Superoxide dismutase activity of Ehrlich ascites tumor cells

A crude extract of Ehrlich ascites tumor cell homogenate was found to contain three distinct bands of superoxide dismutase activity by polyacrylamide gel electrophoresis. Activity bands migrated approximately the same distance and were inhibited by cyanide ions. Isolated mitochondria produced two bands of activity that were also inhibited by cyanid. Ethanol-chloroform treatment of the homogenate had no observable effect on these bands of activity, which suggested that the cyanide-insensitive mitochondrial superoxide dismutase activity in these malignant cells was either present in concentrations below detectable levels or completely absent. Normal liver was used as a control for the detection system.     

Comparative investigation of DNA from mouse and Ehrlich ascites tumor cells

Mouse DNA and DNA of Ehrlich ascites tumor have been comparatively studied in the search for changes in DNA during tumor progression. No differences were found in kinetics of homologous (mouse X mouse) and heterologous (mouse X tumor) DNA reassociation; in thermal stability of homologous and heterologous duplexes of repeated, unique and satellite DNA; in percentage of hybridization with mouse liver heterogenous nuclear RNA; in thermal stability of the RNA.DNA hybrids. The negative results suggest that the considerable evolution of transplantable tumors (both in biological properties and in karyotype) has not been accompanied by alterations of the genome which could be detected by the now available methods of molecular hybridization for studying DNA divergence. In the light of the results obtained the functions ascribed to the mouse satellite DNA are discussed.     

Mechanical stress induces release of ATP from Ehrlich ascites tumor cells

The supernatant from a suspension of Ehrlich cells exposed to centrifugation at 700xg for 45 s induced a transient increase in the intracellular concentration of free, cytosolic Ca2+, [Ca2+]i, as well as activation of an outwardly rectifying whole-cell current when added to a suspension of non-stimulated cells. These effects were inhibited by suramin, a non-specific P2 receptor antagonist, and mimicked by ATP. Reversed phase HPLC analysis revealed that the supernatant from Ehrlich cells exposed to centrifugation contained 2. 6+/-0.2 microM ATP, and that the mechanical stress-induced release of ATP was inhibited by glibenclamide and verapamil, non-specific inhibitors of the cystic fibrosis transmembrane conductance regulator and P-glycoprotein, respectively. After trypan blue staining, less than 0.5% of the cells were unable to extrude the dye. Addition of extracellular ATP induced a suramin-sensitive, transient, concentration-dependent increase in [Ca2+]i, activation of an outwardly rectifying whole-cell current and a hyperpolarization of the plasma membrane. The ATP-induced hyperpolarization of the plasma membrane was strongly inhibited in the presence of charybdotoxin (ChTX), an inhibitor of several Ca2+-activated K+ channels, suggesting that stimulation of P2 receptors in Ehrlich cells evokes a Ca2+-activated K+ current. The relative potencies of several nucleotides (ATP, UTP, ADP, 2-MeSATP, alpha,beta-MeATP, bzATP) in eliciting an increase in [Ca2+]i, as well as the effect of repetitive addition of nucleotides were investigated. The results lead us to conclude that mechanical stimulation of Ehrlich cells leads to release of ATP, which in turn stimulates both P2Y1 and P2Y2 receptors, resulting in Ca2+ influx as well as release and activation of an outwardly rectifying whole-cell current.     

Daunorubicin inhibition of DNA-dependent RNA polymerases from Ehrlich ascites tumor cells

An analysis of data about the +p9 syndrome revealed that this clinical entity may occur in some different genetic forms. The recurrence risk in cases with familial translocations is due to the type of meiotic segregation, 2:2 or 3:1. It was shown a nonrandomness of involvement of chromosomes 15 and 22 in translocations with chromosome number 9.     

T-cell receptor-mediated signal transduction in transformed human T-cells

Carbonated apatite (dahllite) is formed within and between collagen fibrils in the mineralization of connective tissues. However, the mechanism of crystal nucleation at these sites has not been resolved. To identify non-collagenous proteins that may be involved in the nucleation process we have utilized a dissociative extraction procedure to isolate proteins associated non-covalently with the de-mineralized collagen matrix of dentine isolated from tooth roots of adult porcine incisors. Following extraction of dentine fragments with 4M GuHCl (G1-extract) and 0.5M EDTA (E-extract), de-mineralized collagen matrix-associated proteins were isolated with a second series of extractions with 4M GuHCl (G2-extract). Analysis of the G2-extracts on SDS-PAGE revealed two major 32 kDa and 24 kDa protein bands, comprising > 80% of the extracted non-collagenous proteins. The 32 kDa protein was purified by FPLC on hydroxyapatite and Mono Q resins, followed by HPLC reverse-phase chromatography. Small amounts of 26 kDa and 6 kDa proteins, which appear to represent proteolytically processed, disulphide-linked fragments of the 32 kDa protein, co-eluted with the major protein. The 32 kDa protein was identified as lysyl oxidase from amino acid sequence analysis of a 13 kDa CNBr peptide obtained from protein purified by preparative electrophoresis on SDS-PAGE. Fractionation of the 24 kDa protein on FPLC Mono Q resin generated < 5 closely eluting protein peaks. The proteins from these peaks were similar in size, staining properties, amino acid composition and CNBr digestion patterns. Each protein was immunoreactive with antibodies raised against a tyrosine-rich acidic matrix protein (TRAMP), reported previously to co-purify with lysyl oxidase. These studies, therefore, show that lysyl oxidase, which is important in collagen cross-link formation, and proteins with properties of TRAMP, a protein that can modulate collagen fibrillogenesis, are the major proteins in dissociative extracts of de-mineralized porcine dentine.     

Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells

The photosensitizing properties of tetrasulphonated Al- and Zn-phthalocyanines (AlPcS4 and ZnPcS4) in lymphoma cells were studied as a function of the pre/post-illumination incubation time. Photocytotoxicity increased with incubation time, ranging from a transient cell-cycle arrest to cell killing. Under all experimental conditions, the phototoxicity of ZnPcS4 was markedly higher than that of AlPcS4. The primary photoprocesses initiated by metallo-phthalocyanines (MePcS4) in the cells were probed with DMPO/esr spin-trapping techniques. Under all incubation conditions the intracellularly bound MePcS4 sensitized formation of three different types of DMPO spin-adducts: DMPO/OH (hydroxyl radical), DMPO/R (organic carbon-centred radical(s)) and an unidentified simple nitroxyl, referred to as DMPO/ox. The yields of trapped radicals depended on the length of the incubation with the dyes prior to illumination and the formation of spin-adducts was shown to be intracellular. The ability of DMPO to protect cells from the photocytotoxic effects of Al- and ZnPcS4, combined with the generation of carbon-centred spin-adducts is direct evidence for the involvement of free-radical-mediated damage of cellular constituents.     

Altered erythrocyte osmotic fragility in mice with Ehrlich ascites tumor

The osmotic fragility of erythrocytes from mice with Ehrlich ascites tumor was compared with that of erythrocytes from normal control animals. Erythrocytes collected from mice 15 days after the ip injection of tumor cells exhibited a uniform pattern of abnormal resistance to hemolysis in hypotonic saline, with 50% hemolysis occurring at an average saline concentration of 0.44% compared to an average of 0.49% for 11 controls a significant difference (P less than 0.0001). Erythrocytes from mice with this tumor apparently undergo an alteration in some component of the cell membrane that regulates either permeability to cations and water or distensibility of the cell.     

Modification of the Ehrlich ascites tumor cell nuclear lipids

The fatty acid composition of Ehrilich ascites tumor cell nuclei was differend when the tumor-bearing mice were fed diets rich in either coconut or sunflower oil. When coconut oil was fed, the monoenoic fatty acid content of many of the nuclear lipids was increased and their polyenoic fatty acid content was reduced as compared with the sunflower oil diet. By contrast, only small changes were produced in the saturated fatty acid contents of the nuclear lipids. The nuclear membrane choline phospholipid, ethanolamine phospholipid and combined serine phospholipid plus inositol phospholipid fractions exhibited statistically significant changes in fatty acid composition, but the sphingomyelins were not altered appreciably by dietary lipid modification. The fatty acid composition of the small quantity of phospholipids associated with the chromatin was much more resistant to diet-induced mosification. Except for sphingomyelin, the fatty acid composition of the chromatin phospholipids was different from that of the corresponding nuclear membrane phospholipids, containing much larger amounts of fatty acids having less than 16 carbon atoms. The fatty acid compositons of the nuclear triaclglycerols and cholesterol esters, which were associated almost entirely with the chromatin, were modified by the dietary lipid modifications. There were no changes in the DNA, RNA or lipid content of these nuclei. Therefore, this experimental system can be used to prepare mamalian nuclei that differ appreciably only in their fatty acyl composition.     

Purification of a factor from Ehrlich ascites tumor cells specifically stimulating RNA polymerase II

A factor stimulating RNA polymerase II from Ehrlich ascites tumor cells was purified. The final preparation appeared almost homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a molecular weight of 38 000. The endonuclease activity of about 10 mug of purified factor, if any was well below the 10(-5) mug equivalent of pancreatic deoxyribonuclease, indicating that the stimulation of RNA synthesis by this factor was not due to contaminating endonuclease. This factor specifically stimulated RNA polymerase II on native DNA as template and did not affect RNA polymerase I at all. The molecular size of RNA synthesized in the presence of this factor increased markedly compared with that synthetized by RNA polymerase II alone.     

Papaverine-induced changes in physiological characters of Ehrlich ascites tumor cell membranes

Physiological characters of Ehrlich ascites tumor cell membranes were altered by papaverine. The agent induced changes in membrane potential as monitored by cyanine dye (diS-C3-(5)) technique. Papaverine also strongly inhibited increase in membrane permeability to K+ ion induced by lysolecithin. In addition, papaverine inhibited oxygen uptake of the tumor cells and oxidative phosphorylation of their mitochondria, and slightly increased membrane fluidity. The results suggest that papaverine maintains compartmentation of K+ ion, energy metabolism, and membrane fluidity by regulating intracellular mitochondrial metabolism of Ehrlich ascites tumor cells.     

Role of the adenylate deaminase reaction in regulation of adenine nucleotide metabolism in Ehrlich ascites tumor cells

The regulatory properties of adenylate deaminase (EC 3.5.4.6) from Ehrlich ascites tumor cells suggest that the reaction catalyzed by this enzyme serves to protect the cell against sharp decreases in the adenylate energy charge by removing adenosine 5'-monophosphate generated when the rate of utilization of adenosine triphosphate is suddenly increased. The enzyme is effectively inhibited under normal physiological conditions of high energy charge (0.9) and 4 to 5 mM adenine nucleotide pool size. The reaction is sharply activated by a decrease in the energy charge in the physiological range (0.9 to 0.6). At low energy charge (0.6), decrease in the size of the pool causes a marked and nonlinear decrease in the rate of the deaminase reaction. This effect presumably serves to prevent excessive depletion of the adenine nucleotide pool. Calculations based on the kinetic data obtained in this study show that the AMP deaminase reaction can account for the well-established alteration of adenine nucleotide metabolism that is observed following addition of glucose or 2-deoxyglucose to intact ascites cells.     

Selective modulation of non-protein sulfhydryl levels in Ehrlich ascites tumor bearing mice

OBJECTIVES: A total of 97 apparently healthy subjects were studied in order to establish the influence of smoking habits in studies on neurocardiovascular control and the QTc interval duration. MATERIAL AND METHODS: The study group consisted of 37 smokers and 60 non-smokers as the control. A 12-lead electrocardiogram was performed on all subjects to determine the duration of the QTc interval. Other aspects studied include heart rate variability at rest during 150 cardiac cycles using time domain: coefficient of variation and root mean squared successive difference; and frequency domain: low frequency band (0.04-0.15 Hz) and high frequency band (0.15-0.50 Hz), to determine total energy logarithm and maximum energy frequency. Additionally, conventional cardiovascular autonomic function tests, such as orthostasis, Valsalva maneuver and deep breathing were performed. RESULTS: No significant differences were observed in the duration of the QTc interval nor in time and frequency domain parameters, except in the maximum frequency in the high frequency band, which appeared significantly lower (p < 0.05) in smokers when compared to non-smokers (0.28 +1- 0.1 vs 0.33 +/- 0.1 Hz). No modifications were noted in the cardiovascular autonomic function tests applied to smokers and non-smokers, and the QTc interval was not linked to the rest of the variables studied. CONCLUSIONS: To conclude, smoking habits do not seem to have a significant influence in studies addressed to determine the impact of the autonomic nervous systems on cardiovascular control.     

Involvement of protein tyrosine phosphorylation in the effect of green tea polyphenols on Ehrlich ascites tumor cells in vitro

Recently, elucidation has progressed on a crucial role played by dendritic cells (DCs) in the induction of primary antigen-specific immune reactions. Although mature DCs exhibit potent antigen presenting function, DCs are scattered in nonlymphoid organs throughout the body as immature cells that have only minimum antigen presenting function. When they are stimulated to maturate, they increase their expression of class II major histocompatibility (MHC) antigen and several co-stimulatory molecules, resulting in the augmentation of antigen presenting function. Furthermore, these maturated DCs move to the T-dependent areas of secondary lymphoid organs to sensitize naive T cells for these antigens. Therefore, it is important to understand the mechanism to induce the maturation of DCs. Recent progress in the study of DC biology depicts various factors, such as cytokines, bacterial products and haptens, which are responsible for DC maturation. In this paper, the mechanism of DC maturation induced by cytokines and chemicals is described.     

Kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth

The kinetics of cell proliferation and polyamine synthesis during Ehrlich ascites tumor growth were studied. The steady deceleration of the specific growth rate with increasing tumor mass that was observed was attributable to a prolongation of the cell cycle, particularly of the S and G2 phases. The cell cycle time (Tc) was 43.3 hr (TG1 equals 10.8, TS equals 26.8, and TG2 equals 5.7 hr) on the seventh day of growth and 76.0 hr (TG1 equals 14.0, TS equals 52.0, and TG2 equals 10.0 hr) on the tenth day of growth. The growth fraction showed a decrease from 0.77 to 0.60 during the 7- to 10-day tumor growth interval. The cell death rate remained low and essentially unchanged during this period. A high correlation was found between polyamine synthesis (ornithine decarboxylase activity) and the specific growth rate; the correlation coefficient was 0.985. There was also a high positive correlation between the cellular polyamine (spermidine and spermine) and nucleic acid content (spermidine: DNA equals 0.916, spermine: DNA equals 0.947, spermidine:RNA equals 0.907, and spermine: RNA equals 0.881). These observations suggest that there may be a functional coupling between polyamines and nucleic acids, and they support the hypothesis that polyamines play an important role in DNA replication and cell division.     

Sacharose density gradient separation of Ehrlich ascites carcinoma and L-5178Y tumor cells in different cell cycle phases

Optimal conditions were determined for the distribution of Ehrlich ascites carcinoma (EAC) and L-5178Y mouse tumor cells, proliferating in vivo, by their age within the cell cycle by sedimentation in a buffered linear sacharose density gradient. Measurements of cell size, DNA content and incorporation of tritiated thymidine in successive parts of the gradient confirmed the actual separation of cells of different age: in the upper fractions there were cells in G1 phase, in the middle fractions in S phase and in the lower layers of the gradient there were cells in G2 and/or M phase.