bluuebunny/crossref_metadata_2025 huggingface dataset:
Incorporation of fucose and glucosamine into cell bound and medium released macromolecules
Authors: Herrmann Heinz , Havaranis Amalia S. , Doetschman Thomas C.
Published: February 2005

(1) Determinations were carried out on the incorporation of fucose‐6‐[3H] and glucosamine‐6‐ [3H] into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released than into the cell bound macromolecules. (3) The incorporation per microgram DNA increases during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not decrease the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10–30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.
Myonemal contraction of spirostomum. II. Some mechanical properties of the contractile apparatus
Authors: Hawkes R. B. , Holberton D. V.
Published: February 2005

In several respects, notably the high velocity of shortening, Ca2+ dependence, and ATP independence, contraction of Spirostomum resembles the spasmonemal mechanism of the peritrich ciliates. In this report further mechanical properties of the contractile apparatus are described that extend this comparison. The velocity‐load characteristic is more appropriate to an elastomer than to a muscle where contraction force is load‐dependent. Active tension is found to relate linearly to cell length for extensions up to and beyond resting length (lr), an elastic limit is reached around 1.5 lr. At resting length this tension, measured by the deformation of a glass microbalance, is similar to that predicted from consideration of the hydrodynamic forces normally resisting shortening. The tension‐length relation for the unstimulated (passive) cell is also linear between lr and the elastic limit, but is displaced from the active tension‐length curve and is of reduced stiffness. Kinetic studies suggest that maximum tension and maximum velocity coincide. Calculations are presented that support a model of contraction in Spirostomum in which the myonemes behave as a mechanochemical engine powered directly by the chemical potential of Ca2+.
The relation of endogenous adenosine cyclic 3′:5′‐monophosphate to the antagonistic effects of adenosine and colchicine on cell shape
Authors: Yin H. H. , Berlin R. D.
Published: February 2005

Adenosine and colchicine have antagonistic effects on cell shape. When Chinese Hamster lung fibroblasts (CHE36‐6) or SV40 transformed 3T3 (SV3T3) cells are incubated with colchicine (1 μM) for one hour at 37°C, they round up into spheres with short spikes. Cells treated with adenosine (1 μM–4 mM) for one hour become refractile and develop spindly processes. However, when the two compounds are added simultaneously, the characteristic responses to either drug are abolished and the cells appear normal. The counteraction is specific for adenine and its derivatives, adenosine being the most effective of the compounds we tested. Accumulation of colchicine or adenosine is not altered significantly by the presence of the other drug, ruling out decreases in uptake as the basis of the mutual antagonism. The morphological changes can be observed under conditions where there are no changes in intracellular cAMP levels (such as incubation with low concentrations of adenosine or cordycepin, an adenosine analog that cannot be directly converted to cAMP). Colchicine does not alter cAMP content of control or adenosine‐treated cells. These data show that adenine compounds have potent effects on cell shape, and the antagonistic effects of adenosine and colchicine on cell shape are not mediated through changes in intracellular cAMP levels.
Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts
Authors: Fodge D. W. , Rubin H.
Published: February 2005

The multiplication rate of sparse cultures of chick embryo cells is only slightly lower at pH 6.9 than at pH 7.4. There is, however, a marked reduction in the multiplication rate of the pH 6.9 cultures before they reach confluency. Cultures at pH 7.4 continue to multiply beyond confluency with only a slight decrease in the multiplication rate.

Eighty to ninety percent of the glucose taken up by the cells growing at each pH is converted to lactic acid which is released into the medium. Metabolic reduction in pH of the medium is almost entirely accounted for by the amount of lactic acid produced by the cells. Neither the intracellular nor extracellular accumulation of lactic acid nor the accompanying reduction in pH is sufficient to explain density dependent inhibition of the rate of multiplication of chick cells.

The rate of lactic acid production and the multiplication rate of chick cells are independent of glucose concentration in the range of 2–16 mM. In view of the kinetic parameters for the uptake of glucose, this shows that glycolysis is not limited by the rate of glucose uptake and that depletion of glucose from the medium cannot account for the onset of density dependent inhibition of multiplication. However, when cells reach very high population densities, conventional glucose concentrations of 5 mM can be depleted overnight by chick cells. Since the multiplication rate of cells is dependent on glucose concentration when it falls below 2 mM, depletion of glucose may cause some growth inhibition in crowded cultures supplied with conventional medium.
Transient erythropoietic spleen colonies: Effects of erythropoietin in normal and genetically anemic W/Wv mice
Authors: Gregory C. J. , McCulloch E. A. , Till J. E.
Published: February 2005

Properties of the cells (TE‐CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin‐dependent, (2) the population size of TE‐CFU is not erythropoietin‐dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE‐CFU, (4) successful transplantation of TE‐CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE‐CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE‐CFU assay detects a class of early erythropoietin‐sensitive progenitor cells committed to erythropoietic differentiation, rather than “abortive” colony formation by pluripotent stem cells.
Studies on the regulation of the three enzymes of the leloir pathway in cultured mammalian cells II. A Search for quantitative interrelationships between the three enzymes activities
Authors: Stern Elizabeth Sell , Krooth Robert S.
Published: February 2005

Studies on a normal human diploid cell strain revealed that the specific activity of the cell protein, for each of the three enzymes of the Leloir pathway, changed significantly as the cells grew. The kinetics of change in specific activity varied according to the enzyme being studied, and the kinetics for each enzyme varied from experiment to experiment.

Within each experiment, there was no consistent correlation between specific activity for any one enzyme and specific activity for the other two. The ratios between the specific activities did not tend to remain constant as the absolute levels of specific activity changed. Hence, the activities did not behave coordinately. The kinetics of change in these ratios varied from experiment to experiment.

The failure of galactose to stimulate increased cellular activity for the three enzymes (shown in the preceding paper), and the absence of a coordinate relationship between the activities, represent a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.
Effects of metabolites present during growth of Tetrahymena pyriformis on the subsequent secretion of lysosomal hydrolases
Authors: Blum J. J.
Published: February 2005

Tetrahymena were grown in proteose‐peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non‐nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, β‐N‐acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that α‐mannosidase, β‐fucosidase, and β‐galactosidase are secreted into the salt solution and that secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for α‐mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.
Interaction of the fluorescent probe, 1‐anilino‐8‐napthalene sulfonate, with the sulfate transport system of ehrlich ascites tumor cells
Authors: Levinson Charles , Villereal Mitchel L.
Published: February 2005

The addition of the fluorescent dye, ANS, to intact ascites tumor cells results in an enhancement of fluorescence intensity. The increase in fluorescence intensity as a function of time is biphasic which suggests that at least two processes occur. The first associated with the rapid initial rise in fluorescence represents binding to the cell surface while the second or slower phase reflects entrance of ANS into the intracellular phase. The relationship between bound and free ANS in 0.50 mM sulfate medium was used to calculated the apparent dissociation constant of ANS‐membrane complex (Kd = 6.53 × 10−5 M) and the total number of ANS binding sites (4.49 nmoles/mg dry weight).

Kinetic analysis of steady state sulfate transport in the presence and absence of ANS suggests that (1) sulfate exchange can be described by Michaelis Menten type kinetics (Km = 2.05 × 10−3 M), (2) a small fraction of surface associated ANS competitively inhibits sulfate exchange (Ki = 4.28 × 10−6 M) and (3) the transport system has a higher affinity for ANS than for sulfate.

These data are consistent with the hypothesis that inhibition of sulfate exchange is related to the direct, reversible interaction of the negatively charged sulfonate group of ANS with superficial positively charged membrane sites.
ITP pyrophosphohydrolase and idp phosphohydrolase in rat tissue
Authors: Vanderheiden Bernardo S.
Published: February 2005

The existence of a nucleoside triphosphate pyrophosphohydrolase specific for ITP has been demonstrated in the cytosol fraction of a variety of rat tissues. The enzyme, stable to moderate heat treatment, was present in erythrocytes as well as brain, heart, kidney, liver, lung, muscle, ovaries, spleen, testes and thymus. The specific activity of the enzyme ranges from 26 to 150 m̈moles/min/g protein. In addition, evidence is given for a heat labile nucleoside diphosphate (IDP) phosphohydrolase present in most rat tissues, and particularly high in the adrenal (137 m̈moles/min/g protein). An “ITP‐IMP cycle” is proposed as a regulating mechanism for intracellular levels of ATP.
Concurrent development of resistance to 6‐azauridine and adenosine in a mouse cell line
Authors: Hashmi S. , May S. R. , Krooth R. S. , Miller O. J.
Published: February 2005

A variant of the hypoxanthine‐guanine phosphoribosyltransferase deficient, and adenine phosphoribosyltransferase deficient mouse A9 cell line has been obtained by selecting cells which are resistant to 6‐azauridine. These cells are not only resistant to 6‐azauridine (5 × 10−4 M), but also to adenosine (10−3 M). Resistance persists indefinitely even in the absence of both compounds. The resistant cells are killed by 5‐fluorouridine (10−6 M), indicating that the part of the salvage pathway for pyrimidine ribonucleotide biosynthesis which is relevant to the action of 6‐azauridine is intact. The heritable change producing concurrent resistance to 6‐azauridine and adenosine probably involves the de novo pyrimidine biosynthetic pathway.
Hemopoietic colony forming cells in regenerating mouse liver
Authors: Hays Esther F. , Firkin Frank C. , Koga Yoshiko , Hays Daniel M.
Published: February 2005

Cells from regenerating mouse liver removed 2–25 days post 68% hepatic resection have been assayed for in vitro colony forming capacity in soft agar (CFU‐C), proliferative capacity in liquid culture, and in vivo spleen colony forming capacity (CFU‐S).

These studies demonstrated low concentrations of CFU‐C and CFU‐S in normal and sham‐operated liver, with an appreciable increase of both in regenerating liver, reaching maximum values in tissue removed 5–7 days post hepatic resection. Colony formation in agar by regenerating liver cells occurred in the absence of exogenous colony stimulating factor. Separation of liver cells on the basis of adherent properties prior to culture indicated concentration of CFU‐C in the nonadherent fraction, while cells producing colony stimulating factor were concentrated in the adherent fraction. Foci of actively dividing cells of the macrophage and granulocyte series arose in liquid culture from preparations of sham operated and regenerating liver, although total cell formation was greater with regenerating liver. A small proportion of the colonies formed in agar from regenerating liver consisted of cords of epithelioid cells, which resembled hepatocytes and differed from the macrophages or granulocytes found in the majority of colonies, raising the possibility that regenerating hepatocytes form colonies in agar culture.
A comparative study of the effects of lucanthone (miracil D) and actinomycin d on the chinese hamster cells grown in cultures
Authors: Epifanova Olga I. , Makarova G. F. , Abuladze M. K.
Published: February 2005

Chinese hamster cells of an established clone line grown in monolayers were incubated for up to two hours with either lucanthone (0.3–30 m̈g/ ml) or actinomycin D (0.06–0.10 m̈g/ml) and subjected to radioautographic investigations with 3H‐uridine during the period of treatment.

At concentration of 9 m̈g/ml lucanthone selectively inhibited the synthesis of nucleolar (ribosomal) RNA while the extranucleolar RNA synthesis proceeded at a high level. Similar results were obtained with 0.08 m̈g/ml actinomycin D. Protein synthesis and mitotic activity were also affected by lucanthone but the drug did not markedly interfere with DNA synthesis.

Lucanthone appeared to be much less effective in cell killing than actinomycin D and its inhibitory effects on the nucleolar RNA synthesis and other cellular processes proved readily reversible. The results allow to conclude that lucanthone may be useful as a tool for studying RNA synthesis in animal cells.
The stimulation of 2‐deoxy‐D‐glucose transport in control and virus‐transformed cells by ethidium bromide
Authors: Soslau Gerald , Nass Margit M. K.
Published: February 2005

Recently we demonstrated that ethidium bromide altered the plasma and subcellular membrane glycoproteins in control and virus transformed cells. It is reported here that ethidium bromide also stimulated the membrane associated process of sugar transport. The Km of the virus transformed cells and the ethidium bromide treated cells is the same as that of the control cells while the maximum velocity as compared to the control cells is significantly increased. The transport of 2‐deoxy‐D‐glucose was inhibited by glucose, cytochalasin B and neuraminidase but was unaffected by variations in cell density or pH of the incubation medium.
N6‐(Δ2‐Isopentenyl)adenosine, an inhibitor of cellular transport of uridine and cytidine
Authors: Hakala Maire T. , Slocum Harry K. , Gryko George J.
Published: February 2005

N6(Δ2‐Isopentenyl)adenosine (IPAR) inhibited severely the incorporation of uridine and cytidine into S‐180 cells in culture. When IPAR and the nucleosides were simultaneously present in the medium the inhibition was competitive (Ki 3.4 m̈M) and indicated inhibition of transport. However, the inhibition occurred even in the absence of extracellular IPAR if the cells had been preincubated with IPAR. Since 5′‐IPAMP was the product which accumulated in large quantities in S‐180 cells when incubated with IPAR, the effects of this AMP analog on the intracellular metabolism of uridine had to be considered. No direct correlation between the amount of intracellular IPAMP and the degree of inhibition of uridine utilization was observed and the relative distribution of uridine nucleotides in the acid soluble pool of the cells was unaltered in cells treated with IPAR. Also, IPAMP was not an inhibitor of uridine kinase in a cell free system nor was the activity of this enzyme affected by treatment of cells with IPAR. In addition, a profound inhibition of uridine utilization was also observed in a resistant subline of S‐180 cells, which is unable to form IPAMP. These data suggest that IPAMP was not the inhibitory agent. Furthermore, the observation that the inhibition in both sensitive and resistant cells was caused even by a 15‐second exposure to 100 m̈M IPAR, followed by rinsing, suggests that IPAR itself is the effective agent.

It is concluded that IPAR exerts its inhibitory effect on uridine and cytidine utilization by becoming lodged in the cell membrane and thereby preventing the passage of these nucleosides into the cells. It is also shown that the inhibition of uridine and cytidine utilization by IPAR and by other potent nucleoside uptake inhibitors is unrelated to inhibition of growth or of RNA‐synthesis when the cells do not depend on an extracellular source of a nucleoside for growth.
DNA replication in homogenates of Physarum polycephalum
Authors: Brewer E. N. , Ting Pauline
Published: February 2005

Homogenates of Physarum polycephalum incorporate [3H] dATP into nuclear DNA at an initial rate of approximately 15% of the in vivo rate. To attain this level of synthesis, cultures are homogenized in a medium containing Mg++, EGTA, glucose and spermine. Incorporation is strongly stimulated by the addition of ATP and all four deoxyribonucleoside triphosphates to homogenates prior to incubation. Various inorganic cations other than Mg++ either do not affect synthesis or are inhibitory. Incorporation is inhibited by a nonionic detergent, Triton X‐100. DNA synthesis in this cell‐free nuclear system is similar in several respects to that which occurs in vivo: (1) The rate of DNA synthesis in the intact organism at a given time in the mitotic cycle is reflected by the level of synthesis in homogenates prepared from cultures at that time of the cycle; (2) DNA strands labeled in vitro exhibit alkaline sucrose density gradient sedimentation properties similar to those of daughterstrand DNA pulse‐labeled in vivo; and (3) Homogenates of cultures which were pre‐treated with cycloheximide incorporate [3H]dATP at about 60% of the level observed in homogenates of untreated controls.
Development of excitability in embryonic chick skeletal muscle cells
Authors: Kano Masaakira
Published: February 2005

During embryonic and early postnatal development, the chick leg muscle cells undergo a series of changes in their electrical responses in the following sequence: passive response, plateau response, plateau plus spike response and spike response. This suggests that the electrogenetic mechanism of muscles matures during development; a mechanism producing the plateau may first be induced, and then that producing the spike. The plateau is sensitive to manganese or cobalt ions, while the spike to tetrodotoxin. This suggests that the plateau is related to the increase in permeability to calcium ions, while the spike to sodium ions.
Evidence contrary to the protein error hypothesis for in vitro senescence
Authors: Pendergrass William R. , Martin George M. , Bornstein Paul
Published: February 2005

A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucose‐6‐phosphate dehydrogenase (G‐6‐PD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 ± 1%, S.E.), in comparison with that in 19 old cultures (4.9 ± 1%, S.E.). Old cultures had ceased active cell division (49–60 doublings); DNA replication, measured by [3H] thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5–22 doublings) had a [3H] thymidine labeling index of 75–85%. Titration of G‐6‐PD activity in extracts of young and old cells with neutralizing antibody directed specifically against G‐6‐PD failed to detect an increment of enzymatically defective G‐6‐PD in old cells. The thermostability studies were capable of detecting altered G‐6‐PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G‐6‐PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G‐6‐PD from the thermolabile mutant and G‐6‐PD inactivated with N‐ethylmaleimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.
Control of phenylalanine hydroxylase synthesis in tissue culture by serum and insulin
Authors: Tourian Ara
Published: February 2005

Stationary‐phase, minimal deviation hepatoma H4‐II‐E‐C3 cell cultures that are serum‐deprived respond with a biphasic time course of phenyl‐alanine hydroxylase induction when dialyzed fetal calf serum or insulin is added. These two agents induce phenylalanine hydroxylase additively, during both the initial 3‐hour and the delayed 24‐hour phases. The initial phase of induction by insulin is inhibited by cycloheximide but not by actinomycin D. The delayed induction by both dialyzed fetal calf serum and insulin is inhibited by 10−6 M cycloheximide and 0.20 μg/ml actinomycin D.

H4‐II‐E‐C3 cells in culture do not synthesize the factor(s) in serum that induce phenylalanine hydroxylase.
Calcium‐dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor‐promoting phorbol ester (PMA)
Authors: Boynton A. L. , Whitfield J. F. , Isaacs R. J.
Published: February 2005

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing‐medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1–6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 μg/ml of PMA (12‐O‐tetradecanoyl‐phorbol‐13‐acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium‐free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density‐dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the “cycling” state caused by exposure to fresh serum‐containing medium.
Lipid A, the active part of bacterial endotoxins in inducing serum colony stimulating activity and proliferation of splenic granulocyte/macrophage progenitor cells
Authors: Apte Ron N. , Pluznik Dov H. , Galanos Chris
Published: February 2005

An analysis of which component of lipopolysaccharides (LPS), the lipid or the poly saccharide (PS), is active in stimulating the murine granulopoietic system has been performed. LPS with different structures, isolated from different mutant strains of Salmonella and chemical degradation products of lipopolysaccharides have been used. Lipid A obtained by acid hydrolysis of the LPS and complexed to bovine serum albumin (BSA) (lipid A‐BSA) was shown to be active in generating serum colony stimulating factor (CSF) and in increasing the splenic colony forming cells (CFC) levels, although it was less active than the parent LPS. The polysaccharide (PS) showed no significant activity at the concentrations used. LPS (glycolipids) from R mutants of Salmonella minnesota were active to the same extent as the LPS. The fact that even the most defective LPS from the R mutant R595 which contains lipid A and KDO only is a potent endotoxin, points unequivocally, to lipid A, as the active principle in stimulating the granulopoietic system.
Glucose‐6‐Phosphate dehydrogenase activity in a hepatoma cell line: Preliminary evidence for negative genetic control
Authors: De Luca C. , Matheisz J. S.
Published: February 2005

A line of cells, positive for glucose‐6‐phosphate dehydrogenase activity, has been separated from a negative parental stock derived from the H4‐II‐E‐C3 rat hepatoma line. Cytogenetic analyses showed specific marker chromosomes of the parent line were present in the variant. On the other hand, cells of the variant line contained three chromosomes less, on the average, than the total present in parent cells. Karyotypic analyses indicated the diminution in chromosome numbers to be in the subacrocentric group. The appearance of glucose‐6‐phosphate dehydrogenase associated with loss of chromosomes suggests this enzyme is suppressed or under negative control in the parental stock. The consistent loss of chromosomes of the subacrocentric group suggests that the locus bearing regulatory activity is in this specific group.
Regulation of translation in rabbit reticulocytes and mouse L‐cells; comparison of the effects of temperature
Authors: Craig Nessly
Published: February 2005

Various parameters of protein synthesis were analyzed in rabbit reticulocytes exposed to various temperatures for up to five hours. Between 10°C and 40°C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10–24°C; 22 kcal/mole, 24–40°C), as did protein elongation and release (35 kcal/mole, 10–25°C; 12 kcal/mole, 25–40°C). However, the level of polysomes remained essentially unchanged between 0°C and 42°C which implies that the activation energy for polypeptide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that in cultured mouse L‐cells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L‐cells appear to be similar in their temperature dependence of initiation and in their rate of elongation (5–6 amino acids/second at 36°C).
Arrest of 3T3 cells in G1 phase in suspension culture
Authors: Otsuka Haruki , Moskowitz Merwin
Published: February 2005

3T3 cells do not grow in Methocel suspension culture, while other permanent cell lines do. The viability of 3T3 cells in suspension remains unchanged for at least three days with respect to plating efficiency, vital staining and resumption of normal growth when transferred into monolayer culture. When monolayer 3T3 cells in G1 phase are suspended they remain in G1 phase. Cells already in S phase which are suspended complete ongoing DNA synthesis and mitosis and then are arrested in the G1 phase. Progress through the cell cycle is reinitiated after suspended cells attach to a surface. When monolayer cells in late G1 phase (just before entering S phase) are put in suspension cultures they do not initiate DNA synthesis.
Growth of cultured cells from patients with Fanconi anemia
Authors: Elmore Eugene , Swift Michael
Published: February 2005

Fibroblast cultures derived from skin biopsies of patients with Fanconi anemia had doubling times (mean of five lines: 30.3 ± 0.2 hours) significantly longer than randomly selected normal controls (mean of nine lines: 22.9 ± 0.4 hours). Control cultures grew more slowly in the enriched media RPMI 1640 and McCoy's 5A than in MEM, while a culture from a patient with Fanconi anemia grew more slowly only in McCoy's 5A. Differences in growth characteristics between Fanconi anemia and normal cell cultures may be useful in analyzing the metabolic error determined by the Fanconi anemia gene.
The effect of pH on incorporation of galactose by a normal human cell line and cell lines from patients with defective galactose metabolism
Authors: Hill Helene Z.
Published: February 2005

Incorporation of radioactive galactose into TCA‐insoluble material of galactosemic fibroblasts is more sensitive to low pH than is the incorporation by normal human fibroblasts. This study was undertaken to determine (1) whether there was any pH which could correct or counteract the galactosemic defect relative to galactose incorporation, and (2) whether the low pH effect was specific for galactose metabolism or whether general cellular metabolism in galactosemic cells was more sensitive to low pH than that in normal cells.

The pH dependencies of incorporation of radioactive galactose and glucose into cellular macromolecules were investigated in galactosemic and normal cells. Normal cells have a biphasic curve with respect to galactose incorporation with peaks at pH 7.0 and 8.5. Galactosemic cells have only the high pH peak. The maximum incorporation by galactosemic cells was never more than about 30% that seen by normal cells under the conditions of these experiments. Thus manipulation of the pH alone cannot correct the galactosemic defect.

The rate of incorporation of radioactive galactose was studied in normal, galactosemic and galactokinase deficient cells, at pH 7.2 and at pH 6.3. At pH 7.2, galactosemic cells incorporate galactose at a linear rate which is 30 to 40% that of normal cells while incorporation by kinase‐deficient cells is between 5 and 10% of normal. At pH 6.3, the incorporation is also linear. However, galactosemic cells now exhibit the same rate as kinase‐deficient cells in which the low level of incorporation is unaffected by pH.

These results suggest that incorporation of galactose by galactosemic cells at low pH is not due to metabolic death of the cells, but may be due to the inhibition of some specific step or steps along a metabolic route of galactose metabolism other than the Leloir pathway.
Human lymphocyte potassium content during the initiation of phytohemagglutinin‐induced mitogenesis
Authors: Segel George B. , Lichtman Marshall A. , Hollander Marsha M. , Gordon Bruce R. , Klemperer Martin R.
Published: February 2005

The K+ content of human lymphocytes has been examined during the initial 24 hours after exposure of cells to phytohemagglutinin (PHA). We have reconfirmed that lymphocyte K+ exchanges rapidly for extracellular counterions during preparative washing if cells are exposed to PHA. By using a technique to measure cation content which does not require removal of cells from their culture medium, we have shown that K+ does not change for 24 hours following PHA treatment. Previous reports have demonstrated that an enhanced uptake of K+ occurs in lymphocytes treated with PHA. This increased uptake may be a compensatory change for an increased exodus, explaining the failure of K+ to change following lectin treatment.
Induction by short‐chain fatty acids of alkaline phosphatase activity in cultured mammalian cells
Authors: Koyama Hideki , Ono Tetsuo
Published: February 2005

Short‐chain fatty acids, such as propionic, n‐butyric, n‐valeric, isovaleric, n‐caproic, and n‐caprylic acids, induce alkaline phosphatase activity in cultured mammalian cells. Long‐chain fatty acids have no similar effects. With B‐6 cells (mouse × Chinese hamster cell hybrids), n‐butyrate at 2 to 5 mM exhibits the greatest activity. Induction begins exponentially about 24 hours after addition of the fatty acid and continues over 48 hours. Studies on the inducing activity‐structure relationship revealed the necessity of a carboxyl and an ethyl or longer alkyl group.

n‐Butyrate shows a marked synergistic action of induction when added along with other types of inducers: adenosine 3′:5′‐cyclic monophosphate (cAMP) or 5‐bromodeoxyuridine (BrdU). Treatment of other cell lines with either n‐butyrate, cAMP, or BrdU revealed a cell‐type specific response pattern of alkaline phosphatase. The biological significance of this effect of short‐chain fatty acids is discussed.

This study was presented in part at the 45th Annual Meeting of the Japanese Biochemical Society, Tokyo, October, 1972. After submission of this manuscript, the authors noticed a similar study on alkaline phosphatase induction by n‐butyrate, reported by M. J. Griffin et al. ('74) in Arch. Biochem. Biophys., 164: 619–623.
SV40 transformation of mouse brain cells: Critical role of gene A in maintenance of the transformed phenotype
Authors: Anderson Jeffrey L. , Martin Robert G.
Published: February 2005

Brain cells derived from the NIH Swiss mouse strain have been established in tissue culture. Astrocytic neuroglial cells, identified by morphology and staining properties, predominate. The brain cell culture was successfully transformed with SV40 wild type virus and with a representative early (A239) and late (C219) mutant. When subjected to growth analysis the A239 transformant displayed selective loss of six characteristics of the transformed phenotype at the restrictive temperature (40.5°C): doubling time, saturation density, ability to grow in low serum, efficiency of growth on plastic and on normal brain cell layers, and cloning in soft agar. Temperature shift experiments demonstrated the reversibility of the differences in saturation density. T‐antigen was expressed at both temperatures. Alteration in uptake of 2‐deoxyglucose was not a characteristic of the transformed phenotype. The cell lines may be of utility in brain culture work and in studies on the mechanism of SV40 transformation.
Triiodothyronine effects on some electrogenic properties of frog sartorius
Authors: Grossie J.
Published: February 2005

At 21 °C in vitro, 0.2 and 2.0 m̈M of triiodothyronine (T3) produced an increase in resting membrane potential (RMP) of Rana pipiens sartorius when the pH of the external solution was 7.4. The RMP was increased by 2.0 m̈M T3 in the presence of 10−4 and 10−3 M ouabain but not in 10−3 M of 2, 4 dinitrophenol. Small increases in RMP were observed with 2.0 m̈M T3 in solutions with low external Na. At pH 7.1 0.2 m̈M T3 produced a small transient increase in RMP. Membrane resistance (Rm) was found to decline gradually during exposure to 0.2 m̈M at a pH of 7.4. Treatment with 2.0 m̈M T3 at pH 7.4 was accompanied by a transient reduction in Rm. Similar transient changes in Rm were produced by 0.2 and 2.0 m̈M T3 at pH of 7.1. T3 also reduced membrane resistance in isotonic K2SO4 and tris‐buffered Mn (20 mM) solutions indicating that T3 increases potassium permeability. Direct action potentials were studied at pH 7.1. Overshoot, amplitude and rate of rise of the action potential underwent a gradual decrease in the presence of 0.2 m̈M T3 while thresholds remained unchanged. Thresholds were increased during exposure to 2.0 m̈M T3 whereas overshoot, amplitude and rate of rise underwent transient decreases followed by a return toward control levels.
Purine and phosphoribosylpyrophosphate synthesis in differentiating murine virus‐induced erythroleukemic cells in vitro
Authors: Reem Gabrielle H. , Friend Charlotte
Published: February 2005

Phosphoribosylpyrophosphate synthetase activity was determined in Friend virus‐inducted erythroleukemic cells in culture, stimulated to differentiate in the presence of dimethylsulfoxide. The activity of phosphoribosylpyrophosphate synthetase did not decrease in cells which had acquired the specialized function of hemoglobin synthesis, nor was the phosphoribosylpyrophosphate content of untreated erythroleukemic cells significantly different from that of cultures exposed to dimethylsulfoxide for 96 hours.

However, the rate of the early steps of de novo purine biosynthesis as measured by the incorporation of [1‐14C] glycine and [1‐14C] formate into formyglycinamide ribonucleotide, was significantly lower in differentiating cell cultures. The addition of glutamine or ammonia increased glycine incorporation of control cultures, but failed to do so in treated cultures. In the course of the normal development of erythrocytes in vivo, phosphoribosylpyrophosphate synthetase activity is preserved, while the capacity to synthesize purines de novo is lost, as is the activity of the phosphoribosyl‐l‐amine synthesizing enzymes. Our present study suggests that the rate of de novo purine biosynthesis in this erythroleukemic cell line is not limited by the availability of phosphoribosylpyrophosphate, but rather by a decrease in the phosphoribosyl‐l‐amine synthesizing enzymes. These findings provide further evidence that during dimethyl‐sulfoxide‐stimulated erythroid maturation, the same regulatory mechanisms are operative as in normal cellular development, and that ammonia‐dependent purine biosynthesis is subject to the same regulatory mechanisms as is glutamine‐dependent biosynthesis.
Wasting of 18 S ribosomal RNA by human myeloma cells cultured in adenosine
Authors: Bynum John W. , Volkin Elliot
Published: February 2005

When human myeloma cells are pulsed for one hour with 3H‐uridine and chased for six hours in fresh medium containing unlabeled uridine, the processing of 45 S rRNA precursor into the stable 28 S and 18 S rRNA components can be followed. However, when the cells are chased in exogenous adenosine instead of uridine, the accumulation of 18 S rRNA is selectively inhibited. Cells pulsed with 3H‐adenosine and chased in the absence of exogenous nucleosides exhibit normal rRNA precursor processing, while cells pulsed simultaneously with 3H‐uridine and 3H‐adenosine and chased with uridine and adenosine are deficient in labeled 18 S rRNA. Consequently, the inhibition of 18 S rRNA accumulation by adenosine is not an artifact of labeling nor is it relieved by an equal molar concentration of uridine. The wasting of 18 S rRNA in human myeloma cells is similar to that reported to occur in normal lymphocytes during the quiescent state.
Stimulation of ornithine decarboxylase activity in chick fibroblasts by non‐suppressible insulin‐like activity (NSILA), insulin and serum
Authors: Haselbacher Gisela K. , Humbel René E.
Published: February 2005

A factor isolated from human serum (nonsuppressible insulin‐like activity, NSILA) stimulates multiplication of serum‐starved chick embryo fibroblasts and stimulates activity of ornithine decarboxylase (ODC). Physiological doses of NSILA (200 m̈ U/ml) and pharmacological doses of insulin (200 m U/ml) stimulate ODC 4–5‐fold, 10% fetal calf serum about 18‐fold. Combined addition of NSILA and insulin does not result in higher activities, suggesting a common mechanism of action. The increase in cell number obtained with NSILA, insulin or serum parallels the degree of ODC stimulation. Treatment of cells with pronase also stimulates ODC activity.

A sharp increase in ODC activity occurs between 2.5 and 5.0 hours after addition of the growth factors with a peak at 4.0–4.5 hours (“activation period”). As cells leave G1 phase, ODC activity decreases rapidly. To achieve maximal activity of ODC, the growth factors have to be present during the entire “activation period.” The potential to reactivate ODC decreases as cells pass through S phase.

Results obtained using cycloheximide suggest that ODC is translated only in the second half of the “activation period.” Data on effects of dbcAMP and dbcGMP on ODC activation by serum are discussed.
Effect of serum on the growth of balb 3T3 A31 mouse fibroblasts and an SV40‐transformed derivative
Authors: Bartholomew James C. , Yokota Hisao , Ross Priscilla
Published: February 2005

The effect of serum on the growth properties of non‐transformed Balb 3T3 A31 and SV40‐transformed Balb 3T3 A31 was studied. The concentration of serum in the growth medium of non‐transformed cells had little effect on the initial population doubling time, but did regulate the cell density at which the population became quiescent in G1. The doubling time of transformed cells, however, was increased significantly as the concentration of serum was decreased below 4%. This effect on the growth of transformed cells was seen at serum concentrations so low that non‐transformed cells did not complete one population doubling. Flow microfluorometric analysis of these populations indicated that the primary effect of different serum concentrations on the non‐transformed cells was to modulate the average residence time in G1; whereas, all the cell cycle phases of the transformed cells were affected by serum. At saturation densities, the non‐transformed cells became quiescent in G1, but the transformed cells still traversed the cell cycle and their saturation density appeared to be a balance between cell production and cell death occurring primarily in the G1 phase of the cell cycle.
Separation of megakaryocytes from rat bone marrow cells using velocity sedimentation in an isokinetic gradient of ficoll in tissue culture medium
Authors: Pretlow Thomas G. , Stinson Anthony J.
Published: February 2005

A technique for the purification of rat megakaryocytes is described. Velocity sedimentation in a previously described isokinetic gradient of Ficoll (polysucrose) in tissue culture medium was more effective than isopycnic sedimentation for the purification of megakaryocytes and resulted in preparations of megakaryocytes which contained 2.4 ± 0.8% (range 1.85–3.60%) megakaryocytes. Megakaryocytes exhibited a broad range of density between 1.06 and 1.15 gm/ml. The inaccuracy which is inherent in the use of velocity sedimentation without isopycnic sedimentation as a means of particle size analysis is discussed.
Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine‐guanine phosphoribosyltransferase gene
Authors: Graf L. H. , McRoberts J. A. , Harrison T. M. , Martin D. W.
Published: February 2005

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6‐mercaptopurine (6‐MP) or 6‐thioguanine (6‐ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine‐guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities.

The cells from one of these clones, 1020/12, possess 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotitration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme, PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild‐type cells.

The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP “sparing” effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction.

We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.
Density‐dependent regulation of proliferation rate in cultured, androgen‐responsive, tumour cells
Authors: Robinson Jeffery H. , Smith James A.
Published: February 2005

Short‐term cultures of androgen‐responsive Shionogi 115 (S115) cells exhibited density‐dependent regulation of proliferation rate in the presence or absence of testosterone. The average surface area per cell exposed to the growth medium was inversely proportional to population density. By contrast, long‐term cultures (serially passaged in testosterone‐containing medium for several months) did not exhibit density‐dependent regulation of proliferation rate when grown in testosterone‐containing medium. In this medium, cells became elongated and no longer exhibited any obvious decrease in exposed surface area with increasing density. Nevertheless, when subcultured into testosterone‐free medium, these cells reverted to an epithelial morphology and exhibited density‐dependent regulation of proliferation rate. These relationships suggested that the proliferation rate of cells decreased with density in proportion to the decrease in exposed surface area.

Activity of the “A” neutral amino acid transport system decreased with density in proportion to the decrease in proliferation rate whereas activities of the “L” system and sugar transport system(s) did not. Treatment of cells with testosterone for 24 hours increased proliferation rate but did not change activity of the “A” system, indicating that activity of the “A” system changes with density but not with proliferation rate per se. It is suggested that a decrease in exposed surface area of cells produces the change in activity of the “A” system, possibly by changes in activity of the sodium pump.