bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractFreshly 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

AbstractConfluent 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

AbstractClonal 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

AbstractΔ1‐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

AbstractHepatocytes 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

AbstractExposure 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

AbstractThe 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

AbstractThe 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

AbstractThe 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

AbstractFibroblast 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

AbstractWe 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

AbstractUptake 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

AbstractDome 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

AbstractThe 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.