabolism of the mononucleotide by 5′ nucleotidase. An inhibitor of 5′ nucleotidase, thymidine triphosphate, reduced the amount of inosine formed.
Stimulation of macrophage plasminogen activator activity by colony‐stimulating factors
Authors: Hamilton J. A. , Stanley E. R. , Burgess A. W. , Shadduck R. K.
Published: February 2005

Colony‐stimulating factors (CSFs) stimulate granulocyte‐macrophage production from single hemopoietic progenitor cells. Various preparations of purified CSFs of two different subclasses have been shown here to stimulate a plasminogen‐dependent fibrinolytic (plasminogen activator) activity from resident and starch‐induced mouse peritoneal macrophages. Lymphocyte supernatants also stimulate macrophage plasminogen activator (PA) activitty. Since they contain colony stimulating activity, it is possible that one or more sublcasses of CSF in these supernatants is responsible for this effect. Since both colony‐stimulating and macrophage growth activities have been detected at inflammatory sites, these findings could reflect a role for CSF in inflammatory processes.
On the effect of inhibitors of mitochondrial macromolecular‐synthesizing systems and respiration on the growth of cultured chick embryo cells
Authors: Morais Réjean
Published: February 2005

We have found that chick embryo fibroblasts (DEF) cultivated in the presence of tryptose phosphate broth (TPB) are inherently resistant to the growth inhibitory effect of ethidium bromide (EB). As demonstrated by cytochrome oxidase activity and oxygen consumption measurements, analyses of reduced‐minus‐oxidized cytochrome spectra and electron microscopic observations, TPB did not seem to prevent the inhibitory effect of EB on mitochondrial DNA transcription. EB‐treated chick cell populations cultivated in the presence of TPB behave essentially the same as populations treated with chloramphenicol (CAM) and grow with mitochondria devoid of a functional respiratory chain. In contrast to CAM‐trreated CEF populations, however, the respiratory activity of EB‐treated cell populations did not reappear when the cells were shifted back to EB‐free medium. Attempts to demonstrate that TPB confers resistance to the growth inhibitory effect of carbomycin and mikamycin, inhibitors of the mitochondrial protein‐synthesizing system, have failed, the drugs being cytotoxic at doses where protein synthesis on mitoribosomes is not suppressed. On the other hand, the present results demonstrated that chick cell populations proliferate in the presence of the respiratory inhibitors rotenone, antimycin A, amytal and oligomycin whether or not TPB is present in the growth medium.
Identification of a distinct phase during melanogenesis that is sensitive to extracellular pH and ionic strength
Authors: Laskin Jeffrey D. , Mufson R. Allan , Weinstein I. Bernard , Engelhardt Dean L.
Published: February 2005

The cell line B16/C3 will undergo melanogenesis at a specific time after plating. We have found that this time can be modulated by varying the pH of the culture medium. At high pH levels (8.2–8.6) the onset of melanogenesis occurs in 3 or 4 days, while at lower pH (6.7–7.2) it occurs in 7 or 8 days. Furthermore, the time of onset is also sensitive to the extracellular ionic strength. The addition of sodium lactate, sodium chloride, or any other salt tested delays or blocks completely the onset of melanogenesis. These effects are not simply consequence of growth inhibition, nor can they be correlated with patterns of lactate accumulation.

These cells are sensitive to pH or ionic strength after entering the stationary phase just prior to the time of onset of melanogenesis. The existence of a specific pH‐and ionic‐strength‐sensitive phase may provide an important clue to the events responsbile for differentiation in this system.
Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: Possible role of uncharged tRNA in regulating metabolism
Authors: Martin Thomas F. J.
Published: February 2005

Previous studies (J. Biol. Chem, 253: 99–105, 1978) showed that thyrotropin‐releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH4C1) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid‐depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time‐course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid‐depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino‐acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximde can be attributed solely to inhibition of the synthesis of proteins.
Nerve growth factor may stimulate either division or differentiation of cloned C1300 neuroblastoma cells in serum‐free cultures
Authors: Revoltella Roberto P. , Butler Richard H.
Published: February 2005

Two clone of mouse C1300 neuroblastoma cells (clones NB1R and NB6R) bind mouse 2.5S Nerve Growth Factor (NGF) in vitro. The ligand is then capped and internalized by the cells. This step requires active cell processing. In serum‐free or low serum conditions, clear effects of NGF are seen with both clones. Cultured NB1R cells are stimulated, after a lag interval of a few hours, to synthesize DNA and to proliferate, whereas NB6R cells are stimulated to cell differentiation and maintain viability under these conditions much longer than similar cultures in the absence of NGF. Stimulation of clone NB1R occurs within an optimal dose concentration of the same order as that used in the Levi‐Montalcini bioassay with 8‐day‐old chick embryo‐sensitive ganglia; the effects of clone NB6R, however, need higher NGF concentrations. Both effects are protein‐specific since they are inhibited in the presence of added anti‐NGF antibodies. This system could provide a convenient means to study the control of cell division in susceptible malignant neuroblastoma clones and to correlate NGF binding to receptors and biological activity.
Two candidate G1 polypeptides in chick embryo fibroblasts
Authors: Rudert William A. , Graves Mark W. , Chadwick David E. , Lieberman Irving
Published: February 2005

Resting cultures of primary chick embryo cells have been labeled with 3H‐leucine for the first 2 or 3 hours after feeding basal medium or basal medium supplemented with 10% dialyzed serum. The labeling patterns of the 3H‐polypeptides of the soluble cell fraction have been compared by fluorography of two‐dimensional gels. Large and consistent differences are seen in only three of the more than 900 spots that can visualized. This report concerns two of the three spots. The 3H contents of the two polypeptides (41,000 daltons, pI 7.1, designated 41–7.1, and 34,000 daltons, pI 6.2, designated 34–6.2) are increased by serum by about ten‐fold. The highly selective effect of serum on the labeling of the two spots does not appear to be an artifact related to the extractability, solubility, or state of aggregation of the polypeptides. The radio‐intensities of both polypeptides decrease markedly when the cells are labeled later than 3 hours after “shift‐up”.

Drugs that inhibit RNA synthesis and are known to stop the progression of the chick cells through the G1 period, camptothecin, cordycepin and 5,6‐dichloro‐β‐D‐ribofuranosylbenzimidazole, depress, with great specificity, the enhanced labeling of polypeptide 41–7.1 in the stimulated cells, and all but camptothecin have a similar action on polypeptide 34–6.2. A high level of actinomycin D (10 μg/ml), but neither a low level of the drug (0.02 μg/ml) nor 5‐fluorouridine prevents the increased labeling of the two polypeptides in serum‐fed cells. That 5‐fluorouridine enters the chick cells and is converted to its active form is shown by the inhibition of the processing of pre‐ribosomal RNA.

The observations with the RNA inhibitors are at least consistent with the conclusions that the enhanced labeling of the two sports results from increased rates of synthesis of the polypeptïdes that depend upon mRNA production but not on the formation of ribosomal RNAs, and that the polypeptides play a role in the regulation of DNA replication in the chick cells.
Related effects of calcium and serum on the G1 phase of the human WI38 fibroblast
Authors: Tupper Joseph T. , Kaufman Lydia , Bodine Peter V.
Published: February 2005

Deprivation of extracellular Ca or serum inhibits the proliferation of WI38 human diploid fibroblasts. Under these conditions, the cells become quiescent at a point in the cell cycle typical of early G1 or Go phase‐arrested cells. Exit of the cells from this point in the cycle appears to require both the presence of serum and Ca simulataneously. If quiescent cells are serum‐stimulated in low Ca medium (0.01 mM), they do not progress through G1 to the S phase, which normally requires 14–18 hr. However, they remain competent to do so. Addition of Ca for up to 48 hr after serum stimulation results in an equal fraction of the cells progressing G1 phase as compared to the presence of Ca at the time of serum addition. In contrast, if quiescent cells are serum‐stimulated in the presence of Ca, which is then removed, the cells can remain competent to enter S phase for only 10–12 hr. Re‐addition of Ca beyond this time does not allow G1 progression on a normal schedule.

These data suggest that Ca and serum are both required to trigger, in whole or in part, the pleiotypic response. Ca appears also to render the cells competent to enter S phase, but this competence is labile; an observation consistent with the PDGF‐induced competence observed previously in the 3T3 cell. These observations are in contrast to previous data from other cell types which suggest that Ca is required only in late G1 phase for successful entrance to S phase.
Intracellular concentration of potassium and other elements in vaginal epithelial cells stimulated by estradiol administration
Authors: Cameron Ivan L. , Pool Thomas B. , Smith Nancy K. R.
Published: February 2005

Electron probe x‐ray microanalysis was used to measure cytoplasmic elemental content (in mmoles/kg dry weight) of the basal layer of cells of the vaginal epithelium of ovariectomized rats. Measurements were made both before estradiol injection and at 2 hr, 17 hr, and 24 hr after estradiol administration. Mitotic figures first appeared in the basal cell layer at 24 hr. During the course of the study significant time‐dependent differences were seen in the content of all elements measured. A pattern of change in cytoplasmic content was seen for Na, P, S, and Cl; all of which decrease significantly by 17 hr and then return to approximately the nonstimulated concentration by 24 hr. On the other hand K, and to a lesser extent Mg, show an early and continued increase in cytoplasmic content after estradiol injection. Thus, the marked increase in the intracytoplasmic content of K in the estradiol treated cells suggests that K, or the ratio of Na to K, may be directly or indirectly involved in growth stimulation.
Synthesis and degradation of tyrosinase in cultured melanoma cells
Authors: Saeki Hisaaki , Oikawa Atsushi
Published: February 2005

The tyrosinase (EC 1.14.18.1) activity of cultured B‐16 mouse melanoma cells (C2M) in the stationary phase depends greatly on whether the culture medium contains glucose or galactose. The activity in medium cotaining galactose was about ten times that in medium containing glucose at pH 7.2. This difference in tyrosinase activity was concluded to be due to a shift of balance between synthesis and degradation of the enzyme. Experiments were conducted with stationary phase cultures in the presence and absence of cycloheximide.

The melanoma cells did not synthesize tyrosinase in medium containing glucose in the stationary phase. But when they were cultured under identical conditions, except that glucose was replaced by galactose, they continued to synthesize tyrosinase.

The rate of synthesis in medium containing galactose at pH 6.3 was one third of that in the same medium at about pH 7, in which the increase in specific activity of tyrosinase per day was about 30 nmoles/mg cell protein per hr. The rate of degradation of the enzyme was practically the same in medium containing glucose as in medium containing galactose, and largely dependent on the pH of the culture medium. At pH 6.3 the half‐life was about one third of that at pH 7.2, where it was about 1.8 days.

The degradation at acidic pH values was much reduced by ammonium salt and was strongly inhibited by the protease inhibitor, leupeptin.
Regulation of metastatic ovarian cell in serum‐free medium by epidermal growth factor and transferrin
Authors: Turner John T. , Wyche James H.
Published: February 2005

A rat ovarian tumorous cell line (OV1N) has been isolated and established in tissue culture whose growth can be controlled by the addition of epidermal growth factor (EGF) and transferrin to serum‐free medium. EGF at 1 ng/ml (1.5 × 10−10M) stimulates growth about 300% in a six‐day incubation assay. A combination of 1 ng/ml EGF plus 1 μg/ml (1.25 × 10−8M) transferrin stimulates growth 1700% above controls in six days. EGF and transferrin at these concentrations completely replaced fetal calf serum (FCS), which maximally stimulates the cells at 2%. At maximal stimulation (either by FCS or EGF and transferrin) a doubling time of from 16–20 hours is obtained. The growth of OV1N cells was also studied in vivo and compared to another rat ovarian cell line, 31A‐F2. Cells from both cell lines were injected intrasplenically into normal, ovariectomized and hypophysectomized female Fischer 344 rats. Animal data have indicated that 31A‐F2 cells behave more like “normal” ovarian cells in that they grew only in ovariectomized hosts. OV1N cells, on the other hand, grew metastatically in all hosts and killed 36% of those individuals.

The binding of 125I‐EGF was also studied in OV1N and 31A‐F2 cells. OV1N cells bound about eightfold more 125I‐EGF than did 31A‐F2 cells at 37°C. The dose of 125I‐EGF required to specifically saturate 31A‐F2 sites was 10 ng/ml (1.5 × 10−9M) or half‐maximally at 4 ng/ml (6 × 10−10M). Scatchard plots indicate that 31A‐F2 cells contain roughly fourfold less EGF‐specific sites than OV1N cells. Incubation of either OV1N or 31A‐F2 cells with a 100‐fold excess addition of various proteins plus a maximal or a half‐maximal addition of 125I‐EGF demonstrated that unlabeled EGF was most effective in reducing 125I‐EGF binding to both OV1N and 31A‐F2 cell sites. Insulin, ovarian growth factor, transferrin, or their combinations, had minimal, if any effect on 125I‐EGF binding.
Biosynthesis of erythrocyte membrane protein band 3 in DMSO‐induced friend erythroleukemia cells
Authors: Sabban Esther L. , Sabatini David D. , Marchesi Vincent T. , Adesnik Milton
Published: February 2005

The major integral membrane protein of red blood cells, the mouse equivalent of human band 3, was purified and used to raise a specific antiserum. The murine protein resembles its human counterpart in several of its properties, including susceptibility to digestion by chymotrypsin added to intact cells and an ability to bind to concanavalin A. The synthesis of 35S‐labeled band 3 was detected in Friend erythroleukemia cells treated with DMSO by immuneprecipitation followed by SDS gel electrophoresis and fluorography. Induction with DMSO led to a greater than tenfold increase in the synthesis of band 3 and maximal synthesis was reached 3 to 4 days after the beginning of induction.
Purification of human granulocytes by centrifugal elutriation and measurement of transmembrane potential
Authors: Jones G. S. , Van Dyke K. , Castranova V.
Published: February 2005

Viable cell samples containing 93% pure granulocytes were obtained from human blood using the techniques of dextran sedimentation followed by centrifugal elutriation. The resting transmembrane potential (Em) of human granulocytes was estimated using the fluorescent lipophilic cation, Di‐S‐C3(5), from the null point for potassium—i.e., the external K concentration at which there is no change in Em in response to valinomycin (a K ionophore). The Em of human granulocytes, as calculated from the Nernst potential for K at the null point, is approximately – 100 mV. Data indicate that this large transmembrane potential is due in part to the presence of an electrogenic Na‐K pump in human granulocytes which is stimulated by external potassium and inhibited by ouabain.
Protein synthesis and degradation in growth regulation in rat embryo fibroblasts: Role of fast‐turnover and slow‐turnover protein
Authors: Amenta J. S. , Sargus M. J. , Brocher S. C.
Published: February 2005

Cultured rat embryo fibroblasts, when stimulated to grow by the addition of fresh medium containing 10% serum, showed an increase in synthesis of slow‐turnover proteins while maintaining a uniform degradation rate for these proteins. Slow‐turnover proteins with a half‐life of 2.4 days accounted for approximately 95% of the cell protein, while the remaining protein could be described in terms of two fast‐turnover pools. When we labeled cells to limiting levels over a period of 4 days, the fast‐turnover pools became undetectable; with 2‐hour labeling periods, however, 25% of the label entered the fast‐turnover pools. Fibroblasts, stimulated to grow by fresh growth medium, showed proportionate and coordinate increases in synthesis of both fast‐turnover and slow‐turnover proteins during the growth period, both returning to baseline levels on reaching the new steady state. No changes could be detected in degradation of either pool during growth. Fibroblasts placed in a serum‐free medium showed a decrease in cellular protein and an increased degradation of slow‐turnover proteins, while degradation of fast‐turnover proteins remained unchanged. We conclude that the slow‐turnover protein pool forms the bulk of the cell proteins and turns over at a fairly constant rate. Growth stimulation is effected almost entirely by stimulation of protein synthesis in this pool, while decreasing cellular protein growth is a result of enhanced degradation within this pool.
Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell‐line produce transforming factors
Authors: Ozanne Brad , Fulton R. Jerrold , Kaplan Paul L.
Published: February 2005

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat‐1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse cells failed to relase TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat‐1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.
The use of 5‐bromodeoxyuridine and irradiation for the estimation of the myoblast and myocyte content of primary rat heart cell cultures
Authors: Masse M. J. O. , Harary Isaac
Published: February 2005

A method for killing dividing cells (Puck and Kao, '67) was adapted for the elimination of dividing heart muscle cells (myoblasts) in cultures. We have used this method to demonstrate their presence and to estimate their number as well as the number of nondividing heart muscle cells (myocytes) in the neo‐natal rat heart. Cells were cultivated in BUdR (5‐bromodeoxyuridine) 10−4 M for 3 days and then irradiated with long UV light. The selective elimination of dividing cells led to a loss of myosin Ca2+‐activated ATPase in the cultures. This indicates the presence of dividing cells which contain myosin. The percent of ATPase left after irradiation was 32% of the control in cultures derived from 1‐day postnatal rats and 48% in cultures from 4‐day postnatal rats. This reflects an in vivo shift of myoblasts to myocytes in the muscle cell population as the rat ages.
Insulin inhibition of protein degradation in cell monolayers
Authors: Ballard F. J. , Wong S. S. C. , Knowles S. E. , Partridge N. C. , Martin T. J. , Wood C. M. , Gunn J. M.
Published: February 2005

Protein degradation has been measured in confluent monolayers of eleven lines of contact‐inhibited cells and ten transformed lines as the rate of release of trichloroacetic acid‐soluble radioactivity after prelabeling cell protein with [3H]leucine. Insulin, at concentrations from 10−12 M to 10−6 M, has been added at the beginning of the 4‐hour degradation period to detect selective effects of this hormone as an inhibitor of the inducible proteolysis occurring in serumfree medium. In addition insulin binding measurements have been performed on selected cell lines in an attempt to relate receptor properties to insulin action. Substantial effects of insulin are found in most cells with a selective inhibition at low insulin concentrations noted in several of the transformed lines. The difference in insulin sensitivity is not entirely definitive because temperature‐sensitive transformation mutants of NRK cells are not more sensitive to insulin at a temperature where they show the transformed phenotype. Although insulin receptors on different cell lines have similar binding properties, two of the hepatomas used, H35 and MH1C1, show inhibition of protein degradation at insulin concentrations where receptor occupancy is extremely low. Calvarial osteoblast‐like cells have a high rate of protein degradation which can be reduced by growth factors but not by insulin. The lack of an insulin response is a consequence of poor insulin binding to the cells. Insulin binds to the osteogenic sarcoma cells in substantial amounts. However, its normal action to inhibit the induced proteolysis is restricted because with these cells no increase of proteolysis occurs in serum‐free medium. Generally higher rates of protein degradation are observed in the contact‐inhibited lines than the transformed cells. We suggest that this difference may provide a selective growth advantage to transformed cells.
Electrolyte and non‐electrolyte distribution in the ehrlich ascites tumor cells during the cell cycle
Authors: Dupre A. M. , Hempling H. G.
Published: February 2005

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na+, K+, and C1− in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na+ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na+ is completely exchangeable during the entire cell cycle.

The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. 14C‐ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, 14C‐urea distributes in anywhere from 86–100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.
Studies on the control of plasminogen activator production by cultured human embryonic lung cells: Requirements for inhibition by corticosteroids
Authors: Rifkin Daniel B. , Crowe Ruth M.
Published: February 2005

The ability of actinomycin D to interfere with the dexametha‐sone‐mediated inhibition of plasminogen activator (PA) production by human‐embryonic lung (HuEL) cells has been examined. The enzyme produced by HuEL cells in the presence of both dexamethasone and actinomycin D appears to be the product of de novo protein synthesis, as determined by the dependence of PA production on active protein synthesis and the net increase in total PA during the course of an experiment. Inhibition of RNA synthesis must be continuous to maintain PA production in the presence of dexamethasone, since reinitiation of RNA synthesis causes an immediate loss of PA activity in the cells. Cordycepin and α‐amanitin also prevent dexamethasone‐mediated inhibition of PA in HuEL cells, indicating that the RNA whose synthesis must be prevented is of the mRNA class. These experiments imply that PA production in HuEL cells may be under translational as well as transciptional control.
Inhibition of calcium and glucose uptake by murine leukemia L5178Y cells treated with antiserum
Authors: Woodcock‐Mitchell Janet , Yang Tsu‐Ju (Thomas)
Published: February 2005

Studies were performed to determine whether decreases in transport of calcium and glucose might be among the earliest changes triggered by the antigen‐antibody reactions occurring on the cell surface of murine leukemia L5178Y cells after treatment with rabbit antisera. After treatment with antisera, in the absence of complement, these cells exhibited a decreased uptake of 45Ca, 2‐deoxy[3H]glucose, and 3‐0‐methyl[3H]glucose. These changes occurred rapidly, within 2 minutes after the addition of antiserum, in contrast to the previously reported inhibitory effects of antiserum on DNA, RNA, and protein synthesis, which became demonstrable only after 4 to 8 hours. The kinetics of uptake of the radioactive substrates was biphasic, with a very rapid initial uptake followed by less rapid linear uptake. The precise mechanism of cell growth inhibition remains to be elucidated, but one of the initial effects of antiserum treatment may be a perturbation at the cell membrane such that transport of specific nutrients is decreased, resulting in the observed effects on macromolecular synthesis.
Effect of ouabain on growth regulation by serum components in Balb/C‐3T3 cells: Inhibition of entry into s phase by decreased protein synthesis
Authors: Frantz Christopher N. , Stiles Charles D. , Pledger W. J. , Scher Charles D.
Published: February 2005

The effect of inhibition of the cell membrane Na+‐K+ pump on the Balb/c‐3T3 cell growth cycle was studied. Inhibition of the Na+‐K+ pump resulted in a dose‐dependent reduction of intracellular K+ concentration ({K+}i). However, inhibition of protein synthesis in G0/G1 and of subsequent entry into S phase occurred only after {K+}i fell below a critical threshold (50–60 mmoles/liter). Thus, when the {K+}i falls below a critical threshold, protein synthesis is inhibited, preventing cells from entering the S phase.

The platelet‐derived growth factor (PDGF) induces cells to become “competent” to traverse the cell cycle; the platelet‐poor plasma component of serum allows competent cells to progress through G0/G1 and enter S phase. Inhibition of the Na+‐K+ pump did not prevent the induction of competence by PDGF, but it did reversibly inhibit plasma‐mediated events in early G0/G1. Similarly, cycloheximide inhibited plasma‐mediated events but did not prevent PDGF‐induced competence. Thus, protein synthesis may not be required for induction of competence; alternatively, the induction of the competent state may occur in these cells after removal of PDGF and protein synthesis inhibitor. Protein synthesis is required for subsequent plasma‐mediated events in G0/G1.
Cyclic AMP surges during the dihydrotestosterone‐induced growth‐division cycle of rat seminal vesicle cells
Authors: Tsang B. K. , Whitfield J. F. , Rixon R. H.
Published: February 2005

A single subcutaneous injection of the active androgen DHT (5α‐androstan‐17β‐01‐3‐one or 5α‐dihydrotestosterone; 5 mg/Kg body weight) into rats 7 days after orchidectomy stimulated DNA synthesis in the seminal vesicle, which began between 30 and 35 hr after injection and peaked at 48 hr. Mitotic activity began between 35 and 45 hr after DHT injection and peaked around 55 hr. These proliferative responses appeared to be confined mainly to the epithelial cells of the seminal vesicles. The DHT injection did not rapidly (between 5 min and 2 hr) increase the cyclic AMP level, but it did result in two small later surges of the cyclic nucleotide; the first of which peaked between 18 and 28 hr, but well before the onset of DNA synthesis, and the second of which peaked between 48 and 60 hr, when both the DNA‐synthetic and mitotic activities were near or at their peak values.
Defective RNA polymerase II in the G1 specific temperature sensitive hamster cell mutant TsAF8
Authors: Shales Michael , Bergsagel John , Ingles C. James
Published: February 2005

TsAF8 is a temperature‐sensitive (TS) mutant of BHK21 cells that arrests at nonpermissive temperatures in the mid‐G1 phase of the cell cycle. TsAmaR‐1 is a TS for growth mutant of CHO cells with a Ts‐ and α‐amanitin‐resistant (AmaR) RNA polymerase II activity. Hybrid TsAmaR‐1 x TsAF8 cell lines were constructed at permissive temperatures. Such hybrid cells did not grow at nonpermissive temperatures; the two TS mutations did not complement. Two different AmaR derivatives of TsAF8 were isolated. Each contained only AmaR polymerase II activity, indicating that this RNA polymerase II gene locus in TsAF8 is functionally hemizygous, as would be expected for a locus in which the recessive TsAF8 mutation had occurred. One of these AmaR isolates of TsAF8 had a partially reverted TS+ phenotype. Taken together these results suggest that the TS mutation in TsAF8 is in RNA polymerase II.
In vitro development of CFU‐E and BFU‐E in cultures of embryonic and post‐embryonic chicken hematopoietic cells
Authors: Samarut J. , Bouabdelli M.
Published: February 2005

A culture method is proposed for the in vitro development of chicken erythrocytic progenitors. When grown with avian erythropoietin, Colony Forming Unit Erythrocytic (CFU‐E) and Burst Forming Unit‐Erythrocytic (BFU‐E) give rise respectively to erythrocytic colonies and bursts within 3 and 6 days. BFU‐E development is greatly enhanced by pokeweed‐mitogen‐spleen‐cell‐conditioned medium and requires higher erythropoietin concentrations than for CFU‐E. An antigen specific to immature red cells can be detected on CFU‐E but not on BFU‐E, showing that both progenitors represent distinct entities. BFU‐E and CFU‐E are found in embryonic marrow and yolk sac. In the young blastoderm BFU‐E becomes detectable at the primitive streak stage.
Biophysical properties of human astrocytic brain tumor cells in cell culture
Authors: Black Peter McL. , Kornblith Paul L.
Published: February 2005

For malignant cells cultured from a human astrocytoma, electrophysiological characteristics of the plasma membrane included specific resistivity of 446.82 ± 279.5 ohm·cm2, specific capacitance of 0.758 ± 0.52 microfarads/cm2, time constant 0.318± 0.10 msec. The resting membrane potential averaged‐14.07 ± 7.4 mV; the mean input resistance 8.1 ± 4.0 megohms. The average cell area was 1638 ± 585 ±2 for contactual and 1919 ± 989 ±2 for noncontactual cells. Changes in input resistance and resting membrane potential were observed with increasing time in culture, possibly reflecting cell cycling. There did not appear to be electrical coupling in this cell line.
The potentiation of adenine toxicity to Chinese hamster cells by coformycin: Suppression in mutants with altered regulation of purine biosynthesis or increased adenylate‐deaminase activity
Authors: Debatisse M. , Berry M. , Buttin G.
Published: February 2005

When added to medium containing coformycin (2 μM or above), adenine is lethal to Chinese hamster fibroblasts at the concentration inhibiting de novo purine biosynthesis (Debatisse and Buttin, '77b). Rescue by hypoxanthine suggested that cells die of IMP starvation when the analog can turn off deamination of both adenosine and adenylate. As predicted from this hypothesis, two classes of variants resistant to the mixture of coformycin + adenine have been isolated: Class 1 variants have altered control of de novo IMP biosynthesis; they fall into two subclasses on the basis of their resistance to adenosine. Class 2 variants have a 6–10‐fold increased level of AMP‐deaminase (E.C.: 3.5.4.6); their growth in the selective medium is temperature‐dependent, a property accounted for by the observation that cell growth in the presence of coformycin imposes a gradual thermodependent decay of specific AMP‐deaminase activity in both wild‐type and variant lines. This control by coformycin of AMP‐deaminase activity isunaltered in mutants deficient in the four activities of adenosine‐kinase, APRT, HGPRT and deoxycytidine‐kinase. Most of the resistant variants are unstable and exhibit either increased or reduced resistance, depending on prolonged growth in selective or normal medium.