bluuebunny/crossref_metadata_2025 huggingface dataset:
Transformed cells have lost control of ribosome number through their growth cycle
Authors: Stanners C. P. , Adams M. E. , Harkins J. L. , Pollard J. W.
Published: February 2005

Previous studies on the synthesis and function of the protein synthetic machinery through the growth cycle of normal cultured hamster embryo fibroblasts (HA) were extended here to a series of four different clonal lines of polyoma virus‐transformed HA cells. Under our culture conditions, these transformed cells could enter a stationary phase characterized by no mitotic cells, very low rates of DNA synthesis, and arrest in post‐mitotic pre‐DNA synthetic state. Cellular viability was initially high in stationary phase but, unlike normal cells, transformed cells slowly lost viability.

The rate of protein synthesis in the stationary phase of the transformed cells fell to 25‐30% of the exponential rate. Though this reduction was similar to that seen in normal cells, it was accomplished by different means. The specific reduction in the ribosome complement per cell to values below that of any cycling cell seen in normal cells, was not seen in any of the transformed lines. This observation, which implies a loss of normal control of ribosome synthesis through the growth cycle after transformation, was confirmed in normal Chinese hamster embryo fibroblasts and transformed CHO cell lines. Normal control of ribosome synthesis was restored in L‐73 and LR‐73, growth control revertants of one of the transformed CHO lines. The transformed lines reduced their protein synthetic rates in stationary phase either by a greater reduction in the proportion of functioning ribosomes than that seen in normal cells or by a decrease in the elongation rate of functioning ribosomes; the latter effect was not seen in the normal cells.

A model for growth control of normal cells and its derangement in transformed cells is presented.
Regulation of the proliferative response in Rous sarcoma virus transformed chicken embryo fibroblasts by serum and multiplication‐stimulating activity (MSA)
Authors: Knauer Daniel J. , Smith Gary L.
Published: February 2005

A temperature sensitive mutant of Rous sarcoma virus (tsNY68) was used to obtain cultures of quiescent virus‐infected chicken embryo fibroblasts arrested by serum starvation at the non‐permissive temperature. Upon shift to the permissive temperature, these cells enter the replicative cell cycle as evidenced by increases in 2‐deoxyglucose uptake, 3H‐thymidine incorporation and percent labeled nuclei. These changes occur in the absence of serum and the cells become morphologically transformed within eight to ten hours after the temperature shift. Entry into the S phase temporally resembles that of normal quiescent fibroblasts stimulated with serum. This experimental system was used to examine the proliferative response of transformed cells to serum and purified multiplication‐stimulating activity (MSA) during the transition from the resting to the growing state. Data are presented which show that the presence of serum in the medium enhances the proliferative response of quiescent infected cells shifted to the permissive temperature over those shifted in the absence of serum. In contrast, the presence of MSA has no additional effect on the response exhibited by infected cells shifted to the permissive temperature in serum‐free medium. Labeled MSA binding experiments show that this lack of response is not due to a loss of MSA receptors on the cell surface since transformed cells are still capable of binding MSA at the same level as normal cells. The results are consistent with the hypothesis that the set of biochemical events initiated by MSA in normal cells are turned on in infected cells shifted to the permissive temperature by the activation of the src gene product.
MSA stimulation of AIB transport is independent of K+ accumulation in myoblasts
Authors: Merrill Gary F. , Dulak Norman C. , Florini James R.
Published: February 2005

Enhanced uptake of amino acids is frequently associated with hyperplasia in cultured cells; we have recently shown (Merrill et al., '77) that this is true of the stimulation of rat myoblast proliferation in culture by Temin's Multiplication Stimulating Activity (MSA). In many cases the asymmetric distribution of Na+ and K+ across the cell membrane profoundly affects uptake of certain amino acids, so we investigated the possibility that enhanced Na+‐dependent AIB transport was the result of an MSA‐induced increase in K+ accumulation. MSA stimulated the rate of uptake of the potassium analog 86Rb+ 15‐25% within ten minutes; this rate remained elevated for at least seven hours. Effects were limited to the ouabain‐sensitive component of Rb+ uptake. (Our simultaneous measurements of 86Rb+ and 42K+ uptake demonstrated that Rb+ provides a useful qualitative but not an exact quantitative index of K+ uptake.) The stimulation of K+ uptake by MSA did not cause a large increase in total cellular K+ of the myoblasts; after five hours, MSA‐treated cells contained only about 20% more K+ than did corresponding controls (1.21 ± 0.02 vs. 1.01 ± 0.02 μmoles/mg protein, respectively). To investigate whether this small increase in K+ content could be amplified by the cell to account for the 50‐150% stimulation of AIB uptake, we preincubated cells with ouabain for various times and then measured total cell K+ and AIB uptake in the same culture dishes. Under conditions in which the MSA‐stimulated increase of total cell K+ was prevented by ouabain, a substantial stimulation of AIB uptake was still observed. We conclude that MSA stimulation of AIB transport is independent of increased accumulation of K+ in rat myoblasts.
Inhibiton by glucocorticoids and choleragen of the conditional growth of poorly adherent mononuclear phagocytes of newborn hamster liver and lung (Hormonal control of macrophage growth)
Authors: Nozawa Ryushi T. , Yokota Takeshi
Published: February 2005

Conditions for in vitro growth of mononuclear phagocytes from newborn hamster liver and lung were studied. In the primary cultures of liver and lung, round cells outgrew and frequently floated off into the culture medium. They were separated from fibroblast‐like cells adherent to plastic by collecting the medium. The round cells were identified as mononuclear phagocytes on the criteria of phagocytic capacity of heat‐killed bacteria and IgG‐coated erythrocytes, fine cell structure and cytochemistry. The phagocytes that had not been activated previously proliferated for about ten generations in F12 medium supplemented with 10% fetal calf serum depending on a growth factor produced by hamster brain, liver or lung cells. Without the factor, the cells quickly cytolysed. Mononuclear phagocytes from blood had the same characteristics of growth and cytochemistry, but had fewer IgG receptors at the cell surface than similar cells from the liver and lung.

The effects of a variety of chemical compounds on the growth of the liver and lung cells were studied. Insulin stimulated their growth by 20‐30%, but was not replaceable for the growth factor. Glucocorticoids, dexamethasone and hydrocortisone, inhibited the growth of the phagocyates at the physiological concentrations: 3 × 10−9 M and 2 × 10−8 M for 50% inhibition, respectively. Indomethacin, non‐steroid anti‐inflammatory reagent, at 10−8 M to 10−6 M gave no effect. Choleragen that increases the intracellular cyclic AMP level, inhibited the growth at a concentration as low as 5 pg/ml. These data suggest that the growth of mononuclear phagocytes is controlled not only by a growth factor produced by other cells but also by glucocorticoids.
Immortalization of normal liver functions in cell culture: Rat hepatocyte‐hepatoma cell hybrids expressing ornithine carbamoyltransferase activity
Authors: Widman Lawrence E. , Golden Jonathan J. , Chasin Lawrence A.
Published: February 2005

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver‐specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H‐ornithine into protein arginine, and can be selectively grown in arginine‐free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue‐specific phenotypes.
Inhibition of low density lipoprotein uptake in confluent endothelial cell monolayers correlates with a restricted surface receptor redistribution
Authors: Vlodavsky I. , Fielding P. E. , Johnson L. K. , Gospodarowicz D.
Published: February 2005

Binding of either low density lipoprotein (LDL) or Concanavalin A (ConA) to actively growing vascular endothelial cells is associated with a redistribution of the appropriate cell surface receptor sites which form patches and caps. This receptor lateral mobility is greatly restricted when endothelial cells reach confluence and adopt the configuration of a cell monolayer composed of closely apposed and non‐overlapping cells. In this case, although the cells still exhibit specific LDL binding to the appropriate cell surface receptor sites, neither the binding of LDL nor of ConA induces a receptor redistribution. The lack of LDL receptor redistribution correlates with a marked decrease in the rate of LDL internalization. In contrast, no such a density‐dependent changes are observed in cell types which grow on top of each other and form multiple cell layers at confluence. Thus, neither LDL nor ConA induced cap formation in either sparse or confluent smooth muscle cell cultures and the same rate of LDL internalization is observed at both cell densities. Similarly, adsorptive endocytosis of cationized LDL (which enters the cell independently of the LDL receptor sites) was not correlated with a detectable receptor redistribution, nor was it significantly affected by changes in cell density and spatial organization.

The formation of a confluent cell monolayer resting on an underlying basement membrane might therefore provide, via a change in membrane dynamics, a mechanism whereby the endothelium of large blood vessels can function as a protective barrier against the high circulating levels of LDL in plasma.
Regulation of cyclic nucleotide phosphodiesterase forms by serum and insulin in cultured fibroblasts
Authors: Pledger W. J. , Thompson W. J. , Epstein P. M. , Strada S. J.
Published: February 2005

A rapid reduction of cyclic nucleotide phosphodiesterase activity occurs after the replating of confluent cultures of BHK 21 c/13 fibroblasts into fresh medium. This reduction in activity depends on the density to which the cultures are reseeded and the concentration of serum in the medium. Enzyme activity in BHK cells is restored after 24 to 48 hours if cells are diluted into medium containing 10% fetal calf serum or 0.5% fetal calf serum supplemented with insulin (10−6 M), but not into 0.5% serum alone. The restoration in enzyme activity is blocked by cycloheximide or Actinomycin D.

When BHK cells become quiescent by maintenance in 0.5% serum conditions for 48 hours, a rapid (15‐60 minutes) increase in cyclic AMP phosphodiesterase activity occurs when 10% serum is added to the cultures. Enzyme activity is increased even further after 24 to 48 hours in the 10% serum. Cycloheximide or Actinomycin D do not affect the rapid increase in enzyme activity in response to serum, but completely inhibit the long term increase. In contrast to serum, insulin (10−8 to 10−6 M) has no short term effect, but does increase enzyme activity after 24 to 48 hours to levels comparable to those seen with addition of 10% serum. As is the case with serum, this long term effect of insulin on enzyme activity is prevented by inhibitors of protein and RNA synthesis.

Kinetic analyses of cyclic AMP phosphodiesterase activity in homogenates of quiescent BHK cells indicate the presence of only high Km (≃ 20 μM) enzyme activity. Addition of serum or insulin to quiescent cells results in the appearance of apparent low Km enzyme activity in homogenates. Sucrose gradient analysis of BHK cells displays two forms of cyclic AMP phosphodiesterase enzyme activity: a 3‐4 S form and 5‐6 S form. In quiescent cells, the 5‐6 form greatly predominates relative to the 3‐4 form. Addition of serum to quiescent cells results in a rapid appearance of increased 3‐4 S form enzyme activity. Insulin also increases the activity of this higher affinity 3‐4 S enzyme form after 24 to 48 hours in culture. The functional significance of short and long term regulation of cyclic nucleotide phosphodiesterase(s) in cells is discussed.
Growth and G1 arrest of sarcoma virus transformed cells in serum free media
Authors: Moskowitz Merwin , Cheng David K. S.
Published: February 2005

Rous‐sarcoma transformed BHK cells can be continuously cultured in a medium containing Eagle's Minimal Essential Medium, iron and biotin. The rate of cell multiplication increased when serine, or serine plus other non‐essential amino‐acids were added to the medium. With biotin deleted from the medium there is a reduction in DNA synthesis and most cells are blocked in G1.
Early inhibition of phospholipid synthesis in dimethyl sulfoxide (DMSO) treated friend erythroleukemia (FL) cells
Authors: Harrel L. , Lacour F. , Friend C. , Durbin P. , Semmel M.
Published: February 2005

Phosphate metabolism in Friend erythroleukemia cells undergoing DMSO‐induced differentiation was studied. Thirty minutes after the cells were exposed to DMSO in medium at pH 7, an inhibition of 39% in the incorporation of phosphate into phospholipids was observed. This decrease was not due to a change in the precursor pools since phosphate uptake and the phosphorylation of the organic soluble compounds were only inhibited 13%.

Inhibition of phospholipid synthesis preceded inhibition of RNA and protein synthesis and reached a maximum after 24 hours of DMSO treatment. At this time, the phospholipid content of the cells was also decreased as compared to that of the control untreated cells. Phospholipid synthesis remained at a level significantly lower than in the controls over the 4‐day observation period, at which time 85% of the treated cells were benzidine positive.

Separation of the different phospholipids by chromatography on thin layer silicate gel plates showed that, after one hour of DMSO treatment, more than 80% of the radioactivity was in phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine. Phosphatidylethanolamine was the most inhibited. Incorporation of inositol into phospholipid was also significantly decreased. However, there was little variation in the phospholipid composition of the treated and non‐treated cells other than a decrease in the percent of sphingomyelin after 48 hours of DMSO treatment.

These changes in phospholipid metabolism may initiate the first step in the complex differentiation process. The phospholipids are important components of membranes and the inducers are known to influence their fluidity.
On the adaptation of cultured chick embryo cells to growth in the presence of chloramphenicol
Authors: Moris Réjean , Giguére Louis
Published: February 2005

We have found that tryptose phosphate broth (TPB) prevents the inhibitory effect of chloramphenicol (CAM) on the cell proliferation of chick embryo fibroblasts. Study of growth parameters indicated that no lag or adaptation period appeared necessary for TPB‐exposed chick cell populations to grow in the presence of CAM suggesting that a particular cell type was not selected. TPB did not prevent the inhibitory effect of CAM on the mitochondrial protein‐synthesizing system. This was supported by cytochrome oxidase activity measurements, studies on the incorporation of 35S‐methionine into mitochondrial proteins, electron microscopic observation of alterations in mitochondrial structure. Oxygen consumption was reduced by 95% and cyanide, 2‐4‐dinitrophenol, and salicylhydroxamic acid do not significantly affect the residual respiration. Analyses of reduced‐minus‐oxidized‐cytochrome spectra of CAM‐treated chick cells demonstrate the disappearance of the absorption bands of cytochromes aa3, b559, c1, and c. The presence of a type b cytochrome with maxima at 552 and 557 nm was observed. The results obtained indicate that long‐term cultures of CAM‐treated chick embryo cells cultivated in the presence of TPB grow with mitochondria devoid of a functional respiratory chain.
The cyclic AMP‐dependent initiation of DNA synthesis by T51B rat liver epithelioid cells
Authors: Boynton A. L. , Whitfield J. F.
Published: February 2005

The brief rise in the cellular cyclic AMP content which occurs late in the prereplicative phases of rat hepatocytes in vivo and T51B rat liver epitheloid cells in vitro seems to be necessary for the initiation of DNA synthesis. Thus, the extracellular calcium‐deprivation in T51B rat liver cells in culture which induces a late G‐1 block is rapidly reversible (cells surge into S phase within one hour) either by creating a cyclic AMP surge by the addition of calcium or 3‐isobutyl‐1‐methyl xanthine (a cyclic 3′, 5′‐nucleotide phosphodiesterase inhibitor) or by the exogenous addition of low concentrations of cyclic AMP itself (i.e., 10−8‐10−5 M). On the other hand, prevention of the calcium‐induced cyclic AMP surge by imidazole (a cyclic 3′, 5′‐nucleotide phosphodiesterase activator) blocked the initiation of DNA synthesis by the calcium‐deprived T51B cells.
Concanavalin A is mitogenic for resident peritoneal macrophages
Authors: Wang Ai‐Lan , Basch Ross S.
Published: February 2005

Con A, a known T‐cell mitogen, is also mitogenic for resident peritoneal macrophages. The stimulated cells morphologically resemble macrophages and are actively phagocytic. The concentration of con A (30 μg/ml) required to stimulate 3H‐TdR incorporation is ten times that required for T‐cell activation. Con A must be present throughout the entire culture period to produce the maximum effect, and con A‐depleted supernatant fluids from con A‐stimulated cells cannot replace the con A requirement. Stimulation of 3H‐TdR incorporation occurs after a 48‐hour lag period and is maximal on the fifth to seventh day of culture. At the peak of the response, 20–30% of the macrophages can be stimulated to incorporate 3H‐TdR, but little or no increase in the total number of cells present in the culture occurs. This and pulse‐chase experiments indicate that only a single cycle of replication occurs in the stimulated cells. Con A‐responsive peritoneal macrophages appear to be a distinct subpopulation and might play a different role in the interaction with T cells and B cells in the immune response than the con A‐non‐responsive cells.
Properties and separation of T lymphocyte growth stimulatory activity (TL‐GSA) and of granulocyte‐macrophage colony stimulatory activity (GM‐CSA) produced separately from two human T lymphocyte subpopulations
Authors: Wu A. M.
Published: February 2005

We have previously identified two stimulatory activities affecting blood cell maturation in PHA‐stimulated human lymphocyte conditioned medium (PHA‐LyCM). One was granulocyte‐macrophage colony stimulatory activity (GM‐CSA), and the other was T lymphocyte growth stimulatory activity (TL‐GSA) in suspension culture. In this paper we have shown that although both activities can be produced from purified non‐adherent human T lymphocytes, they are produced from two distinct subpopulations. The production of these activities was greatly enhanced by T cell mitogens. Both protein factors were relatively heat stable (56°, 30 minutes), were sensitive to trypsin treatment and were specific for primate blood cells. These two activities were fractionated by means of ammonium sulfate precipitation, Sephadex G‐150 gel filtration, DEAE cellulose and Con A‐Sepharose column chromatographies. MW of the major peak estimated from the elution volume of gel filtration in the presence of 0.5 M NaCl was 40,000 for GM‐CSA and 13,000 for TL‐GSA. Results from Con A‐Sepharose column showed that while about 70% of TL‐GSA was bound to Con A, less than 25% of GM‐CSA was bound. These observations show that the majority of TL‐GSA and GM‐CSA were separable by these two conventional column chromatographic methods.
Regulation of the G1 → S phase transition in chick embryo fibroblasts with α‐keto acids and L‐alanine
Authors: Groelke John W. , Baseman Joel B. , Amos Harold
Published: February 2005

Temporal inhibition of protein synthesis with cycloheximide prevents subsequent insulin, but not serum‐stimulated DNA synthesis in G1‐arrested chick embryo fibroblasts (CEF). The inhibition is measured by the incorporation of 3H‐thymidine into acid insoluble material and confirmed by chemical estimate of the DNA content of inhibited and uninhibited cells. Cycloheximide treatment is without effect if the cell cultures are maintained at 4° C while exposed to the drug. Several α‐keto acids (pyruvate, oxaloacetate, α‐keto‐butyrate) at 0.5‐1 mM concentrations restore DNA synthesis in previously inhibited cells when combined with insulin. L‐alanine (D‐alanine is inert) is even more effective than the keto acids in stimulating DNA synthesis after cycloheximide treatment. Clucose transport was unaffected by cycloheximide treatment while lactate levels in medium from inhibited, insulin‐stimulated CEF were reduced 70% compared to uninhibited counterparts. We speculate that cycloheximide treatment may lead to the decay of a glycolytic enzyme which compromises the ability of inhibited cells to synthesize pyruvate from glucose, and thus induces an exogenous requirement for α‐keto acid or L‐alanine. A serum component(s) with a molecular weight of about 100 permitted insulin‐stimulated DNA synthesis in inhibited cells.
Growth‐promoting actions of extracts from mouse submaxillary glands on human endothelial cells in culture
Authors: Johnson Alice R. , Boyden Nit Ton , Wilson Carol M.
Published: February 2005

Extracts of submaxillary glands from two different strains of inbred mice were mitogenic for human endothelial cells in culture. The mitogenic activity of extracts from glands of males of the SWR/J and C57BL/10J strains were equivalent, and the growth stimulating effect was unrelated to renin or esteroproteolytic activity. Mitogenic activity in extracts from SWR/J females was less than that from males, and extracts from C57BL/10J females were inactive. The polypeptide growth factors, epidermal (EGF) and fibroblast (FGF) growth factors, also stimulated replication of endothelial cells. Cells from either umbilical arteries or veins responded to submaxillary extracts, EGF, or FGF with a similar increase in cell number, increase in protein and enhanced uptake of 3H‐thymidine. The proliferative response was associated with decreased activity of angiotensin I converting enzyme which is localized on the endothelial surface. Nerve growth factor (NGF) was not mitogenic for endothelial cells. Extracts of submaxillary glands from male mice of either strain contained approximately 20 times more EGF than extracts from females, as determined by immunodiffusion. Mitogenic activity of the extracts was completely inhibited by antiserum to EGF, suggesting that the active component of these preparations is EGF.
An X‐ray microanalysis survey of the concentration of elements in the cytoplasm of different mammalian cell types
Authors: Cameron Ivan L. , Pool Thomas B. , Smith Nancy K. R.
Published: February 2005

Electron probe energy dispersive X‐ray microanalysis was performed on freeze‐dried tissue sections. The dry weight concentration of elements (mmole/kg dry weight) was measured in the cytoplasm of several cell types from adult mice and rats. This comparative investigation showed: (1) That the energy dispersive X‐ray spectrum of element concentration from the cytoplasm of a specific cell type allows one to distinguish this specific cell type from other cell types with considerable accuracy. (2) That there is a relationship between the concentration of the various elements and the ultrastructural features of the cytoplasmic regions being analyzed. For example, areas rich in ribosomes are also rich in P, K and Mg. (3) These data support the idea that K is directly involved in the control of protein synthesis. The catalog of element concentrations in the cytoplasm of 13 cell types from both mice and rats should be of value to others who seek to answer various questions about these cell types.
Protein synthesis in transformed 3T3 cells permeabilized by exogenous ATP
Authors: Kitagawa Takayuki
Published: February 2005

Exogenous ATP has been shown to cause a rapid and reversible increase in permeability in transformed 3T3 cells (3T6 and SV3T3) but not in untransformed 3T3 cells. The cells remain viable, but lose intracellular acid‐soluble pools. Treatment of transformed cells with ATP greatly reduces incorporation of 14C‐leucine into protein, which is restored by the incubation of the cells with Dulbecco's modified Eagle's medium or by the external additions of certain ions and energy sources. tRNA is not required for the restoration of protein synthesis. In the permeabilized cells the energy for protein synthesis can be provided by glycolysis, oxidative phosphorylation, or direct addition of ATP. These studies demonstrate the usefulness of this method for studying the control of metabolism and macromolecular synthesis in monolayer cultures of transformed mammalian cells.
Concanavalin A induces an intraluminal alkalinization of thymocyte membrane vesicles
Authors: Mikkelsen Ross B. , Schmidt‐Ullrich Rupert , Wallach Donald F. H.
Published: February 2005

Weak acid distribution methods demonstrate that mitogenic levels of concanavalin A induce an intravesicular alkalinization of isolated thymocyte membrane vesicles. Experiments with chemical reagents that crosslink the high affinity concanavalin A receptor and extensive correlation with known cellular events suggest that a “membrane Bohr effect” may participate in the initiation of mitogenesis.
Formation of low molecular weight RNA species in HeLa cells
Authors: Eliceiri George L.
Published: February 2005

It has been previously shown that newly synthesized nuclear low molecular weight RNA species C and D are first detected in the cytoplasm for a few minutes before they are finally found in the nucleus. The following are some of the observations made in the present study, regarding the formation of C and D RNA: (1) The 5′ end cap ribose methylation of the C RNA precursor is complete in its cytoplasmic stage; the internal ribose methylation of the precursor seems to be completed about the time of its apparent transition from cytoplasm to nucleus. (2) The few nucleotides lost from the D RNA precursor during maturation seem to be excised sometime near its apparent cytoplasmic → nuclear transition. Newly synthesized C RNA also appears to lose some of its non‐conserved nucleotides about the time of that transition, while the other extra nucleotides are lost later, in the nucleus. (3) The maturation of C and D RNA is inhibited early during suppression of protein synthesis by cycloheximide, while their synthesis is not. (4) The cytoplasmic precursors of C and D RNA are not associated with ribonucleoprotein particles as large as those reported for mature C and D RNA, although they do not appear to be free in the cytoplasm. (5) When the cellular UTP pool is depleted by exposure of the cells to amino sugars, and the synthesis of C, D, and other RNA species decreases, the level of[3H]uridine labeling of C and D RNA increases, while that of 4 S and 5 S RNA does not. These data are compatible with the existence of more than one nuclear UTP pool.
Changes in the cell cycle during culture of mouse‐Chinese hamster cell hybrids
Authors: Graves Jennifer A. Marshall , Koschel Kenneth W.
Published: February 2005

Cell cycle studies, using PLM analysis, were carried out on a mouse‐Chinese hamster cell hybrid and its derivatives which stably retained all parental chromosomes during the year of study. Parameter estimates were obtained from the PLM curves, using conjugate gradient curve fitting procedures. The hybrid initially grew very slowly, and all phases (especially G1) were longer than those of either parent. During propagation, mean generation time decreased progressively, and the phase times approached those of the mouse parent (which had the longer G1 and S). DNA replication could be scored separately in mouse and hamster chromosome sets; initially termination was highly asynchronous, but during growth asynchrony was progressively reduced as DNA synthesis in the hamster set was prolonged.

We conclude that cell hybrids may undergo progressive modifications of the cell cycle, even in the absence of significant chromosome segregation, and suggest that such changes may at least partly account for the great variety of relationships between the growth rates and phase times of parent and hybrid cells which have been reported. Because of the complexity of these changes in the cycles of interspecific cell hybrids, we believe that somatic cell genetic analysis of the regulation of the cell cycle would be more usefully applied to intraspecific hybrids whose parents differ in only one specific cycle characteristic.
Fetal hemoglobin synthesis in culture of early erythroid precursors (BFU‐E) from the blood of normal adults
Authors: Vainchenker William , Dubart Anne , Bouguet Jacqueline , Testa Ugo , Tsapis Andréas , Tonthat Hoi , Henri Annie , Monplaisir Nicole , Beuzard Yves , Rochant Henri , Rosa Jean
Published: February 2005

BFU‐E from the blood of 14 normal adults have been grown by the plasma clot technique. The hemoglobins synthesized in burst colonies were purified from other proteins by affinity chromatography on Sepharose‐haptoglobin. The radioactivity incorporated in the globin chains was estimated by CM‐cellulose chromatography in urea. The number of bursts scored at the 14th day of culture fluctuated between 50‐130 (average 86, s: 29) for 106 mononuclear plated cells. A constant reactivation of fetal hemoglobin was found (from 1.4% to 11%, mean value 5.8%, s: 3.07), but was lower than previously described, mainly because of the highly selective purification of Hb. This reactivation of fetal hemoglobin was not dependent upon the concentration of erythropoietin (from 1 U/ml to 6 U/ml) nor on the purity of the erythropoietin preparations (from 6 U/mg of protein to 70 000U/mg of protein). In addition, the same subject exhibited a constant proportion of Hb F synthesized in culture over a period of time up to 6 months. A positive correlation exists between the proportion of Hb F in culture and that of F cells present in the blood, with the exception of two subjects. Such findings suggest that Hb F in culture is a characteristic of each individual and that this reactivation often represents an amplification of the Hb F synthesis in vivo.
Pactamycin resistance in CHO cells: Morphological changes induced by the drug in the wild‐type and mutant cells
Authors: Gupta Radhey S. , Siminovitch Louis
Published: February 2005

Stable mutants resistant to pactamycin (PacR), a polypeptide chain initiation inhibitor, have been selected in a single step in Chinese hamster ovary (CHO) cells. The sensitivity of protein synthesis in mutant cell extracts to pactamycin indicates that resistance involves an alteration in the permeability of this drug. The failure of PacR mutants to show cross‐resistance to other compounds provides further indication that the lesion is presumably specific for pactamycin. Cell hybrids formed between PacR × PacS lines show intermediate sensitivity towards pactamycin, suggesting that the PacR lesion behaves codominantly under these conditions. In the presence of subinhibitory concentrations of pactamycin, CHO cells, which are normally short, polygonal and disoriented, became greatly elongated and aligned themselves in parallel fashion to produce highly oriented colony morphologies, reminiscent of normal diploid fibroblasts. This effect of pactamycin on cellular morphology was seen much more clearly with the PacR mutants, although somewhat higher concentrations of the drug were required to produce this change.
Secretion and partial degradation of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) of mouse L‐P3 cells
Authors: Tsuneoka K. , Shikita M.
Published: February 2005

Secretion of a granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) was accomplished by L‐P3 cells in culture with a serum‐free medium. Cell proliferation per se was not requisite for the production of GM‐CSF; the cells continued secreting GM‐CSF even after their growth had been suspended. The amount of GM‐CSF accumulated in the conditioned medium was reasonably accounted by the daily rate of production, and the addition of a proteinase inhibitor such as leupeptin and pepstatin did not result in greater accumulation of GM‐CSF in the culture. It is thus postulated that there is no significant proteolytic inactivation of the secreted GM‐CSF in the culture. However, when partially purified GM‐CSF preparation was chromatographed on a gel‐filtration column in the presence of 0.1% Triton X‐100, a derivative of the GM‐CSF was yielded which had been diminished in the molecular weight and altered in the isoelectric point. On the other hand, when leupeptin was included in the solution during production and isolation of the factor, the yielded GM‐CSF did not manifest such a detergent‐induced transformation and maintained its isoelectric point at pH 3.5. It is thus assumed that, in the presence of the detergent, GM‐CSF suffers deterioration by an endogenous proteinase and releases a sialoglycopeptide fragment without loosing its colony‐stimulating activity.
A study of metabolic cooperation with rat peritoneal macrophages
Authors: Kane A. B. , Bols N. C.
Published: February 2005

Rat peritoneal macrophages were studied for their ability to undergo metabolic cooperation. Macrophages were unable to cooperate with human fibroblasts. This was true for macrophages which had been activated in vivo as well as for macrophages treated with various agents in vitro. Macrophages were also unable to undergo metabolic cooperation with rat fibroblasts or with other macrophages. In contrast, rat reticular cells, mesothelial cells, and fibroblasts were able to cooperate with human fibroblasts.
Fibronectin from aged fibroblasts is defective in promoting cellular adhesion
Authors: Chandrasekhar Srinivasan , Millis Albert J. T.
Published: February 2005

Late passage fibroblasts show decreased cell‐substrate adhesion. We provide evidence that the reduced adhesion is due to a defect in the adhesive glycoprotein fibronectin. Late passage cells become more adhesive in culture media that has been conditioned by the growth of early passage cells. Analysis of fibronectins purified from early and late passage cell conditioned media indicates that there are striking differences in their abilities to promote cell adhesion. Young cell fibronectin supports the maximal adhesion of both young and old cells. However, old cells require quantitatively more fibronectin. In contrast, old cell fibronectin is less effective in supporting the adhesion of either cell type. In addition, neither cell type achieves a normal morphology in the presence of old cell fibronectin. The results support the conclusion that the fibronectin released by late passage cells is defective and does not support normal cell‐substrate interactions.
A critical temperature transition of K+‐Na+ exchange in human lymphocytes
Authors: Negendank William , Shaller Calvin
Published: February 2005

Human lymphocytes were equilibrated for 48 hours at 5‐6 mM K+ex over a range of temperatures between 0 and 37°C, and at 5°C over a range of external K+ levels between 0 and 32 mM. Cell K+ and Na+ contents are normal between 37 and 10′. Below 10′ there is a critical thermal transition in ion contents centering around 3°C. This and the steep sigmoidal isotherms of K+ and Na+ at 5°C confirm the cooperative nature of ion exchange. At 0′, cell K+ is maintained at a concentration that is seven to eight times that of the external medium. Isotopic K+ influx shows smaller, rapidly‐exchanging, and larger, slowly‐exchanging fractions. The latter, which correspond to the saturable, sigmoidal components of cell K+, are slowed by decreasing temperature. Although there is a critical temperature transition of K+‐Na+ exchange, there is no corresponding transition for isotopic K+ exchange, which has an activation energy of 11.6 kcal/mole. The combined ion content and flux data are readily understood by reference to two major concepts of the association‐induction hypothesis: that of rapid solute exclusion from cell water existing in a state of polarized multilayers, and that of solute accumulation limited by adsorption onto and desorption from fixed anionic sites that interact with one another in a critical, cooperative fashion.
Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes
Authors: Kelley Darshan S. , Shull James D. , Potter Van R.
Published: February 2005

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2‐amino [1‐14C]isobutyric acid (AIB) and N‐Methyl‐2‐amino [1‐14C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium‐dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane‐1‐carboxylic acid, 1‐ [carboxyl‐14C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium‐independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium‐dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3‐fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8‐fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.
The regulation of RNA metabolism in suspended and reattached anchorage‐dependent 3T6 fibroblasts
Authors: Benecke Bernd‐Joachim , Ben‐Ze'ev Avri , Penman Sheldon
Published: February 2005

Suspending anchorage‐depent fibroblasts in methocel results in marked reduction in a number of macromolecular metabolic processes. These are restored when cells make contact with a solid substrate and are allowed to spread. The response of 3T6 cells to suspension culture and their recovery upon reattachment has been extensively studied. (Benecke et al., '78; Farmer et al., '78). In these cells, message production and its turnover rate both drop abruptly when cells are suspended, while protein synthesis declines very slowly but extensively. Recovery of protein synthesis in 3T6 cells only requires surface contact and not cell spreading and proceeds rapidly, reaching control values within a few hours. The recovery of messenger RNA production in spread cells is much slower, and commences about 18 hours after cells are replated.

The present report extends the studies on RNA regulation in suspended and reattached 3T6 fibroblasts. All RNA synthesis systems respond to cell suspension. Production of small nuclear RNA species is shown to behave very similarly to the production of messenger RNA, suggesting these may be coordinately regulated. Ribosomal precursor RNA synthesis ceases promptly upon cell suspension and recovers only slowly after reattachment. The control of ribosome formation appears independent of the regulation of protein synthesis in this system. The products of polymerase III including transfer RNA, 5S RNA, and species K and L are all regulated together and show a distinct pattern of behavior unlike that of any other RNA species. Their formation is rapidly inhibited at the beginning of suspension, but then recovers to the level of control cells, even during continued suspension culture. This unusual property of polymerase III RNA species is quite different from the behavior of the small nuclear (snRNA) RNA species, which strengthens the previous suggestion that these two classes of RNA molecules are formed by different mechanisms and regulated independently.
Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation
Authors: Ley K. D.
Published: February 2005

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [3H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G1 traverse. In cells synchronized in G1 by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS‐polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [3H] leucine into mitochondrial polypeptides after reversal of G1‐arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.
Nature of permeability changes in membrane of HeLa cells adsorbing sendai virus
Authors: Fuchs Pinchas , Gruber Eva , Gitelman Janina , Kohn Alexander
Published: February 2005

Adsorption of Sendai virus to HeLa cells induced in them an increased permeability to K+, Na+, Ca++, deoxyglucose, but not to fluorescein. The stimulation of uptake of 42K was temperature‐dependent, did not occur below 15°C, and was not inhibited by ouabain.

The virus‐induced increase in the uptake and release of 42K and of 3H deoxyglucose could not be mimicked by treatment of cells with linoleic acid, a procedure which increased the fluidity of the cellular membranes. The stimulatory effect of 0.5 mM ATP on the release of deoxyglucose was enhanced several fold in the presence of Sendai virus. These results seem to indicate the possible involvement of membranal enzymes such as e.g. protein kinase in the permeability changes induced by Sendai virus.
Electron probe analysis of cultured vascular smooth muscle
Authors: James‐Kracke M. R. , Sloane B. F. , Shuman H. , Karp R. , Somlyo A. P.
Published: February 2005

Mitochondrial and cytoplasmic composition were determined with electron probe analysis in freeze‐dried guinea pig aortic smooth muscle cells cultured on stainless steel grids. The mitochondrial calcium content in normal cells was low: not significantly different from that detected in the cytoplasm. Mitochondrial calcium granules were found in less than 3% of the cells, and in these the cytoplasmic K/Na ratio was reduced, indicating that they were damaged. There were no major differences between the cytoplasmic concentrations of K, Cl, Ca, Mg, and S of cultured cells and those previously found in adult vascular smooth muscle (Somlyo et al. '79). There was no evidence of nuclear Na or Ca sequestration in cultured cells, and the transmitochondrial Na, Cl, and K gradients were small.

Attempts to selectively remove adhering, extracellular ions with a 2‐second wash with isotonic ammonium acetate were unsuccessful because they were accompanied by loss of cell K.
Age‐related decrease in repair of oxidative damage to surface sulfhydryl groups on rat adipocytes
Authors: Hughes B. A. , Roth G. S. , Pitha J.
Published: February 2005

Adipocytes were isolated from the Wistar and Sprague‐Dawley strains of rats of different ages. An impermeable reagent, mercury‐[3H] dextran, was used to quantitate the sulfhydryl groups on the surface of these cells. The application of this reagent after the pre‐reduction of cells with 2‐mercaptoethanol yielded a measure of the total sulfhydryl and disulfide groups located there. Aging was found to significantly decrease the ratio of the number of exofacial sulfhydryl groups to the total number of exofacial sulfhydryl and disulfide groups.
Lack of selective killing by steroids in normal and malignant cells
Authors: Weichselbaum R. R. , Little J. B. , Nove J. , Hellman S. , Piro A. J.
Published: February 2005

Using a colony formation assay, the cytotoxic effects of steroids and an anit‐steroid on an established human breast tumor line and two human diploid fibroblast strains were studied. Experiments involving 17 a‐estradiol, 17 β‐estradiol, dexamethasone, cortisone, dihydrotestosterone, and the anti‐estrogen Tamoxifen showed no killing at concentrations below 10–7M following a 24‐hour exposure to these agents. A maximum of 80% killing was observed at 10–5M with dexamethasone in one fibroblast strain and at the same dose of 17 β‐estradiol in the breast tumor line. The extent of killing observed is insufficient to account for many of the clinical remissions observed with steroid therapy. The data also suggest that at therapeutic doses, there is no selective killing of malignant cells by these agents.
Membrane‐associated ribosomes in producing and nonproducing mouse myeloma cells
Authors: Cieplinski William , Scharff M. D.
Published: February 2005

Mouse myeloma cell lines synthesize large amounts of immunoglobulin on their membrane‐associated polyribosomes. Variants which no longer synthesize immunoglobulins have been studied and shown to have the same number of tightly bound membrane‐associated polyribosomes as the parental cell lines. These polyribosomes are still active in the synthesis of nonimmunoglobulin proteins.
Increased neurite development and plasminogen activator expression by exposure of human neuroblastoma cells to a plasminogen‐deficient growth medium
Authors: Becherer P. R. , Wachsman J. T.
Published: February 2005

Growth of human neuroblastoma strain SK‐N‐SH in a plasminogen‐deficient medium results in about a 40% increase in the number of differentiated cells (cells with a neurite‐like process at least 50 μm in length) and about a five‐fold increase in the amount of plasminogen activator liberated per cell. Plasminogen deficiency has no effect on the growth rate of SK‐N‐SH cells. These results are consistent with the hypothesis that plasminogen activator is involved in neuroblast development.