bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells
Authors: Hyslop Paul A. , Hinshaw Daniel B. , Schraufstätter Ingrid U. , Sklar Larry A. , Spragg Roger G. , Cochrane Charles G. 
Published: February 2005

AbstractThe effects of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]i) was investigated utilizing the intracellular fluorescent calcium chelator “Quin 2.” [Ca++]i rose from approximately 150 nM to >2 μM over a time course that was strongly dependent on the concentration of H2O2 used (5 × 10−5 to 5 × 10−3M). After exposure of P388D1 cells to 5 × 10−3 M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spetcroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points >45 min after exposure to H2O2 the influx of extracellular 45Ca and net intracellular Ca++, Na+ and K+ rapidly increased. Half times for H2O2 catabolism by the cells varied from about 8 min at 5.0 × 10−4 M H2O2 to 14.0 min at 5.0 × 10−3 M. When the total [Ca++]i‐buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68‐fold (1.1 × 102 ‐ 7.6 × 103 amol per cell), the rate of rise of [Ca++]i was depressed by only 1.6‐fold following exposure to 5 mM H2O2. During the rise of intracellular [Ca+++]i, cell morphology was observed by both light and scanning electron microscopy and revealed that “surface blebs” appeared during this phase of injury. Both the rise in [Ca+++]i and “blebbing” were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51Cr from the cells. From these results we conclude the following, (1) H2O2 exposure induces a dose‐dependent disturbance of intracellular calcium homeostatis; (2) the rise in[Ca+++]i is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; (3) the rate of rise of [Ca+++]i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; (4) during the early phase (<30 min) of rise of [Ca+++]i, only intracellular calcium is involved in the response; (5) these events occur concomitantly with gross morphological changes to the plasma membrane; and finally, (6) these events precede loss of integrity of the plasma membrane as a permeability barrier.
L‐929 cells under hyperosmotic conditions: Volume changes
Authors: Clegg James S. 
Published: February 2005

AbstractVolume changes resulting from the addition of sorbitol to the environment of mouse L‐929 cells were evaluated from cell diameter measurements. Over periods of 1 hour or less, this solute was effectively excluded from intracellular water. The reduction in cell volume was inversely related to sorbitol concentration up to levels of about 0.6 molal, above which no further significant reduction occurred. Reduced cell volumes were maintained for at least 1 hour without measurable volume regulation. The percentage of volume lost was independent of the initial cell volume and was quickly regained when physiological conditions were restored. However, cell volume was influenced strongly by cell density or by some variable related to it. L‐cells store surface area when dehydrated, apparently by means of plasma membrane convolutions and microvilli, based on the rapid kinetics of reversible volume changes and on observations from scanning electron microscopy. These results are related to current views on the nature of intracellular organization.
Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: Relationship to senescence
Authors: Hornsby Peter J. , Aldern Kathy A. , Harris Sandra E. 
Published: February 2005

AbstractDuring cellular senescence, non‐clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin‐ or prostaglandin E1− stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH‐stimulated cAMP production rates were measured in clones immediately after isolation, during long‐term growth following isolation, and after subcloning. ACTH‐stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/106 cells/min, whereas mass cultures at this PDL showed ∼50 pmol/106 cells/min. Forskolin‐stimulated cAMP production rates in clones varied only over the range of 59–119 pmol/106 cells/min and showed no correlation with the ACTH‐stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP‐inducible 17α‐hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub‐subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eightfold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.
Intracellular distribution of active and inactive transglutaminase in stimulated cultured C6 glioma cells
Authors: Korner G. , Bachrach U. 
Published: February 2005

AbstractThe cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum‐containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post‐stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detercted 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post‐stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serumcontaining medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.
Murine macrophage heparanase: Inhibition and comparison with metastatic tumor cells
Authors: Savion Naphtali , Disatnik Marie‐Helene , Nevo Zvi 
Published: February 2005

AbstractCirculating macrophages and metastatic tumor cells can penetrate the vascular endothelium and migrate from the circulatory system to extravascular compartments. Both activated murine macrophages and different metastatic tumor cells (B16‐BL6 melanoma; ESb T‐lymphoma) attach, invade, and penetrate confluent vascular endothelial cell monolyaer in vitro, by degrading heparan sulfate proteoglycans in the subendothelial extracellular matrix. The sensitivity of the enzymes from the various sources degrading the heparan sulfate proteoglycan was challenged and compared by a series of inhibitors. Activated macrophages demonstrate a heparanase with an endoglycosidase activity that cleaves from the [35S]O4 = ‐labeled heparan sulfate proteoglycans of the extracellular matrix 10 kDa glycosaminoglycan fragments. The macrophages do not store the heparanase intacellularly but it is instead found pericellularly and requires a continuous cell‐matrix contract at the optimal pH for maintaining cell growth.The degradation of [35S]O4=‐labeled extracellular matrix proteoglycans by the macrophages' heparanase is significantly inhibited in the presence of heparan sulfate (10 μg/ml), arteparon (10 μg/ml), and heparin at a concentration of 3 μg/ml. In contrast, other glycosaminoglycans such as hyaluronic acid, dermatan sulfate, and chondroitin sulfate as well as the specific inhibitor of exo‐β‐glucuronidase D‐saccharic acid 1,4‐lactone failed to inhibit the degradation of sulfated proteoglycans in the subendothelial extracellular matrix. Degradation of this heparan sulfate proteoglycan is a two‐step sequential process involving protease activity followed by heparanase activity. However, the following antiproteases—α2‐macroglobulin, antithrombin III, leupeptin, and phenylmethylsulfony fluoride (PMSF)—failed to inhibit this degradation process, and only α1‐antitrypsin inhibited the heparanase activity.B16‐BL6 metastatic melanoma cell heparanase, which is also a cell‐associated enzyme, was inhibited by heparin to the same extent as the macrophage heparanase. On the other hand, heparanase of the highly metastatic variant (ESb) of a methylcholanthrene‐induced T lymphoma, which is an extracellular enzyme released by the cells to the incubation medium, was more sensitive to heparin and arteparon than the macrophages' heparanase, inhibited at concentrations of 1 and 3 μ/ml, respectively. These results may indicate the potential use of heparin or other glycosaminoglycans as specific and differential inhibitors for the formation in certain cases of blood‐borne tumor metastasis.
Regulation of amino acid transport in isolated rat hepatocytes during development
Authors: Leoni S. , Spagnuolo S. , Dini L. , Devirgiliis L. Conti 
Published: February 2005

AbstractThe effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2‐amino [1‐14C]isobutyric acid (AIB) or N‐methyl 2‐amino [1‐14C]isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre‐ and postnatal development. The data obtained show that the Na+‐dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AIB and MeAIB uptake enhancing the Vmax, while the Km was unchanged. This effect was evident in cells from adult and 18–20‐day‐old fetuses, while no response was present before the 18th day of fetal life and in the perinatal period. Actinomycin D or cycloheximide abolished this hormone‐dependent increase. A decrease in AIB and MeAIB transport after incubation in an amino acid‐rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the releas from transinhibition of the transport.
Differentiation‐associated decrease in muscarinic receptor sensitivity in human neuroblastoma cells
Authors: Heikkilä Jari E. , Scott Ian G. , Suominen Liisa A. , Åkerman Karl E. O. 
Published: February 2005

AbstractMuscarinic receptor‐linked increases in intracellular free Ca2+ as measured with quin‐2 and Ca2+ release from monolayers of cells have been measured in the human neuroblastoma cell line SH‐SY5Y. Induction of differentiation with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) leads to a decrease in the sensitivity of the cells to low concentrations of agonists with respect to the induced increase in cytosolic free Ca2+ and stimulation of Ca2+ efflux. No decrease in agonist binding affinity was observed when the displacement of a labelled antagonist, 3H‐NMS, by a non‐labelled agonist was studied.
Role of Na‐K ATPase in regulation of resting membrane potential of cultured rat skeletal myotubes
Authors: Brodie Chaya , Bak Asia , Shainberg A. , Sampson S. R. 
Published: February 2005

AbstractThe role of Na‐K ATPase in the determination of resting membrane potential (Em) as a function of extracellular K ion concentration was investigated in cultured rat myotubes. The Em of control myotubes at 37°C varied as a function of (K+)0 with a slope of about 58–60 mV per ten‐fold change in (K+)0. Inhibition of the Na‐K pump with ouabain or by reduced temperature revealed that this relation consists of two components. One, between (K+)0 of 10 and 100 mM, remains unchanged by alterations in enzyme activity; The second, between (K+)0 of 1 and 10 mM, is related to the amount of Na‐K pump activity, the slope decreasing as pump activity decreases. Indeed, with complete inhibition of the Na‐K pump, Em does not change over the range of (K+)0 1 to 10 mM. Measurements of 86Rb efflux and input resistance of individual myotubes showed that membrane permeability does not change as (K+)0 increases from 1 to 10 mM but increases as (K+)0 increases further. Monensin, which increases Na ion permeability, increases Em at values of external K+ below 10 mM, and is without effect at higher values of K+ concentration. The effect of monensin is blocked by ouabain. Tetrodotoxin, which blocks voltage‐dependent Na+ channels, decreases Em at low (2–10 mM) K+. We conclude that changes in Em as a function of extracellular K+ concentration in the physiological range are not adequately explained by the diffusion potential hypothesis of Em, and that other theories (electrogenic pump, surface‐absorption) must be considered.
The effects of sulfhydryl modifying reagents on nonhormonal and hormonally regulated hexose transport in cultured human skin fibroblasts
Authors: Germinario Ralph J. , Vlachopoulou Fotini 
Published: February 2005

AbstractThe effects of various sulfhydryl modifying reagents on hexose transport in cultured human skin fibroblasts were studied. H2O2 was observed to have no effect on 2‐deoxy‐D‐glucose transport in serum‐starved glucose‐fed cells. The elevation of hexose transport rates in cells by glucose deprivation, insulin, or serum stimulation rendered them sensitive to H2O2. Hexose transport in glucose‐deprived cells was inhibited 51–55% by 1–2 mM H2O2, while hexose transport in insulin or serum‐stimulated glucose‐fed cells was inhibited 45% and 46%, respectively. H2O2 inhibition was blocked or reversed by 8 mM dithiothreitol. N‐ethyl‐maleimide (NEM), a permeant, sulfhydryl reagent, elicited effects on hexose transport similar to those effected by H2O2 (i.e., in glucose‐deprived and insulin‐stimulated cells, inhibition of hexose transport was 44% and 23%, respectively). Impermeant sulfhydryl reagents such as dithio(bis)nitrobenzoic acid (DTNB) and N‐iodoacetyl‐N'‐(5‐sulfo‐1‐naphthylethylenediame (1,5,‐I‐AEDANS)) had no inhibitory effect on hexose transport under any conditions (i.e., glucose‐fed, glucose‐deprived, and insulin‐stimulated cells). DTNB and 1,5‐I‐AEDANS afforded no protection from the action of H2O2 on hexose transport. The data suggest that the sensitive sites are thiol in nature and are located at an intramembrane or intracellular site and probably not exofacial.
2‐Mercaptoethanol acts at the restricted stage of interleukin‐2 dependent lymphocyte proliferation
Authors: Sugama Kazushige , Namba Yuziro , Hatanaka Masakazu , Hanaoka Masao 
Published: February 2005

AbstractAn interleukin‐2 (IL‐2) dependent murine cell line (TN‐9) which could be grown continuously with the crude culture supernatant of concanavaline A‐stimulated rat or mouse spleen cells could not synthesize DNA in the culture medium supplemented with partially purified or recombinant IL‐2. The cell growth was restored by adding another factor obtained from the same crude culture supernatant. This factor, physicochemically inseparable from serum albumin, was also obtained from the culture medium added with 2‐mercaptoethanol (2‐ME) and incubated at 37°C for 24 hr without the cells. By the experiments using semi‐synchronized cell population, it was demonstrated that 2‐ME or 2‐ME carrying protein acted at the restricted process(es) of cell proliferation which occurred between IL‐2‐acting stage and the initiation of DNA synthesis.
Coordinate expression of c‐myc, c‐myb, and histone H4 genes in reversibly differentiating HL 60 cells
Authors: Brelvi Zamir S. , Studzinski George P. 
Published: February 2005

AbstractThe expression of oncogenes c‐myc and c‐myb in human leukemic cells HL 60 was compared to the expression of histone H4 gene, known to be cell‐cycle dependent. Steady‐state levels of mRNA transcribed from these genes were determined by simultaneous hybridization of Northern transfers with four probes, and the rates of gene expression were measured by nuclear transcription (“run‐on”) assays. Expression of genes c‐myc, c‐myb and histone H4 varied coordinately and in parallel with the rates of DNA synthesis, while the rates of total and ribosomal RNA synthesis, the expression of gene c‐Haras, unrelated to proliferation of these cells, and gene p 72, a constitutively expressed human gene, were unchanged. Further, the levels of c‐myc and c‐myb mRNA but not p 72 mRNA were higher in cell populations enriched for S phase cells. Thus, transcription of genes c‐myc and c‐myb in HL 60 cells appears to be linked to DNA replication in a manner previously demonstrated for core histone gene expression.
Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes
Authors: Roederer Mario , Bowser Robert , Murphy Robert F. 
Published: February 2005

AbstractThe temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11°C; acidification was limited to pH 6–6.5 at temperatures between 13°C and 17°C. In contrast, a rapid drop to pH 6–6.5 followed by acidification to pH 5–5.5 was observed at temperatures above 19°C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98:1757–1762). The timing of exposure of material internalized by fluid‐phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37°C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13° and 21°C, the rate of hydrolysis was reduced to 31–44% of that at 37°C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20°C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low‐density organelle containing lysosomal enzymes.
Thrombin chemotactic stimulation of HL‐60 cells: Studies on thrombin responsiveness as a function of differentiation
Authors: Bar‐Shavit R. , Hruska K. A. , Kahn A. J. , Wilner G. D. 
Published: February 2005

AbstractThrombin, a major procoagulant enzyme and growth factor, is also selectively chemotactic for monocytes and macrophages but not for neutrophils. This effect stands in contrast to other well‐known chemotactic agents such as fMet‐Leu‐Phe, C5a fragments, and LTB4, which stimulate directed cell movement in both cell types, and have important physiological implications. The human leukemic cell line HL‐60, which is capable of differentiating either along granulocytic or monocytic lineages, was therefore used to explore the development of this selective monocyte/macrophage chemotactic response to thrombin. Esterolytically inactive DIP‐α‐thrombin, as well as the thrombin‐derived chemotactic peptide CB67–129, elicits a dose‐dependent chemotactic response in HL‐60 cells differentiated to monocytelike cells by treatment with 1,25(OH)2D3 (HL‐60/mono), whereas no such response is evident in either undifferentiated HL‐60 cells or in cells differentiated into granulocytes by treatment with DMSO (HL‐60/gran). Similarly, early events which characterize stimulation of inflammatory cells by chemotactic agents are also evident, but only in monocyte‐differentiated cells. In HL‐60/mono, thrombin selectively stimulates rapid cytosolic Ca2+ elevation as well as rapid cytoskeletal association of cytosolic actin. Following thrombin stimulation, maximal actin association in these cells occurs within 30 sec (declining to basal levels at the end of 5 min), and maximal Ca2+ elevations are also evident within 15–20 sec, suggesting a temporal relationship between these two events. Thus, the events accompanying stimulation of HL‐60/mono by thrombin are characteristic of those seen following stimulation of inflammatory cells by chemotaxins, with a major difference being the selectivity of thrombin as a chemotaxin for cells of macrophage/monocytic lineage. The selective chemotactic responsiveness of HL‐60/mono to thrombin appears to relate to the development of specific receptors on these cells as part of monocytic differentiation: HL‐60/mono (but HL‐60/gran nor undifferentiated HL‐60) are capable of significant specific 125‐I‐labeled α‐thrombin‐binding (ka∼20 nM), and possess an estimated 400,000 thrombin‐binding sites per cell. Our finding further suggest that the thrombin response of HL‐60 and particularly the expression of thrombin receptors on these cells may serve as a useful model system for exploring the biology of monocyte/macrophage differentiation.