bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitogenesis in normal human fibroblasts by polyinosinic · polycytidylic acid and other synthetic acidic polymers: Enhancement of action by glucocorticoids
Authors: Barrack Evelyn R. , Hollenberg Morley D.
Published: February 2005

The ability of specific synthetic polyelectrolytes to act as mitogens for quiescent normal human fibroblasts in cultures is described. Of several acidic polymers tested, polyinosinic acid*polycytidyli acid (poly I *poly C) and dextran sulfate were the most effective in stimulating 3H‐thymidine incorporation (2‐ to 10‐fold). The concentration for a half‐maximal effect (ED50) was 0.4 μg/ml (0.8 nM) for poly I *poly C, and 1.7 μg/ml (3.4 nM) for dextran sulfate. Single‐stranded polyinosinic acid or polycytidylic acid had no effect. The time course of stimulation of DNA synthesis by these acidic polymers was similar to that for naturally occurring mitogens such as epidermal growth factor, beginning at about 18 hours and reaching a maximum rate 26 to 30 hours after the addition of polymer. Glucocorticoids that have an 11‐β hydroxyl group (e.g., dexamethasone) had no effect on DNA synthesis alone, but enhanced several‐fold the mitogenic activity of poly I * poly C or dextran sulfate; the ED50 for dexamethasone was 0.75 ng/ml (1.9 nM). Glucocorticoids with an 11‐keto group were inactive in this respect. The labeling index following treatment of cultures with poly I *poly C and dexamethasone was 14%, compared with a labeling index of 25% following stimulation by fetal calf serum. The extent of stimulation of DNA synthesis by poly I *poly C and dexamethasone was comparable to that induced by epidermal growth factor. It appears that both the poly I *poly C and dexamethasone are required for only a short period of time (approximately 3 hours) in order to produce maximal stimulation of DNA synthesis 30 hours later.
Transport of L‐alanine in cultured human fibroblasts: Evidence for two kinetically distinguishable systems
Authors: Feneant M. , Moatti N. , Lemonnier F. , Lemonnier A.
Published: February 2005

The transport of L‐alanine in human diploid fibroblasts was investigated. Transport measurements were performed on subcultures between the third and eighth passages with subconfluent cells growing on glass coverslips. Kinetic analysis of approximate initial rates of transport at substrate concentrations from 0.05 to 10 mmole/liter indicate the presence of two distinguishable systems. The high affinity system has a Km of 0.24 mmole/liter and a Vmax of 6.4 nmole/100 μg protein/2 min. For the low affinity system, the contribution of the high affinity system to the uptake must absolutely be taken into account. The Km and Vmax values, obtained by using a computer program, are a Km of 15.0 mmole/liter and a Vmax of 14.7 nmole/100 μg protein/2 min. For alanine concentrations below 1 mmole/liter, the contribution of the Na+‐independent uptake is less than 10%, and the kinetic constants of the high affinity system are in the same range if this contribution is taken into account. On the contrary the influence of a diffusion‐like process is more significant on the low affinity system whose Km is about 49 mmole/liter after subtraction of the Na+‐independent uptake from the experimental velocities. Inhibition studies were performed with NCH3‐alanine. They permitted us first to confirm the existence of system A in cultured human fibroblasts in agreement with two recent works and second to show how this system contributes to L‐alanine uptake. This contribution seems very small in low concentrations but it rises as the concentrations increase.
Clonal variation in cultured neuroblastoma cells. II. The relationship of increased intracellular cyclic amp content to increased anchorage requirement for growth and flattened morphology
Authors: Lanks Karl W. , Lombardo Joseph M.
Published: February 2005

Flat variant clones were isolated from both the N18 neuroblastoma cell line and from a subclone of the parent line exhibiting the typical round cell morphology. Several revertant clones exhibiting the round parental morphology were also isolated from one of the flat variant clones. The flat variants exhibit decreased cloning efficiency in suspension and increased amounts of myosin heavy chain when compared to the round cell clones. Intracellular cAMP levels were increased from two‐ to fivefold over those in clones representative of the parent line and in the round cell revertants. Treatment with the phosphodiesterase inhibitor RO20‐1724 increased cAMP levels and reduced suspension cloning efficiency without altering doubling time or attached cloning efficiency. Increasing cAMP levels of two of the round cell clones by treatment with the phosphodiesterase inhibitor caused increased flattening of the cell body and increased myosin heavy chain content. Thus, even though increased cAMP level may be sufficient to explain the reduced cloning efficiency of the flat variants, it is not the sole cause of the flat morphology.
Elemental content in the nucleus and in yolk and yolk‐free cytoplasm of developing Rana pipiens oocytes: An X‐ray microanalysis study
Authors: Labadie D. R. L. , Pool T. B. , Smith N. K. R. , Cameron I. L.
Published: February 2005

Energy dispersive x‐ray microanalysis measured the Na, K, Cl, P, Mg, S, and Ca contents (mM/kg dry weight) in the nucleus, yolk‐free cytoplasm, and yolk platelets of Rana pipiens oocytes quick frozen in the ovary. The data revealed that significant content changes occur in frog oocytes during intraovarian growth. All elements but Ca changed in content in the nucleus and cytoplasm, while in the yolk platelet only Na content did not change. A nucleus to cytoplasm K gradient develops and increases in magnitude during oocyte growth. The data from this and previous reports lead to the hypothesis that intra‐oocytic water and elements undergo changes in state during oocyte growth and that three subcellular Na compartments exist.
Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation
Authors: Morstyn George , Nicola Nicos A. , Metcalf Donald
Published: February 2005

More than 20% of human granulocyte‐macrophage and eosinophil colony‐forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental‐conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony‐forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence‐activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony‐forming cells. Two colony‐stimulating factors fractionated from HPLCM (CSFβ and CSFα) have been shown previously to stimulate the day 7 and day 14 colony‐forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte‐macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.
A granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) produced by carrageenin‐induced inflammatory cells of mice
Authors: Shikita M. , Tsuneoka K. , Hagiwara S. , Tsurufuji S.
Published: February 2005

Seven days after subcutaneous injection of 2% carrageenin solution in mice, the induced inflammatory tissue was isolated and treated with 0.1% trypsin. When the dispersed cells were incubated in a nutrient medium containing 5‐‐20% calf serum, the cells grew adhering to the culture‐dish surface and colony‐stimulating factor (CSF) was accumulated in the medium gradually. Addition of lipopolysaccharide (Escherichia coli endotoxin) in the cell culture did not enhance the CSF production. It was shown by isoelectrofocusing that the majority of the produced CSF was an acidic molecule (pI = 3.8), while the treatment of this CSF with neuraminidase yielded a less acidic CSF species (pI = 5.1). Upon gel‐filtration chromatography in the presence of 6 M guanidine HCl, the CSF exhibited an apparent molecular weight of 42,000. On the other hand, polyacrylamide gel‐electrophoresis in the presence of 0.1% sodium dodecyl sulfate gave a molecular weight estimate of 65,000. Microscopic examination of the bone marrow cell culture showed that about one‐third of the colonies were granulocytic. Addition of prostaglandin E1(PGE1) in the bone marrow cell culture significantly inhibited the action of the CSF, while prostaglandin D2 was less inhibitory than PGE1. The result suggests that the cells isolated from the inflammatory tissue are capable of producing CSF, which may have a role for proliferation and maturation of the mononuclear phagocytes at the site of inflammation.
The relationship of serum fibronectin and cell shape to thrombin‐induced inhibition of dna synthesis in human fibroblasts
Authors: Hall William M. , Ganguly Pankaj
Published: February 2005

In a previous report it was shown that inhibited DNA synthesis and altered morphology resulted when human fibroblasts (HF) were plated in 3% fetal calf serum (FCS) medium preincubated with thrombin (Hall and Ganguly, 1980a). This was in contrast to the stimulatory effects of this enzyme when added to cells several hours after subculture. Those observations suggested that thrombin may act upon serum components of the growth medium necessary for initial culture establishment following cell plating. In this report, the relationship of serum fibronectin (FN) to this thrombin‐mediated inhibitory phenomena was investigated. It was found that the development of altered morphology and inhibited DNA synthesis could be completely prevented by the addition of this glycoprotein to medium preincubated with thrombin. Cell shape and DNA synthesis appeared to be closely related and both parameters showed a dose‐dependent sensitivity to added fibronectin. To further investigate this, a technique was developed in which cell shape could be selectively varied and DNA synthesis measured in the absence of serum or thrombin. These studies indicated that cell shape was closely related to DNA synthesis and morphologies identical to that seen in thrombin‐treated medium were produced. As observed in the thrombin system, normal cellular appearance and DNA synthesis could be restored by the addition of fibronectin. The results of this work suggest that thrombin acts upon medium components necessary for normal morphological development, possibly fibronectin, in cells following subculture. Inhibited DNA synthesis and growth seem to arise as a direct consequence of this effect.
Multiple mechanisms of growth inhibition by cyclic AMP derivatives in rat GH1 pituitary cells: Isolation of an adenylate cyclase‐deficient variant
Authors: Martin Thomas F. J. , Ronning Susan A.
Published: February 2005

GH pituitary cells have been widely utilized for studies of hormone response mechanisms. Studies reported here were motivated by the desirability of isolating characterized GH clones defective in cyclic AMP synthesis or action. Spontaneously occurring GH1 cell variants resistant to the growthinhibitory effects of cyclic AMP analogs were isolated. Characterization of four variants showed that these were deficient in adenosine kinase and had acquired resistance to the cytotoxic effects of purine nucleoside derivatives formed in the culture medium. A second‐stage selection was undertaken with mutagenized adenosine kinase‐deficient cells. One 8 Br cAMP‐resistant variant was found to have normal cyclic AMP‐dependent protein kinase activity but exhibited altered adenylate cyclase activity. Activation of cyclase activity by fluoride, guanyl nucleotides, cholera toxin, and hormone (VIP) was subnormal in the variant. Mndependent cyclase activity was also subnormal, suggesting that the 8 Br cAMP‐resistant variant may have a deficiency in the catalytic moiety of adenylate cyclase.

Surprisingly, adenosine 3′ :5′ ‐monophosphate and 5′ ‐monophosphate derivatives were found to be equally potent in growth‐inhibiting adenosine kinasedeficient cells. Cross‐resistance to 8 Br AMP was observed in the 8 Br cAMP‐resistant variant. We conclude that cyclic AMP derivatives inhibit growth of GH cells by an unanticipated mechanism that is, nonetheless, related to endogenous cyclic AMP synthesis.
The influence of cellular proliferative history on the susceptibility to oncogenic transformation
Authors: Chan Gerald L. , Little John B.
Published: February 2005

C3H mouse 10T½ clone 8 cells were serially transferred from passage 11 to 15 with 5 × 104 cells seeded per 60‐mm dish at each passage. One group of cells was passaged as soon as confluence was reached. Two other groups were kept for 3 or 6 days in confluence at each passage before subculture. Cloning efficiency was found to increase progressively with passage of all three groups. At the 15th passage, cells from all three groups were harvested just prior to confluence, irradiated with ultraviolet light, and assayed for clonogenic survival and malignant transformation. Survival response was the same for all three groups, but cells which were kept constantly proliferating in previous passages were found to be much more susceptible to transformation. These results suggest that the susceptibility of these cells to transformation is influenced by their proliferative history; in particular, intermittent growth quiescence in previous passages decreased this susceptibility.
The effects of alterations in the external sodium concentration on human leucocyte sodium and potassium transport in vitro
Authors: Hilton P. J. , Johnson Valerie E. , Jones R. B. , Patrick J.
Published: February 2005

Human leucocytes incubated in tissue culture fluid of low‐sodium concentration (2 mM; iso‐osmolarity maintained with choline chloride) reached a new equlibrium within 1 hour and lost approximately 25% of intracellular potassium and 70% of intracellular sodium. The rate constant for ouabainsensitive sodium efflux fell by more than 50% and the ouabain‐insensitive rate constant increased nearly threefold in the low‐sodium medium. Total sodium efflux fell in proportion to internal sodium whereas ouabain‐insensitive sodium efflux remained unchanged. A reduction in external sodium from 140 to 2 mM was associated with a 75% fall in sodium influx. In the low‐sodium medium ouabainsensitive potassium influx exceeded ouabain‐sensitive sodium efflux and no ouabain‐sensitive potassium efflux could be demonstrated. Ouabain‐insensitive potassium influx and that portion of potassium efflux which is dependent on external potassium fell in parallel in low‐sodium cells, suggesting reduced activity of a ouabain‐insensitive K:K exchange system.
Uptake of putrescine by 3T3 and SV3T3 cells and its effect on ornithine decarboxylase activity
Authors: Bethell Delia R. , Pegg Anthony E.
Published: February 2005

The addition of 50 μM exogenous putrescine to Swiss 3T3 cells during serum stimulation resulted in a sixfold increase in intracellular putrescine content and prevented the rise in ornithine decarboxylase activity normally seen in response to serum. SV3T3 cells had putrescine levels ten times higher than those in the 3T3 cells. When SV3T3 cells were exposed to 50 μM putrescine, the intracellular content increased less than twofold. Despite the high levels of intracellular putrescine, ornithine decarboxylase was stimulated by addition of serum to SV3T3 cells. These results indicate that the system by which ornithine decarboxylase activity is regulated by its product is much less sensitive in the transformed SV3T3 cells. The ability of the 3T3 cells to take up putrescine changed with changes in the growth state, increasing when growth was stimulated by serum. These changes were manifested by changes in the maximum rate of transport (Vmax) rather than the Km. The increased transport rate was prevented when protein synthesis was inhibited by cycloheximide. The uptake of putrescine by SV3T3 cells did not change significantly under any of the conditions tested and the activity of this system in the SV3T3 cells was less than that of the 3T3 cells during exponential growth. This distinguishes the putrescine uptake system from those for amino acid uptake and suggests that the activity of the system may be regulated coordinately with the need for polyamines for cell growth. Finally, cadaverine (1,5‐diaminopentane), a diamine not usually present in fibroblasts, was taken up much more slowly than putrescine and uptake was not saturable under the conditions tested. The slower uptake may account for the lesser ability of cadaverine to reduce ornithine decarboxylase activity in the 3T3 cells.
Effect of dexamethasone, ammonium lons, and serum‐deprivation on intracellular proteolysis in cultured muscle cells
Authors: Mayer M. , Chaouat M. , Hadar R. , Nissan S. , Lernau O. Z.
Published: February 2005

Intracellular proteolysis was measured in primary cultures of newborn rat skeletal (gastrocnemius) and cardiac muscle cells by release to the medium of trichloroacetic acid‐soluble label from cells grown in the presence of 14C‐labeled phenylalanine. Exposure of the cultured cells to 10−7M dexamethasone for 5 days starting at day 0 of culture resulted in an enhancement of proteolysis in skeletal muscle but not in cardiac muscle cells. Dexamethasone did not affect cell viability measured by release of label from cells preloaded with Na2 51CrO4, release of creatine phosphokinase, and release of lactic dehydrogenase into the culture medium. Incorporation of 3H‐thymidine into the cells increased during the first 3 to 4 days of culture and subsequently decreased, indicating that cell proliferation ceases at that time. When the exposure to dexamethasone was started on day 4 of culture, i.e., at a postmitotic stage, and continued for 4 days, proteolysis was again found to increase in skeletal but not cardiac cells, thereby suggesting that the response to the hormone is independent of the proliferative state of the culture.

Ammonium chloride at a concentration of 10 mM produced a 50% reduction of the basal proteolysis in cultures of skeletal muscle cells and did not affect proteolysis in cardiac muscle cells. Exposure to ammonium chloride did not prevent the dexamethasone‐induced increase of proteolysis in skeletal muscle cells. Serum‐deprivation induced enhanced proteolysis which was not affected by NH4Cl in both cell types. It is concluded that the differential responses of the two cultures to dexamethasone reflects the sparing of heart proteins and concomitant wasting of skeletal muscle proteins by glucocorticoid hormones in vivo, and that the enhancement of proteolysis by the glucocorticoid hormone or by serum‐deprivation is not sensitive to the lysosomotropic agent NH4Cl. Thus, while a lysosomal‐autophagic enzyme system is responsible for almost half of the basal proteolysis, the accelerated proteolysis occurs via ammonium chloride‐insensitive enzymes.
The activation of cloned macrophages by concanavalin A for tumoricidal effect: Assessmetn of tumor cell cytotoxicity by a clonogenic assay
Authors: Yen Shing‐Erh , Thomasson David L. , Stewart Carleton C.
Published: February 2005

The activation of pure populations of cloned peritoneal macrophages for tumor cell cytotoxicity by Con A was demonstrated using a recently developed tumor cell clonogenic assay. Cloned macrophages were rendered cytotoxic by Con A at concentrations above 20 μg/ml. The tumor cell cytotoxicity was caused mainly by the tumoricidal activity of the Con A‐activated cloned macrophages. Increasing the Con A‐activation time from 24 hours to 48 hours and 72 hours heightened the cytotoxic activity of cloned macrophages. Cloned macrophages incubated with con A for only 2 hours possessed no cytotoxic effect. Culture fluid from cultures of activated macrophages exerted no tumor cell cytotoxicity. Alpha‐methyl‐D‐mannoside, a specific receptor‐binding inhibitor for Con A, was capable of blocking the activation of macrophages for cytotoxicity at 0.01 M concentration.
Effects of isolation and culture on prostaglandin synthesis by porcine aortic endothelial and smooth muscle cells
Authors: Ager Ann , Gordon John L. , Moncada Salvador , Pearson Jeremy D. , Salmon John A. , Trevethick Michael A.
Published: February 2005

Freshly isolated neonatal porcine aortic tissue (smooth muscle with or without endothelium present) produced ∼ 30 ng/mg wet tissue of 6‐oxo‐prostaglandin F1α (the stable hydrolysis product from prostacyclin) and ∼ 15 ng/mg of prostaglandin E2, as measured by radioimmunoassay after 24 h incubation in culture medium. Primary cultures of porcine endothelial and smooth muscle cells (isolated by enzymic digestion of aortic tissue) exhibited the same pattern of prostaglandin production, but absolute values were greater than for fresh tissue, particularly in the case of endothelium. Subcultures of endothelium produced smaller amounts of prostaglandins, although the pattern remained similar. In contrast, subcultures of smooth muscle cells produced a greater total amount of prostaglandins than did primary cultures, and the main product was prostaglandin E2. Experiments with [14C] prostaglandin H2 or [14C]arachidonic acid confirmed that aortic tissue, cultured endothelium, and primary cultures of aortic smooth muscle cells synthesized prostacyclin, and demonstrated that subcultured smooth muscle cells enzymically isomerised prostaglandin H2H to prostaglandin E2. Kinetic studies showed that prostaglandin production by cultured vascular cells was transiently increased by subculture or changing the growth medium, and that production per cell declined with increasing cell density. The change in pattern of prostaglandin production during culture was shown to be due to a rapid decline in the rate of prostacyclin production (which apparently began immediarely after tissue isolation), together with a more gradual rise in prostaglandin E2 production. These results indicate that the amounts and ratios of prostaglandins produced by vascular endothelial and smooth muscle cells are geatly affected by the conditions used to isolate and culture the cells; vascular cells in vivo may similarly alter their pattern of prostaglandin production in response to local cahnges in their environment.
Synthesis and distribution of cytoskeletal elements in endothelial cells as a function of cell growth and organization
Authors: Savion N. , Vlodavsky I. , Greenburg G. , Gospodarowicz D.
Published: February 2005

Confluent cultures of adult bovine aortic endothelial (ABAE), correal endothelial (BCE), and fetal bovine heart endothelial (FBHE) cells form a monolayer of highly flattened, closely apposed, and nonoverlapping cells. In ABAE and BCE cultures, this is associated with a 50‐fold decrease in the rate of DNA synthesis and correlates with a 14‐fold decrease in protein synthesis. In contrast, in confluent FBHE cultures only partial decreases in the rates of DNA synthesis (6‐fold) and protein synthesis (3‐fold) are observed. FBHE cells therefore fulfill the morphological, but not the biochemical, criteria for confluent cultured endothelial cell monolayers. The appearance of the cytoskeletal elements actin, tubulin, and vimentin in sparse and confluent cultures of endothelial cells has been analyzed by two‐dimensional gel electrophoresis and immunofluorescence. Sparse versus confluent ABAE, FBHE, and BCE cultures showed no changes in their relative rates of synthesis or cellular content of tubulin. Actin behaved similarly to tubulin in FBHE and BCE cultures, while in ABAE cultures a small increase (3‐fold) in its relative rate of synthesis was observed in confluent versus sparse cultures. BCE cultures showed no change in the rate of synthesis of vimentin, but the cellular content of vimentin was markedly increased when cultures reached confluence. When the distribution of vimentin in both sparse and confluent BCE cultures was analyzed by immunofluorescence, in both cases it appeared distributed throughout the cytoplasm as thin fibers and bundles of fibers. In confluent ABAE cultures, both the relative amount and biosynthetic rate of vimentin increased by 15‐fold. This increase in the intracellular accumulation of vimentin correlated with its immunofluorescent distribution within the cells. While in sparse cultures, vimentin appeared to be distributed as thin fibers, in confluent cultures thick curl‐like fibrous bundles could be seen distributed throughout the cytoplasm and organized in a perinuclear ring. In contrast, in FBHE cultures no significant changes in the distribution and organization of rate of synthesis of vimentin were observed.
Plasminogen activator: The major secreted neutral protease of cultured skeletal muscle cells
Authors: Festoff Barry W. , Patterson Michael R. , Romstedt Karl
Published: February 2005

Clonal mouse skeletal muscle cells which differentiate in culture and from synpases with neuronal cells were found to secrete high levels of protease activity as measured with an 125I‐fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n‐ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean typsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occured with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady‐state level of cell‐associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromusclar contacts in experimental and pathologic denervation.
Stimulation of the hexosemonophosphate‐pentose pathway by pyrroline‐5‐carboxylate in cultured cells
Authors: Phang James M. , Downing Sylvia J. , Yeh Grace Chao , Smith Robert J. , Williams Jeffery A. , Hagedorn Curt H.
Published: February 2005

Δ1‐Pyrroline‐5‐carboxylic acid, an intermediate in the interconversions of proline, ornithine, and glutamate, is a potent stimulator of glucose oxidation through the hexosemonophosphate‐pentose pathway. The effect is observed in cultured human fibroblasts, Chinese hamster ovary cells (CHO‐K1), and rabbit kidney cells (LLC‐RK1). In human fibroblasts, the magnitude of the stimulation of the hexosemonophosphate‐pentose pathway is dependent on the concentration of added pyrroline‐5‐carboxylate and the effect is observed over a wide range of glucose concentrations. The mechanism of the effect is related to the generation of oxidizing potential in the form of NADP+ by pyrroline‐5‐carboxylate reductase concomitant with the conversion of pyrroline‐5‐carboxylate to proline. In LLC‐RK1 cells, a cell line unique in having proline oxidase activity, proline also stimulated hexosemonophosphate‐pentose pathway activity. Although pyrroline‐5‐carboxylate markedly stimulated the hexosemonophosphate‐pentose pathway, it had no effect on glucose metabolism in the Embden‐Meyerhof pathway or the tricarboxylic acid cycle. Since the hexosemonophosphate‐pentose pathway is a source of ribose‐5‐phosphate, the precursor of phosphoribosyl pyrophosphate, the effect of pyrroline‐5‐carboxylate on the hexosemonophosphate‐pentose pathway may link amino acid and nucleic acid metabolism.
Protein synthesis in different populations of rat hepatocytes separated according to density
Authors: Smith‐Kielland Anne , Bengtsson Gunnar , Svendsen Lene , Mørland Jørg
Published: February 2005

Hepatocytes were isolated from fasted rats by a two‐step Ca++‐free/collagenase perfusion method. The cells were subjected to centrifugation under mild conditions at 12°C in a linear metrizamide gradient (1.075–1.12 gm/cm3). The cells were distributed in the gradient a bell‐shaped manner. According to their position in the gradient the cells were divided in five different population. The heaviest population was omitted from the subsequent evaluation because it contained a high proportion of dead cells. The activity of alanine aminotransferase increased with increasing cell density indicating that the lightest cell population was enriched in perivenous cells, whereas the heaviest cell population had an excess of periportal cells. Protein synthesis was more rapid in the light (perivenous) cell population than in the heavy (periportal) cell population as measured by means of incoporation of radioactively labeled valine into protein. The distribution measured in vitro indicated approximately 80% higher rates in perivenous cells. On the other hand, the synthesis and secretion of export proteins were similar in all cell populations regardless of their density. Protein degradation measured as appearance of free valine in cell media was higher in the light (perivenous) cell population than in the other populations. Thus protein metabolism seemed to be faster in the light cell population.
Retinoic acid‐promoted expansion of total cellular ATP pools in 3T3 cells can mediate its stimulatory and growth inhibitory effects
Authors: Rapaport Eliezer , Schroder Edward W. , Black Paul H.
Published: February 2005

Exposure of Swiss 3T3 cells to micromolar quantities of β‐all‐trans‐retinoic acid (RA) results in either inhibition of growth or stimulation of cellular responsiveness to mitogens, depending on the length of treatment. Inhibition of growth is produced by treatment of the cells with RA for at least 48 hours. The total cellular pools of adenosine 5′‐triphosphate (ATP) are markedly increased after 48‐hour RA treatment and dose dependence studies show a correlation between the expanded ATP pools and the inhibitory effects. The expansion of total cellular ATP pools by retinoic acid occurs throughout the cell cycle and parallels the cell cycle‐dependent fluctuations in total cellular ATP pools of untreated cells. Studies of [3H]thymidine incorporation and labeling indices in intact cells and [3H]dTTP incorporation and labeling indices in isolated nuclei of RA‐treated and control cultures suggest that cellular acid‐soluble nucleotide pools mediate the inhibition of DNA replication in the 48‐hour‐RA‐treated cells. The stimulatory activity of RA for mitogenic responsiveness is demonstrated by treatment of G0/G1‐arrested 3T3 cells with micromolar levels of RA for a maximum of 18 hours resulting in the potentiation of phorbol myristate acetate (PMA)‐stimulated transition into S phase of the cell cycle. Marked increases in total cellular ATP and UTP pools are produced by 18‐hour treatment of G0/G1‐arrested cells with RA, before their exposure to PMA.
Comparison of the effects of insulin and H2O2 on adipocyte glucose transport
Authors: Ciaraldi Theodore P. , Olefsky Jerrold M.
Published: February 2005

The abilities of insulin and the insulin mimickers spermine and H2O2 to stimulate 3‐O‐methyl glucose transport in isolated rat ft cells were stuided in an attempt to determine possible common mechanisms of action. All three agents caused a seven‐ to 12‐fold stimulation of initial rates of glucose transport with insulin being the most effective agent. Insulin and spermine displayed similar time courses for the onset of their stimulation of transport; an initial lag before any effect was seen and then a gradual rise until the full effect was reached. The time course of H2O2 activation of glucose transport was different since stimulation was seen at the earliest time point tested and then gradually rose to the maximal effect. Trypsinization of cells removed insulin receptors and rendered the cells insensitive to insulin but not to spermine or H2O2. However, trypsinization did alter the time course of H2O2 action, causing an initial lag phase to appear and a general slowing of the activation kinetics. Pretreatment of cells with 2,4‐dinitrophenol to lower ATP levels prevented the stimulatory effects of insulin and the mimickers. All three of the agents revealed a similar temperature dependency for stimulated glucose transport, resulting in linear Arrhenius plots with activation energies of 10.2–11.4 kcal/mole. These results show that (1) H2O2 does not act directly on the glucose transport system of rat adipocytes and (2) insulin and H2O most likely act through a common energy‐dependent biochemical pathway to stimulate glucose transport, but H2O2 enters the stimulus‐response sequence distal to the initial steps in insulin action.
Myeloperoxidase‐mediated oxidation of methionine
Authors: Tsan Min‐Fu
Published: February 2005

The myeloperoxidase‐mediated oxidation of methionine was studied using a purified canine myeloperoxidase preparation. The system required the simultaneous presence of myeloperoxidase, H2O2, and a halide anion. When 0.1 mM H2O2 was used, the system has a pH optimum of approximately pH 5–5.5. Bromide and iodide were much more effective than chloride in the myeloperoxidase‐mediated oxidation of methionine. Horseradish peroxidase was unable to oxidize methionine whether chloride or iodide was used. In contrast, lactoperoxidase oxidized methionine in the presence of iodide but not chloride.

Based on studies of (1) the effect of various inhibitors and singlet oxygen quenchers, as well as (2) the effect of D2O on the oxidation of methionine, by the myeloperoxidase system, OCl−, or methylene blue, it was shown that the oxidation of methionine by the myeloperoxidase system was not mediated by OCl− or 1O2. The mechanism of the myeloperoxidase‐mediated oxidation of methionine remains unclear. However, it may be one mechanism by which the myeloperoxidase system damage microorganisms.
Effect of glucose uptake on growth rate of mouse 3T3 cells
Authors: Romano Antonio H. , Connell Nancy D.
Published: February 2005

The objective of this investigation was to determine whether the rate of glucose uptake by mouse 3T3 cells was a primary determinant of growth rate. The experimental approach was to control the rate of glucose uptake into intracellular pools by supplying this sugar at varying concentration in minimal Eagle's medium with dialyzed serum in the absence and presence of 6‐deoxy‐D‐glucose, a metabolically inert homomorphic analog of D‐glucose that competitively inhibits the uptake of D‐glucose. Total hexose (D‐glucose and 6‐deoxy‐D‐glucose) concentration was maintained at the physiological concentration of 5.5 mM, in order to maintain saturation and maximum activity of the D‐glucose transport system; thus the flux of D‐glucose into the cell was controlled by adjusting its concentration relative to its competing nonmetabolizable analog. It was found that even when the concentration of D‐glucose was reduced to 0.7 mM, one eighth of the “normal” level of 5.5 mM. and 6‐deoxy‐D‐glucose was present in sevenfold excess (4.8 mM), conditions under which glucose uptake was reduced to 20% of that shown by cells in the presence of 5.5 mM D‐glucose, and intracellular pools of glucose and phosphorylated sugars derived from glucose were reduced to approximately 14% of normal, there was not a significant decrease in growth rate. These data support the view that the rate of glucose uptake is not a primary determinant of growth rate under the usual conditions of cell culture.
Regulation of growth of human diploid fibrobalasts by factors elaborated by activated lymphoid cells
Authors: Korotzer Terry L. , Page Roy C. , Granger Gale A. , Rabinovitch Peter S.
Published: February 2005

Fibroblast growth and synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin‐P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose‐dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80°C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (α‐LT). It was not cytotoxic, as shown by the 51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0 or G1 stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA Synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.
Dissociated occurrence of single‐gene mutation and oncogenic transformation in C3H 10T½ cells exposed to ultraviolet light and caffeine
Authors: Chan Gerald L. , Little John B.
Published: February 2005

We examined the relationship of cytotoxicity, mutagenesis, and malignant transformation by measuring in parallel clonogenic survival, mutation to ouabain resistance, and malignant transformation in cultured C3H mouse 10T 1/2 cells. Exposure of caffeine alone for 48 hours was cytotoxic and induced transformation in a dose‐dependent manner. However, this same treatment did not induce any detectable ouabain‐resistant mutants. When caffeine was present for 48 hours immediately following UV irradiation, alkaline sucrose gradient sedimentation of DNA showed that postreplication repair was inhibited. This inhibition of repair was correlated with reduced survival and inhibition of mutation induction, but the transformation frequencies were either unaltered or potentiated, depending on the UV dose and caffeine concentration. Thus, these experiments demonstrate that gene mutation and malignant transformation in 10T 1/2 cells can be dissociated. We suggest that the mechanism of transformation of 10T 1/2 cells is nonmutagenic in nature.
Effects of hormones and amino acid depletion on the kinetic parameters of amino acid uptake in monolayer cultures of rat hepatocytes
Authors: Kelley Darshan S. , Campbell H. A. , Potter Van R.
Published: February 2005

Uptake of 2‐aminoisobutyric acid (AIB) at concentrations of 0.1 mM to 30 mM was examined in sodium‐containing and sodium‐free media in hepatocytes pretreated without hormones (control), with hormones, or with amino acid depletion. Results show that 1‐minute but not 4‐minute rates can be taken as initial rates for the total or sodium‐dependent transport of AIB. The data for the 1‐minute sodium‐dependent transport of AIB were analyzed by a comptuer program and also by Eadie‐Hofstee and Lineweaver‐Burk plots, and a single saturable system was found. In the control cultures, the saturable system had a Km of 1‐2 mM AIB and a Vmax of 1.2 nmoles AIB/mg protein/minute. There was an increase in the Vmax of two to three‐fold after pretreating the cultures with insulin or amino acid depletion, three to four‐fold with glucagon, and six to seven fold with glucagon + dexamethasone.
Lysosomotropic agents and inhibitors of cellular transglutaminase stimulate dome formation, a differentiated characteristic of MDCK kidney epithelial cell cultures
Authors: Slaughter Robert S. , Smart Colleen E. , Wong Dai‐Shan , Lever Julia E.
Published: February 2005

Dome formation is a manifestation of transepithelial fluid transport in cell culture, a differentiated characteristic of transporting epithelia. A dramatic increase in numbers of domes in confluent MDCK kidney epithelial cell cultures was noted after addition of Friend cell inducers such as hexamethylane bisacetamide (HMBA) (Lever, 1979b). In the present study, we show that primary amines such as methylamine, ethylamine and dansyl cadaverine also stimulate dome formation. These compounds largely prevented the marked decrease in numbers of spontaneously occurring domes which occurred when cultures were switched from medium containing 10% serum to medium containing serum concentrations below 0.2%. Many of these primary amines are not only lysosomotropic agents but also potent inhibitors of transglutaminase activity when assayed in MDCK cell extracts, at concentrations correlating with those effective in stimulation of dome formation. Other lysosomotropic agents such as chloroquine and secondary and tertiary amines stimulated dome formation yet did not inhibit transglutaminase. Induction of domes by HMBA differed in several properties from that stimulated by amines and did not involve fluctuations in transglutaminase activity. These findings suggest that lysosomal functions modulate serum stimulation of dome formation in epithelial cells by a pathway distinct from that triggered by HMBA.
Volume regulation by human lymphocytes: Characterization of the ionic basis for regulatory volume decrease
Authors: Cheung Roy K. , Grinstein Sergio , Dosch Hans‐Michael , Gelfand Erwin W.
Published: February 2005

The mechanism of volume regulation in hypotonic media was analysed in human peripheral blood mononuclear (PBM) cells. Electronic cell sizing showed that hypotonic swelling is followed by a regulatory volume decrease (RVD) phase. This was confirmed by both electron microscopy and by cellular water determinations. The rate of regulatory shrinking was proportional to the degree of hypotonicity in the 0.5–0.9 X isotonic range. Cell viability was only marginally affected in this range. The content of cellular K+ decreased during RVD, while Na+ content remained unchanged. Similarly, the efflux of 86Rb (used as a K+ analog) increased upon dilution, whereas 22Na efflux was not altered. 86Rb uptake was enhanced by hypotonic stress and both ouabain‐sensitive and ‐insensitive components were affected. A ouabain‐sensitive stimulation was also seen in Na+‐ free media. Ouabain partially inhibited RVD only if added to the cells hours before hypotonic challenge. A normal shrinking response was observed in K+‐free media, and also in Na+‐free media when Li+, choline+, or Tris+ were the substitutes. In high K+ or Rb+ hypotonic media shrinking was absent and a second swelling phase was observed. Cs+ displayed an intermediate behavior, with shrinking observed at lower dilutions and secondary swelling at higher ones. The direction and magnitude of the response also changed when the external K+ concentration was varied and, with 50 mM K+, no regulatory volume change occurred following hypotonic stress. These findings suggest that RVD occurs largely by a passive loss of cellular K+, resulting from a selective increase in permeability to this ion. In addition, the (Na‐K) pump appears to be activated upon cell swelling by a mechanism other than Na+ entry into the cell, but this activation is not essential for RVD.
Effect of reduction of culture medium sodium, using different sodium chloride substitutes, on the proliferation of normal and rous sarcoma virus‐infected chicken fibroblasts
Authors: Balk Samuel D. , Polimeni Philip I.
Published: February 2005

We have substituted choline chloride, tetramethylammonium chloride, sucrose, or glucose for culture medium sodium chloride. When culture medium sodium is reduced below physiological levels (143 mM) by replacement of graded concentrations of sodium chloride with equivalent concentrations of choline chloride, normal fibroblasts approach proliferative inactivity in the presence of 90 mM Na, while their Rous sarcoma virus (RSV)‐infected counterparts proliferate actively; both normal and neoplastic cells die with further sodium reduction. When culture medium NaCl is replaced with tetramethylammonium chloride, however, both normal and RSV‐infected fibroblasts alike approach proliferative inactivity in the presence of 110 mM Na and both die off in the presence of 90 mM Na. When culture medium NaCl is replaced with sucrose or glucose yet another set of resuls is obtained: both normal and RSV‐infected fibroblasts proliferate at reduced, although significant, rates in the presence of 42 mM Na. It is clear from our experimental results that the effects of reduction of culture medium sodium on cell proliferation differ markedly with the use of different sodium chloride substitutes. Caution must be exercised, therefore, in drawing inferences concerning the role of sodium in mitogenesis from experimental studies based on the tactic of reduction of external sodium.
Effect of cocultivation of the levels of spontaneous and induced sister chromatid exchanges in chinese hamster cells
Authors: Rainaldi Giuseppe , Luccini Fulvio , Mariani Tullio
Published: February 2005

The level of sister chromatid exchanges, both spontaneous and induced by UV light or ethylmethanesulphonate (EMS), in Chinese hamster V79/AP4 cell line, is partially reduced upon cocultivation with human cells. Normal and xeroderma pigmentosum (XP) cells are equally effective. On the basis of these observations, cocultivation may represent a good tool for investigating the SCE's formation processes.
Multihormonal induction of α2u‐globulin in an established rat hepatoma cell line
Authors: Widman Lawrence E. , Chasin Lawrence A.
Published: February 2005

A subclone of the FU5‐5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein α2u‐globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 × 10–6 of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4–8 × 10–3. Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of α2u–globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum‐free medium raised the RSR to only 3 × 10–5; exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6–20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t½1/2 15 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 × 10–8 M and 3 × 10–11M, respectively. Higher concentrations of insulin, although still in physiological range (10–9M), inhibited induction. At yet higher insulin levels, beyond the physiological range, α2u‐globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for α2u‐globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with α2u‐globulin induction and for genetic approaches to the problem of multihormonal regulation of gene expression.