bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic heterogeneity of phosphoglyceride classes and subfractions during cell cleavage and early embryogenesis: Model for cell membrane biogenesis
Authors: Barassi Carlos A. , Bazán Nicolás G.
Published: February 2005

During the early developmental stages of the toad, Bufo arenarum, Hensel. up to the stage of gill circulation (150 hr of development at 20–25°C) the total phospholipids content as well as that of phosphoglycerides of choline and of ethanolamine were found unchanged. The subfraction of both phosphoglycerides were separated according to the number of double bonds on silver‐ion chromatography and were also found to be unchanged up to the tail bud stage. The distribution of non‐polar side chains in the subfractions varied in both phosphoglycerides showing a structural heterogeneity. In the phosphatidylethanolamines predominate the polyenoic containing subfractions.

In contrast with the constant concentration of polar lipids, during early embryogenesis a steady increase in 32P incorporation into phospholipids takes place when oocytes labeled during oogenesis are used. These changes were also correlated with the DNA content up to gill circulation stage.

It is proposed that most of the nascent membrane polar lipids during early embryogenesis may be derived from a storage site through an active and specific intracellular redistribution process. At the arrival of the polar lipid to the nascent membrane a change in their covalent structure by introduction of a phosphorylbase from a highly labeled pool may explain the raise in specific activity. This change may be necessary to make possible the assembly of the lipid into the membrane structure.
Further studies on the factor in lung‐conditioned medium stimulating granulocyte and monocyte colony formation in vitro
Authors: Sheridan J. W. , Metcalf D. , Stanley E. R.
Published: February 2005

The CSF's extracted from 6‐hour “in vivo” post‐endotoxin mouse lung tissue and 48‐hour “in vitro” lung‐conditioned medium appear to be glyco‐proteins, for like human urinary CSF they were inactivated by proteases and altered in charge but not inactivated by sialidase.

Neither endotoxin injection nor length of in vitro incubation significantly affected the apparent S20 W (approximately 2.OS) of the bulk of the CSF in mouse lung‐conditioned medium. The lower apparent molecular weight of CSF in mouse lung‐conditioned medium contrasted with that of monkey lung‐conditioned medium (S20 W approximately 3.3S). CSF's in both monkey and mouse lung‐conditioned media exhibited a much higher apparent MW on gel nitration than one would expect from their sedimentation behavior, a characteristic generally observed with most unpurified CSF preparations.

Mouse lung‐conditioned medium CSF was antigenically distinguishable from other CSF's. Failure to demonstrate this form of CSF in the tissue serum or urine of normal or endotoxin‐injected mice suggests that this type of CSF might normally lack in vivo significance.
Purified multiplication‐stimulating activity from rat liver cell conditioned medium: Comparison of biological activities with calf serum, insulin, and somatomedin
Authors: Smith Gary L. , Temin Howard M.
Published: February 2005

Multiplication‐stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum‐free medium conditioned by the growth of a line of rat liver cells (CRL), The biological activities of purified CRL MSA for chicken embryo fibroblasts were compared with those of calf serum to determine which activities are important for the stimulation of DNA synthesis and mitosis. In a balanced salt solution, only glucose and amino acids were needed in addition to purified CRL MSA to stimulate DNA synthesis maximally. Purified CRL MSA stimulated the rates of uptake of glucose and α‐aminoisobutyric acid. Only the stimulation of the rate of glucose uptake appeared to be a primary response to purified CRL MSA since the stimulation was not inhibited by actinomycin D or cycloheximide. The stimulation of the rate of uptake of α‐aminoisobutyric acid was inhibited by actinomycin D.

CRL MSA differed from calf serum in its inability to commit cells irreversibly to synthesize DNA after the removal of CRL MSA and in its lack of the ability to stimulate the migration or prolong the survival of chicken embryo fibroblasts.

Comparative studies indicated that purified CRL MSA had functional similarities to insulin and somatomedin. CRL MSA may be representative of a family of small polypeptide hormones having insulin‐like activity which are involved in the control of cell multiplication.
Adenosine diphosphate and calcium stimulation of respiration in mitochondria from alloxan diabetic rats
Authors: Garrick Rita Anne , Hall James C.
Published: February 2005

Liver mitochondria from normal and alloxan diabetic rats, isolated in 0.25 M sucrose, were assayed with an oxygen electrode for ADP/O and Ca+2/O ratios, respiratory ratio, and respiratory control index. Mitochondria were incubated with two substrates, succinate and β‐hydroxybutyrate; two types of ionic media, Na+ medium (Na+ the major monovalent cation) and K+ medium (K+ the major monovalent cation); and two respiratory stimulants, ADP (352 μM) and Ca+2 (187 μM). Significant differences between respiratory rates and ADP/O ratios were dependent upon the substrate and ionic medium employed. The results confirm previous studies which showed no alteration in ADP/O ratio but decreased State 3 respiratory rates under similar conditions of K+ medium with ADP stimulation in the diabetic. Furthermore, the State 3 respiration was prolonged compared to normal. Ca+2 stimulation was the same in normal and diabetic mitochondria in K+ medium. Studies in Na+ media revealed more significant differences in RCI's, respiratory rates, and ADP/O ratios that were substrate dependent as well as ion dependent. The results from these various studies can be accounted for by an hypothesis linking mitochondrial K+ interaction with alterations in the diabetic mitochondria.
Biochemical criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice. The production of acetylcholine esterase and creatine phosphokinase by teratoma cells
Authors: Levine Arnold J. , Torosian Michael , Sarokhan Alan J. , Teresky Angelika K.
Published: February 2005

When embryoid bodies are grown in suspension culture in vitro, they undergo only a limited amount of morphological development. When these same embryoid bodies are permitted to attach to the surface of a culture dish, a wide variety of new morphological cell types appear.

Suspension cultures of embryoid bodies do not contain significant detectable levels of acetylcholine esterase or creatine phosphokinase. These same enzymes however are produced in cell cultures derived from embryoid bodies attached to the culture dish surface. Polyacrylamide gel electrophoresis has been employed to demonstrate that the electrophoretic form of creatine phosphokinase produced by teratoma cells in culture is the brain form of the enzyme.

Solid transplantable tumors containing only embryonal carcinoma cells (stem cells) do not contain either of these enzymatic activities. Well differentiated transplantable teratomas contain both enzymes.
Morphological criteria for the in vitro differentiation of embryoid bodies produced by a transplantable teratoma of mice
Authors: Teresky Angelika K. , Marsden Margery , Kuff Edward L. , Levine Arnold J.
Published: February 2005

Embryoid bodies are produced when a transplantable testicular teratoma from strain 129 mice is serially passaged in the peritoneal cavity of these mice. These bodies are roughly spherical containing two morphologically distinct cell types. Scanning and transmission electron microscopy have been employed to show that the endodermal cells of embryoid bodies, like those of the mouse embryo, are on the outer surface and have highly convoluted surfaces containing numerous microvilli‐like projections. The inner, embryonal carcinoma cells, are the pluripotent stem cells of this tumor.

Intracisternal A‐type particles have been observed in electron micrographs and are almost exclusively located in the endodermal cells of the embryoid bodies. The A‐type complement‐fixing antigen has been identified in extracts prepared from this tumor.

When embryoid bodies are placed in culture and allowed to attach to the surface of a petri dish, a large number of new morphologically distinct cell types appear. Attachment to the petri dish surface is required for the generation of these new cell types. Cells of similar morphology in culture, display a distinctly “clonal” distribution on the petri dish surface.
Effect of hyperthermia on protein biosynthesis in L5178Y murine leukemic lymphoblasts
Authors: Fuhr J. E.
Published: February 2005

The effects of elevated temperatures upon protein biosynthesis were determined in L5178Y murine leukemic lymphoblasts. The rate of protein synthesis was inhibited proportionately to the increase in temperature. Efforts were made to determine the mechanism of heat inactivation of protein synthesis by studying the requirements for recovery of activity after the cells were returned to 37°C. The ability of actinomycin to block the recovery process suggests that elevated temperatures destroy or inactivate a species of RNA required for protein synthesis. Loss of RNA during heating of the cells is apparently at least partially dependent on protein synthesis, since the presence of cycloheximide during heat shock, is capable of ameliorating the effects of short duration heat treatment.
The transport of sulfate ions across the membrane of the ehrlich ascites tumor cell
Authors: Levinson Charles , Villereal Mitchel L.
Published: February 2005

The applicability of the membrane fixed charge hypothesis to anion transport in Ehrlich ascites tumor cells was studied by investigating the dependence of steady state sulfate transport on the extracellular pH, chloride and sulfate concentration. When the extracellular sulfate was maintained at 10 mM both cellular sulfate and sulfate transport increased with decreasing pH and chloride concentration. The dependence of sulfate transport on the cellular sulfate concentration suggests a saturation phenomenon.

The relationship between sulfate transport and cellular sulfate was also studied as a function of extracellular sulfate, both in the presence and absence of chloride. In both cases, sulfate transport is a saturable function of the cellular sulfate. However, in the presence of chloride the maximal flux is twice that in its absence. The discrepancy between the maximal fluxes suggests that the transport system mediates chloride‐sulfate exchange in addition to sulfate self exchange. Unidirectional sulfate effluxes into chloride and sulfate‐free medium; into 50 mM sulfate medium or 50 mM chloride medium were: 0.38, 1.95 and 3.91 nmoles/107 cells min‐1, respectively. These results indicate that in the absence of either sulfate or chloride the net efflux, of sulfate is low. However, chloride or sulfate on the trans side of the membrane is effective in accelerating unidirectional sulfate efflux. Taken together, the results of this investigation cannot be explained in terms of the membrane fixed charge hypothesis. Rather, they support the contention that sulfate transport across the tumor cell membrane is a carrier‐mediated process.
Phosphorylation but not transport of sugars is enhanced in virus‐transformed mouse 3T3 cells
Authors: Colby C. , Romano A. H.
Published: February 2005

The transport and phosphorylation of 2‐deoxy‐D‐glucose are separate and sequential events in both normal and virus‐transformed 3T3 cells. The apparent enhancement of 2‐dOG uptake by 3T3 cells accompanying virus transformation is not due to an effect on the transport process but to enhanced phosphorylation by intracellular kinases. Phosphorylation of 3‐O‐methyl‐D‐glucose does not occur in these cells. Both the rate and extent of transport of this glucose analog is the same in normal cells, SV40 virus‐transformed cells and sarcoma virus‐transformed cells. The appropriateness of using 3‐O‐MeG for studies of the glucose transport system of animal cells is examined and discussed.
Adaptation of potassium metabolism and restoration of mitosis during prolonged treatment of mouse lymphoblasts with ouabain
Authors: Cuff Joyce M. , Lichtman Marshall A.
Published: February 2005

The effects of ouabain on the growth of murine lymphoblasts in vitro have been studied. Exposure of cells to ouabain (0.1 mM) initially inhibited 86Rb+ uptake rate, reduced the intracellular potassium concentration, and decreased population growth rates. Continued exposure to the same ouabain concentration resulted in an increase of 86Rb+ uptake rate, intracellular potassium content and population growth rates to control values (adaptation). When treated cells were resuspended in medium free of ouabain after 12 to 15 hours of ouabain treatment, 86Rb+ uptake rates and intracellular potassium levels exceeded those of untreated cells. Adaptation was inhibited by cycloheximide (3 μg/ml) and by actinomycin D (0.05 μg/ml). Kinetic analysis of transport suggested that while the total capacity of the Na+, K+ transport system increased, the affinity for both the cation (86Rb+) and ouabain decreased.
The effect of potassium on the cell membrane potential and the passage of synchronized cells through the cell cycle
Authors: Stambrook Peter J. , Sach Howard G. , Ebert J. D.
Published: February 2005

The cell membrane potential of cultured Chinese hamster cells is known to increase at the start of the S phase. The putative role of the cell membrane potential as a regulator of cell proliferation was examined by following the cell cycle traverse of synchronized Chinese hamster cells in the presence or absense of high exogenous levels of potassium. An increase in external potassium levels results in a depressed membrane potential and a reduced rate of cell proliferation. A potassium concentration of 115 mM was used in experiments with synchronized cells since at that level cell proliferation is almost completely halted, recovery of growth is rapid and complete, and the membrane potential is reduced to a level well below that normally found in cells in the G1 phase.

A mitotic population was divided into four aliquots and plated in either control medium or medium containing 115 mM K+. Cells placed directly into high K+ medium were retarded in their exit from mitosis and displayed a delayed and abnormal entry into the S phase. If control medium was added after two hours, cell cycle traverse was normal, but delayed by two hours compared to control cells. If the mitotic cells were plated directly into control medium and two hours later were shifted to high K+ medium, the cells entered the S phase in the absence of the normally observed increase in membrane potential and proceeded to the next mitosis normally. It was concluded that the increase in membrane potential observed at the start of the S phase in isolated synchronized cells is not a requirement for the initiation of DNA synthesis. In addition, sensitivity to the high potassium regimen was found at two different times during the cell cycle. In one case, cells were impeded in their transit through mitosis. Such cells displayed an altered chromosome structure which may account for the partial mitotic block. In the second case, synchronized cells displayed a sensitivity to the high potassium regimen in early G1 which appeared to be separate from the block in mitosis and independent of a change in the membrane potential.
Dissociation by cytochalasin B of movement, DNA synthesis and transport in 3T3 cells
Authors: Brownstein Barbara L. , Rozengurt Enrique , de Asua Luis Jimenez , Stoker Michael
Published: February 2005

Cytochalasin B was used as a tool to study the inter‐relationships between cell movement, the reinitiated DNA synthesis and the enhanced transport of specific small molecules stimulated by serum in quiescent 3T3 cells. Cytochalasin at concentrations of less than 1 μg/ml inhibits serum‐stimulated movement within the monolayer and migration into a wound. Even at ten times this concentration there is little effect on the increase in DNA in the culture, indicating that movement away from neighboring cells is not required for the initiation of DNA synthesis.

While DNA synthesis is not inhibited by concentrations of cytochalasin up to 10 μg/ml, the increased thymidine transport which is associated with the onset of the S phase of the cell cycle is inhibited and DNA synthesis cannot be measured by the labelling of nuclei with radioactive thymidine.

Cytochalasin has a differential effect on the early transport changes produced by serum addition. Glucose transport is inhibited by low concentrations of the drug (< 1 μg/ml) while the enhanced uptake of phosphate and uridine is unaffected by a 10‐fold increase in concentration. Although the doses of cytochalasin required for 50% inhibition of hexose uptake and of cell movement are the same, no causal relationship between sugar transport and locomotion can be demonstrated.

Cytochalasin affects membrane functions in at least two different ways. The drug inhibits the uptake of glucose directly but affects only the S‐phase associated increase in thymidine transport.
Control of normal differentiation of myeloid leukemic cells. VI. Inhibition of cell multiplication and the formation of macrophages
Authors: Lotem Joseph , Sachs Leo
Published: February 2005

D+ but not D− myeloid leukemic cells can be induced by the appropriate conditioned medium or by serum from endotoxin treated mice, to undergo cell migration in agar, cell attachment to the surface of a Petri dish and differentiation to mature macrophages and granulocytes. Inhibition of cell multiplication by cytosine arabinoside, hydroxyurea, mitomycin C, thymidine, 5‐bromodeoxyuridine, 5‐iododeoxyuridine, 5‐fluorodeoxyuridine or actinomycin D, but not by vinblastine or cycloheximide, induced cell migration, cell attachment to the Petri dish and the formation of macrophages in D+ cells. There was no induction of cell migration or formation of macrophages and a much lower induction of cell attachment in D− cells. The induction of these changes in D+ cells required protein synthesis and the inhibitors showed the same toxicity for D+ and D− cells. The results indicate, that the inhibitors induced specific surface membrane changes in D+ but not in D− cells.
Somatic genetic analysis of cyclic AMP action: Characterization of unresponsive mutants
Authors: Bourne Henry R. , Coffino Philip , Tomkins Gordon M.
Published: February 2005

N6,O2′‐dibutyryl adenosine 3′,5′‐monophosphate kills cultured mouse lymphosarcoma cells, but not resistant mutants derived by a single‐step clonal selection. Resistant clones lack the cyclic AMP binding proteins present in wild type, cyclic AMP sensitive clones. Both endogenous cyclic AMP, accumulated in response to isoproterenol or cholera toxin, and exogenous dibutyryl cyclic AMP induce cyclic AMP phosphodiesterase, slow growth, and eventually kill wild type cells. In the resistant mutants, however, the endogenous and exogenous cyclic nucleotides appear to be completely inactive. These results indicate that an intracellular receptor for cyclic AMP, previously shown to be associated with a cyclic AMP‐dependent protein kinase, mediates cyclic AMP's regulation of growth and phosphodiesterase synthesis.
Difference in transport of leucine in attached and suspended 3T3 cells
Authors: Otsuka Haruki , Moskowitz Merwin
Published: February 2005

3T3 cells in subconfluent culture take up leucine through a transport system which has a relatively high affinity for leucine (M system). When the culture becomes confluent, the M system is turned off and leucine is transported by another system which has a low affinity for leucine (S system). The M system is reactivated by transferring the cells into subconfluent cultures.

In suspension cultures 3T3 cells, initiated from confluent cultures, the M system is not activated and leucine is transported by the S system. In cells suspended from subconfluent culture, the M system continues to operate at a high level for four hours and then is gradually turned off.

Tumor virus transformed 3T3 cells (SV3T3 and Py3T3) grow quite well in suspension culture and transport leucine both in monolayer and suspension through a high affinity system, with a high Vmax value. A derivative of 3T3, 3T3/41, which grows in suspension much more slowly than tumor virus transformed 3T3 cells, also takes up leucine through a high affinity transport system both in monolayer and suspension but its Vmax value is lower than that of the transformed cells.
The inhibition of DNA‐dependent RNA synthesis in yoshida ascites cells by bovine follicular fluid
Authors: Moore G. P. M. , Lintern‐Moore Sue , Peters Hannah , Byskov Anne Grete , Andersen Mette , Faber M.
Published: February 2005

Follicular fluid aspirated from large cow follicles inhibits endogenous, DNA‐dependent RNA polymerase activity in Yoshida ascites cells. The inhibitory component of follicular fluid is probably a protein and appears to affect specifically the activity of the nucleoplasmic polymerase II.
Early cellular responses to diverse growth stimuli independent of protein and RNA synthesis
Authors: Rubin H. , Koide T.
Published: February 2005

Serum, elevated pH, excess Zn++, 9,10 dimethyl‐1,2 dibenzanthracene (DMBA) and insulin accelerate the progress of growth‐inhibited chick embryo cells into the S‐period of DNA synthesis. A comparative study was made of their capacity to elicit other cellular responses within two hours after their application. All the agents studied stimulated the uptake of the glucose analogue 2‐deoxy‐D‐glucose (2‐dGlc). Elevated pH elicited a more striking increase than the other agents in the uptake of the amino acid analogue α‐amino isobutyric acid (AIB). The application of subtoxic concentrations of Zn++ or DMBA did not stimulate the uptake of uridine by cells nor its incorporation into RNA when tested at 2 hours. However, it was found that the stimulation of uridine utilization did occur but was delayed several hours. Similarly, the accelerated onset of DNA synthesis was also delayed for several hours by these agents. Insulin acted like serum in stimulating the utilization of 2‐dGlc, AIB and uridine. Serum and DMBA were particularly effective in stimulating the utilization of choline. It was concluded that the utilization of 2‐dGlc, uridine and thymidine are affected similarly by all the agents, but that there may be differential effects in the utilization of AIB and choline.

The inhibition of RNA synthesis by actinomycin D did not prevent the relative stimulation of 2‐dGlc, AIB and choline utilization by serum and pH treatment. The inhibition of protein synthesis by cycloheximide did not prevent the relative stimulation of 2‐dGlc and choline utilization by serum and pH treatment. It partially blocked the increased uptake of AIB and had erratic effects on the utilization of uridine. It was concluded that neither RNA nor protein synthesis is required for some, if not all, the early responses to growth stimuli measured here. The inhibited cell appears to be a poised system which carries out a programmed array of reactions characteristic of the cell type following perturbation by a variety of unrelated agents. In vivo specificity is provided by the physiological reagents available (i.e., hormones) and their capacity to interact with different cell types.
Effect of cell trypsinization on nuclear proteins of WI‐38 fibroblasts in culture
Authors: Maizel Abby , Nicolini Claudio , Baserga Renato
Published: February 2005

When resting confluent monolayers of WI‐38 fibroblasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non‐trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin‐associated proteins and most of them are non‐histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non‐histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.
The effects of metabolic inhibitors on the synthesis of inducible tyrosine aminotransferase in cultured hepatoma cells
Authors: Dethlefsen Lyle A.
Published: February 2005

The effects of actinomycin‐D and 3′‐deoxyadenosine (cordycepin) on the steroid‐mediated induction of tyrosine aminotransferase (TAT) synthesis have been reexamined in view of recent reports that the primary inhibitory action of these compounds may affect synthesis of proteins as well as RNA. The present results confirm that cordycepin blocks the steroid‐mediated induction of TAT in rat hepatoma cells (HTC), but unlike actinomycin‐D, cordycepin neither increases nor maintains the levels of TAT found in HTC cells preinduced with dexamethasone. Indeed, cordycepin added to preinduced cells, either in the presence or absence of steroid, causes a prompt decline in TAT activity. These data also confirm that both actinomycin‐D and cordycepin have an early inhibitory effect on protein synthesis, but the cordycepin effect is observed sooner and the extent of inhibition is greater. When actinomycin‐D and cordycepin are added simultaneously to preinduced cells with the steroid removed, the actinomycin‐D produced maintenance of preinduced levels of TAT persists. Also, the inhibition of protein synthesis in cultures with both inhibitors approaches that for the cells treated with actinomycin‐D alone instead of cordycepin alone. These data suggest that cordycepin inhibits TAT synthesis in preinduced cells by its inhibition of protein synthesis, and this inhibitory effect of cordycepin is blocked by actinomycin‐D. It is possible that actinomycin‐D does this by preventing the incorporation of cordycepin into RNA. However, regardless of the correctness of this speculation, the multiple effects of cordycepin indicate that this inhibitor cannot be used either to prove or rule out the post‐transcriptional model for regulation of gene expression. Also, this requirement that protein synthesis must continue in order to maintain pre‐induced levels of TAT is inconsistent with the assumption that the maintenance of these induced TAT levels by actinomycin‐D is due to inhibition of TAT degradation.
The role of adenosine 3′,5′‐monophosphate in the transformation of cloudman mouse melanoma cells
Authors: Lincoln Thomas M. , Vaughan Gerald L.
Published: February 2005

The role of adenosine 3′,5′‐monophosphate (cyclic AMP) in the regulation of mouse melanoma cell growth and differentiation was investigated. A variant melanoma (Cloudman S91‐F) which displays a greater degree of transformation than the parental cell (Cloudman S91) was isolated. A correlation between cyclic AMP metabolism and transformation was made. Dibutyryl cyclic AMP depressed cell growth and increased pigmentation in both parental and variant cell lines. The parental cell line, however, was more responsive to melanocyte‐stimulating hormone (MSH) which was found to affect cell growth and pigmentation by increasing cyclic AMP levels. The more transformed S91‐F cell line contained lower levels of cyclic AMP than the parental cell line, and this fact correlated well with the higher degree of growth and lesser degree of pigmentation in the variant. Enzymatic analysis revealed that the hydrolysis of cyclic AMP in both cell lines was similar, while the adenylate cyclase activity of the variant cell line was lower than that of the parental cell line. Lineweaver‐Burk plots demonstrated that the Km′s for the enzymes in the two cell lines were the same but that the Vmax of the S91‐F cell line was significantly less than that of the S91 cell line. Thus, the lesion in the S91‐F cell which is responsible for its more transformed characteristics seems to be one which affects adenylate cyclase at the level of the cell membrane.
Polysomes from cultured muscle cells: The cell‐free synthesis of myosin
Authors: Tepperman Katherine , Essien Francine , Heywood Stuart M.
Published: February 2005

The results reported here have shown that there are significant differences between polysome patterns obtained from cultured cells and from freshly isolated muscle tissue. Polysomes from embryonic homogenates show different patterns with different levels of myosin synthesis, but this does not appear to be the case with cultured cells. Experiments utilizing cell‐free protein synthesizing systems indicate that the polysomes isolated from myoblast cultures can synthesize myosin at levels similar to those obtained from myotube cultures, suggesting that the myoblasts contain significant amounts of the messenger RNA for myosin. In contrast, the polysomes isolated from BrdUrd‐inhibited cultures synthesize a comparatively low level of myosin. These findings illustrate a significant difference between myoblasts and BrdUrd‐inhibited cells.
Transformation of cells by rous sarcoma virus: Cytoplasmic vacuolization
Authors: Bader Artrice V. , Bader John P.
Published: February 2005

Chick embryo cells transformed by the Bryan “high titer” strain of Rous sarcoma virus (RSV‐BH) are heavily vacuolated. A variety of microscopic techniques have been used in demonstrating that the vacuoles are cytoplasmic, bounded by membrane, and are composed largely of water. Proteins, lipids, glycoproteins, glycolipids, glycosaminoglycans, glycogen, and nucleic acids were undetectable in the vacuoles.

Physiological requirements for development of the vacuoles, and reversal of vacuolization, were examined in cells infected with a virus mutant, RSV‐BH‐Ta, which induces reversible temperature‐dependent transformation. Na+ was the only component of the cell culture medium found essential for both the development and reversal of vacuoles. Glucose depletion or dinitrophenol treatment inhibited vacuolization, suggesting a possible energy requirement in the vacuolization process. Ouabain, an inhibitor of Na+−K+ ATPase, enhanced vacuolization, but a variety of other substances affecting cell surface components were inactive. Two sugars, glucosamine and mannosamine, prevented the disappearance of vacuoles.

The observations suggest that cellular vacuolization may be a normal physiological response to an increase in water and Na+, and, in the specific case of transformation by RSV‐BH, may be relevant to the physiological basis for malignancy.
Enhancement of phenotypic expression in cultured malignant liver epithelial cells by a complex medium
Authors: Martyn Gunn J. , Shinozuka Hisashi , Williams Gary M.
Published: February 2005

The ability of two media identically supplemented with serum to maintain functional and structural differentiated properties of cultured malignant liver epithelial cells was compared. Reuber H35 hepatoma cells grown in a complex medium, Medium E, possessed the gluconeogenic enzyme phosphoenolpyruvate carboxykinase and bile canaliculi. When grown in Eagle's Minimal Essential Medium, the activity of phosphoenolpyruvate carboxykinase was reduced and bile canaliculi were not formed. This decrease in properties was restored when cells maintained in Minimal Essential Medium were returned to Medium E. It was concluded that the enhanced phenotypic expression was probably due to enrichment of the nutrient environment.
Serum stimulation of Phosphate uptake into 3T3 cells
Authors: Hilborn David A.
Published: February 2005

The stimulation by calf serum of phosphate uptake into 3T3 cells results from a change in maximum velocity of the transport process with no change in the Michaelis constant. Only arsenate among a series of inorganic structural analogs of phosphate inhibited phosphate uptake indicating a high specificity for the process. The arsenate inhibition was competitive in nature. Papaverine, theophylline, and protaglandin E1, drugs known to maintain high intracellular levels of cAMP, had little effect on serum stimulated phosphate uptake. The phosphate uptake stimulating factor(s) in serum could be distinguished from the 3T3 cell survival and migration factors by stability characteristics, but this factor(s) could not be completely separated from a uridine uptake stimulation activity or growth promoting activity using a variety of serum fractionation procedures. Only partial stimulation of the uptake process was achieved with any one serum fraction indicating a multiplicity of serum components is probably involved in this process. Because of the rapidity of serum activation of phosphate uptake and its apparent independence of intracellular cyclic nucleotide levels, it is suggested that serum factors may stimulate phosphate uptake by inducing structural changes in the phosphate carrier system.
Relationship between membrane potential and external potassium in human glioblastoma cells in tissue culture
Authors: Manuelidis Laura , Manuelidis Elias E. , Prichard J.
Published: February 2005

Cells from a human glioblastoma (TC 526) maintained in tissue culture for ten years had a mean membrane potential of 27 ± 0.9 mV at an external potassium concentration [K0] of 5.3 mM. When [K0] was varied between 2.5 and 5.3 mM, membrane potential changes were close to those predicted by the Nernst equation. At higher [K0], the Nernstian slope was approached only in the presence of 10−5 M ouabain, which did not affect membrane potential at a [K0] of 5.3 mM. An electrogenic sodium pump activated by high [K0] could explain these findings; such a mechanism has been demonstrated in other tissues.
Reversible release of chick embryo fibroblast cultures from density dependent inhibition of growth
Authors: Hovi Tapani , Vaheri Antti
Published: February 2005

Initiation of proliferation in density‐inhibited chick embryo fibroblast cultures induced by insulin or trypsin was partially reversed by replacing the medium with supernatants from parallel non‐stimulated cultures. Growth stimulation by neuraminidase, pokeweed mitogen, bacterial lipo polysaccharide or purified tuberculin was less, or not at all, affected by this procedure. Medium change per se caused some proliferation in non‐stimulated cultures.

Increased rate of sugar uptake in insulin‐stimulated cultures returned to the level of that in non‐stimulated cultures within a few hours after medium change. This reversion took place apparently irrespective of the phase of the cell cycle. Replacing the medium with supernatants from non‐stimulated cultures induced a rapid decline in subsequent thymidine incorporation during the first S‐phase, and completely abolished the second peak of DNA synthesis. The fraction of cells irreversibly committed to mitosis increased when the time after stimulation increased. Less than three hours' incubation with insulin or trypsin was needed to initiate proliferation of a significant fraction of the cell population. It is concluded that reversion of the initiated cycle of a given cell is no more possible after the cell has entered the S‐phase.
Myonemal contraction of Spirostomum. III. The thermal dependence of contraction, relaxation and excitation‐contraction coupling
Authors: Hawkes R. B. , Holberton D. V.
Published: February 2005

A microphotometric technique that displays rapid length changes of Spirostomum has been used to follow the variation with temperature of three kinetic parameters of myonemal contraction: contraction rate, relaxation rate and stimulus duration at threshold. In each case the exponential form of the relationship indicated that the gross rate constant might be equated with the limiting rate constant, k, of a driving chemical reaction, and from standard expressions of chemical kinetics the change in activation free energy appropriate to this reaction has been computed.

The thermal dependence of contraction is described by an activation enthalpy (ΔLH‡) of 21.7 kcal mol−1, and the activation entropy (ΔLS‡) of 26.8 e.u. is consistent with a model of contraction requiring neutralization of fixed myonemal charges by divalent cations.

The analysis of thermal dependence of relaxation gives a negative activation entropy, a result predicted for a rate‐limiting reaction involving dissociation of a neutral molecule. On the other hand, values of ΔLS‡ and ΔLH‡ for relaxation fall close to an isokinetic correlation drawn in the literature from analysis of the thermal dependence of ciliary beat frequency in different organisms, and for which breakdown of an ATP‐ATPase complex could be the common rate‐limiting reaction.

ΔLS‡ for stimulus duration suggests that the rate‐limiting step in excitation‐contraction coupling is a reaction between ions of like charge, or ion pair formation from a neutral molecule.
Effect of exogenous atp on sodium transport in mammalian red cells
Authors: Romualdez A. , Volpi M. , Sha'afi R. I.
Published: February 2005

The effect of exogenous adenosine triphosphate (ATP) and other nucleotides on the transport of Na in various mammalian red cells has been studied. While they have no effect on the transport of Na in human and cat red cells, in dog red cells adenosine and its mono‐, di‐, and triphosphorylated forms were found to increase Na‐influx. Of these, ATP has the most striking effect, causing a more than 8‐fold increase at a concentration of 0.6 mM and exerting this effect at a dose range of 10−5 to 10−3 M. The effect of ATP is rapid (>5 minutes) and can be reversed by washing or the addition of calcium or magnesium. In contrast to the adenosine series other phosphorylated nucleotides (GTP, CTP, UDP, GDP and cAMP) have no effect. The well known volume dependent Na‐transport in these cells is reversed in the presence of 0.6 mM ATP. It is suggested that ATP acts on passive cation movements either by chelation of membrane charge of by a direct interaction with membrane proteins and may be involved in the volume regulation of cation transport in the dog erythrocyte.
The influence of dissolved oxygen on the mitogen responses of mouse lymphocytes
Authors: Kilburn D. G. , Morley M. , Yensen J.
Published: February 2005

Mouse spleen cells incubated at either 30 mm Hg dissolved oxygen partial pressure (pO2) or 160 mm Hg did not differ significantly in their survival or in their proliferative response to the mitogens concanavalin A, phytohemagglutinin or bacterial endotoxin in a subsequent static culture. If the mitogen was added prior to exposing the cells to the controlled dissolved oxygen environment the incorporation of thymidine was always substantially lower in cells held at 160 mm Hg pO2. This toxic effect of oxygen increased with the time of incubation and was not reversed by the inclusion of 2‐mercaptoethanol in the medium. Incubation of cells with mitogen in the absence of oxygen had no deleterious effect on the proliferative response. A comparison of DNA, RNA and protein synthesis in Con A stimulated cells indicated that the predominant effect of oxygen was on DNA synthesis.
Methionine metabolism in BHK cells: Selection and characterization of ethionine resistant clones
Authors: Caboche Michel
Published: February 2005

The selection of clones resistant to methionine antagonists was undertaken on baby hamster Kidney cells grown in a methionine free medium, supplemented with homocystine, folic acid and hydroxo‐B12. Clones resistant to 30 μg/ml ethionine were isolated after mutagenesis at an induced mutation frequency of 2.3 × 10−5. An ethionine resistant clone, ETH 304, was extensively studied. The resistant cells excreted methionine in the culture medium and the intracellular pools of methionine and SAM were two to five times greater in the resistant clone than in the wild type cells. A semidominant ethionine resistant phenotype was observed in hybrids between the wild type and this resistant clone.

Measurement of the specific activity of menadione reductase, B12 methyl‐transferase and ATP: L‐methionine S‐adenosyl‐transferase in crude extracts of the wild type showed a repressive action of methionine on the level of the three enzymes. However, the ethionine resistant clone ETH 304 was not modified in this function. Menadione reductase is feedback‐inhibited by SAM in wild type cells. The enzyme of the ethionine resistant clone was significantly less sensitive to SAM. When a comparison of thermal stability was made between the wild type and ethionine resistant clone enzymes, it was found that the thermal stability of the latter was modified. Three other ethionine resistant clones, independantly isolated, were similarly affected in the properties of menadione reductase.

These results suggest that the pathway of re‐use of S‐adenosyl homocysteine, produced during methylation reactions, is highly regulated by methionine and SAM.
A comparative study of superoxide dismutase activity in polymorphonuclear leukocytes, monocytes, and alveolar macrophages of the guinea pig
Authors: Rister Manfred , Baehner Robert L.
Published: February 2005

Superoxide dismutase, an enzyme which catalyzes the dismutation of superoxide radical formed during the univalent reduction of oxygen, was quantitated by observing the inhibition of cytochrome C reduction in three cell fractions in guinea pig peritoneal PMNs and monocytes and compared to alveolar macrophages. No differences were found in the 16,000 × g pellets containing mitochondria, membranes, and granules and representing 96% of total SOD activity in PMNs and monocytes but only 48% total SOD activity in alveolar macrophages. The 100,000 × g microsomal pellet of alveolar macrophages contained 8% of total SOD activity and two‐five times more activity than the respective fractions from monocytes and PMNs. However, there was 70 times more SOD in the 100,000 × g supernatant from alveolar macrophages containing 44% of total enzyme activity than in the same fraction of PMNs and monocytes containing less than 2% total SOD activity. SOD activity is mainly located in the 16,000 × g particulate fraction of PMN and monocytes but more equally distributed between the particulate fractions and cytosol of alveolar macrophages.
Flow microfluorometric analysis of sperm DNA content: Effect of cell shape on the fluorescence distribution
Authors: Gledhill Barton L. , Lake Suzanne , Steinmetz Lloyd L. , Gray Joe W. , Crawford James R. , Dean Phillip N. , Van Dilla Marvin A.
Published: February 2005

The DNA content of individual sperm from populations of acriflavine‐stained cells was investigated by analysis of fluorescence frequency distributions obtained with high‐resolution flow‐systems instruments. Sperm with spherical or cylindrical heads from three mollusk species produce narrow, symmetric fluorescence distributions. Flat sperm heads from six eutherian species produce asymmetric distributions consisting of a peak with a lateral extension to higher fluorescence values. The unexpected shape of these distributions was shown to be due to the flat geometry and high refractive index of the sperm heads in conjunction with the orthogonal axes of flow, excitation, and detection in the flow‐systems instruments. The theoretical and experimental results indicate that the lateral extension can be eliminated either by controlling the sperm orientation with planar flow conditions or by accounting for sperm orientation by means of orientation sensing.
Regulation of cellular growth by sodium pump activity
Authors: Shank Brenda Buckhold , Smith Nancy Egan
Published: February 2005

Cellular growth has been found to be directly related to the amount of sodium pumping activity in mouse lymphoblasts (L5178‐Y) cultured in varying concentrations of the cardiac glycoside, ouabain. No short‐term adaptation (within one generation) occurred; i.e., neither growth rate nor (Na+ + K+)‐ATPase activity increased in cells cultured for 1–2 days in ouabain.

Growth inhibition commenced after two hours, occurring concomitantly with decreased 3H‐leucine incorporation into protein. The time course of this inhibition of protein synthesis, measured by leucine incorporation was similar to, but slightly slower than the time course of the dissipation of the sodium gradient. On the other hand, 3H‐thymidine incorporation is unaffected by ouabain treatment over the same period. The uptake of 3H‐alanine, a neutral amino acid thought to be transported via a Na+‐dependent carrier, was depressed concurrently with the sodium gradient dissipation. It is suggested, therefore, that ouabain inhibition of cellular growth results primarily from the dissipation of the sodium gradient leading to decreased Na+‐dependent transport of amino acids (e.g., alanine) and, therefore, decreased protein synthesis, as observed by leucine incorporation.

A sensitive and rapid method for determining ouabain inhibition of cell volume regulation is also described, which may prove potentially useful for assaying Na pump activity.
Pleiotropic phenotype of colchicine‐resistant CHO cells: Cross‐resistance and collateral sensitivity
Authors: Bech‐Hansen N. T. , Till J. E. , Ling V.
Published: February 2005

Colchicine resistant (CHR) mutants of CHO cells with reduced permeability to colchicine display extensive cross‐resistance to a number of apparently unrelated compounds including puromycin, daunomycin, emetine, ethidium bromide and gramicidin D. A positive correlation was observed between the level of cross‐resistance and the relative hydrophobicity of these compounds. The mutants also showed increased (collateral) sensitivity to local anaesthetics (procaine, tetracaine, xylocaine and propanolol), steroid hormones (1‐dehydrotestosterone, corticosterone and 5β‐pregnan‐3,20‐dione) and some Triton × compounds. In general, the degree of the pleiotropic response (cross‐resistance or collateral sensitivity) correlated with the degree of colchicine resistance in mutant lines. These results are consistent with the pleiotropic phenotype being the result of the same mutation(s) which confer colchicine resistance and support a model for resistance in which the reduced permeability is assumed to be the result of an alteration in the modulation of the fluidity of the surface membrane.
Steroids and hematopoiesis I. The effect of steroids on in vitro erythroid colony growth: Structure/activity relationships
Authors: Singer Jack W. , Samuels Alan I. , Adamson John W.
Published: February 2005

When substituted steroids of several classes are added to cultures of rat bone marrow cells in the presence of erythropoietin a consistent enhancement of the number of colonies of hemoglobin synthesizing cells is obtained. Maximum steroid effectiveness was found to be between 10−6 and 10−7 M. Representative compounds of several classes of steroids were examined for their ability to enhance colony growth, including δ 4‐estrenes, δ 4‐androstenes, 5α‐H androstanes and estranes, 5β‐H estranes, pregnanes and androstanes. While testosterone and its 5α‐H derivatives had little or no activity, many synthetic derivatives of testosterone were highly active in increasing erythroid colony growth. All 5β‐H androstanes, estranes, and all but one 5β‐H pregnane were active. Cortisol consistently inhibited colony growth and estradiol and progesterone had no significant effect.