Transport of calcium ions by Ehrlich ascites-tumour cells.

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.     

Comparison of Ehrlich ascites tumour and mouse liver cells by analytical subcellular fractionation combined with a sensitive computational method for data analysis

A simple method of analytical subcellular fractionation, combined with a sensitive computational method for data analysis and presentation, has been used to reinvestigate the distribution and relative amounts of several enzymes in the cytoplasmic and plasma membranes of two different cell types: one is a neoplastic, transformed cell type (Ehrlich ascites tumour cells), the other an untransformed, highly differentiated cell type (liver hepatocytes plus Kupffer and endothelial cells). In general the distribution of the enzymes in particular membranes is similar in the two cell types, however the relative amounts differ. Ehrlich ascites tumour cells have a higher specific activity of galactosyltransferase and ouabain-sensitive (Na,K)ATPase, while liver cells have higher glucose-6-phosphatase, 5'-nucleotidase and succinate dehydrogenase activity. These differences appear to be correlated with morphological and, in some cases, functional differences between the two cell types.     

Role of acetoin on the regulation of intermediate metabolism of Ehrlich ascites tumor mitochondria: its contribution to membrane cholesterol enrichment modifying passive proton permeability

Acetoin, an unusual metabolite of highly glycolytic mammalian tumor cells, is synthesized from decarboxylated pyruvate and active acetaldehyde in mitochondria. It plays important roles in the regulation and detoxification of pyruvate metabolism through pyruvate dehydrogenase. We show in this report the inhibitory effect of acetoin on succinate oxidation by Ehrlich tumor cell mitochondria, and thus its regulatory role on intermediate metabolism. Acetoin utilization by Ehrlich mitochondria may lead to small quantities of citrate formation which increase the already increased cholesterol synthesis of cancer cells. Membranes, in particular the inner mitochondrial membrane, flooded with cholesterol, show a proton passive permeability twice as low as that of control mitochondrial membranes, a feature that may be related to drastic changes in membrane potential-dependent metabolism of cancer cells.     

Lipidg Peroxidation in Liver and Ehrlich Ascites Cell Mitochondria

Ehrlich ascites cell mitochondria are highly resistant to lipid peroxidation as compared to liver mitochondria from host animals. Succinate protects mitochondria from peroxidative damage, proteins from crosslinks, enzymes from inactivation of the enzymes and membranes from permeability changes. The sensitivity of Ehrlich ascites cell mitochondrial membranes to lipid peroxidation is significantly increased in sub-mitochondrial particles. Lipid peroxidation in tumour mitochondrial membranes can not be diminished by succinate as effectively as in liver mitochondria. Ascites cell mitochondria seems to be protected very efficiently from peroxidative damage by a glutathione-dependent mechanism.     

Cloturin: effect on energy-producing processes in Ehrlich ascites and P388 murine leukaemia cells.

The main purpose of the present investigation was to study the effect of cloturin on aerobic glycolysis, endogenous and exogenous respiration and the level of ATP in both Ehrlich ascites carcinoma (EAC) and P388 murine leukaemia cells incubated in vitro. Also its effect on the level of total (T-SH) and non-protein (NP-SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis was found only in P388 cells after 60 min of cloturin action. Cloturin inhibited both endogenous and exogenous respiration of EAC with succinate as substrate. Cloturin decreased the level of ATP after 2 h incubation in both types of tumour cell. The level of NP-SH was decreased more than that of T-SH in both types of cell.     

The effect of polycations on cell membrane stability and transport processes

Summary The interaction of poly-l-lysines of different molecular weights (PL) with Ehrlich ascites tumor cells was studied experimentally with respect to cell surface binding, cell electrophoresis, cytotoxicity and membrane permeability. Although they decrease the net negative charge of Ehrlich ascites cells similarly at low PL concentrations, low molecular weight PL was less cytotoxic and less damaging to the potassium transport mechanism than was high molecular weight PL. At certain PL concentrations, membrane damage was reversible on reincubation in PL-free media. The amount of bound polylysine as determined with fluorescent labeled polylysine was compared by electrophoresis to the amount of polylysine expressed on the electrokinetic surface. The results indicated that only a small fraction of polylysine bound to Ehrlich ascites tumor cells was electrokinetically detectable. The adsorption of polylysine to Ehrlich ascites tumor cells was not describable by the usual adsorption isotherms. It is suggested that the same number of monomeric lysine units of high and low molecular weight PL are adsorbed at the cell electrokinetic surface, but cytotoxicity is dependent on molecular weight. Although the negative charge of human red blood cells could be reversed at low PL concentrations, no such effect could be observed for ELD (a subline of Ehrlich ascites carcinoma) cells even at high PL concentrations. The relationship of PL binding to the stimulation of macromolecular uptake is discussed.     

The effect of ascitic fluid on the growth of Ehrlich tumor and Lewis carcinoma]

In the study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (m.m. less than 15 kDa) on the growth of Ehrlich and Lewis carcinoma it was found that the ascitic fluid significantly decreased the size of Ehrlich tumor (by more than 50% on day 9-25 after the tumor cell inoculation). It also reduced Lewis carcinoma tumor volume by more than 30% during 3 weeks after the tumor cells inoculation. Dialysate of Ehrlich tumor cells significantly inhibited the growth of Ehrlich tumor too. It is suggested that this test-system simulates inhibition of a small tumor by a big tumor in vivo.     

VARIATION OF CELL KINETIC PARAMETERS IN RELATION TO THE GROWTH RATE OF THE EHRLICH ASCITES TUMOR

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Variation of cell kinetic parameters in relation to the growth rate of the Ehrlich ascites tumor.

A multicompartmental model of the cell cycle and proliferation kinetics was used to analyse the time-course behavior of the cell cycle time, the growth fraction, and the cell loss rate during Ehrlich ascites tumor growth. The growth rate of Ehrlich ascites tumor cells as the tumor aged was significantly influenced by change in the cell cycle time.     

Electrophoretic mobility of mouse cells and homologous isolated nuclei

The electrophoretic mobilities of Ehrlich ascites, sarcoma 37 ascites, mouse liver cells and their isolated nuclei were measured under similar environmental conditions. No differences in mobility were detected between cells and homologous nuclei from the same cell population and it was concluded that their surface charge densities were probably the same. The effect of neuraminidase on Ehrlich ascites and liver cells and nuclei was also determined; neuraminidase reduced the mobility of Ehrlich ascites cell nuclei as well as cells. The reduction in mobility of cells and nuclei prepared by a sucrose method was the same; however, the reduction in mobility of citric acid prepared nuclei was less than that of citric acid treated cells. The reduction in mobility of both liver cells and nuclei was small or insignificant. It is suggested that although cells and nuclei have similar electrophoretic mobilities, possibly different groups contribute to their surface charge.     

血清抑癌多肽作用机制的初步探讨——对癌细胞膜及其骨架的影响

本文研究血清抑癌多肽(SCSP)对癌细胞表面的效应和其表面蛋白质分子的侧向扩散及其内的F-actin的变化.扫描电镜观察结果是SCSP作用后,艾氏腹水癌细胞表面的微绒毛变为平坦而光滑的结构.从荧光漂白恢复技术测得的结果是上述癌细胞表面蛋白质分子的侧向扩散系数(?)降低.用Rhodamine—Pha-lloidin标记上述癌细胞得知其内的F—actin增加.从上述结果推测,在SCSP杀伤艾氏腹水癌细胞过程中,使F—actin在癌细胞内重组,有更多的F—actin与癌细胞质膜内大分子紧密相连,使癌细胞表面分子的活动性降低,故在癌细胞表面的结构变平,其蛋白质分子的扩散受限制而变慢.     

Lipid peroxidation and cytotoxicity of Ehrlich ascites tumor cells by ferric nitrilotriacetate

Ferric nitrilotriacetate, which causes in vivo organ injury, induced lipid peroxidation and cell death in Ehrlich ascites tumor cells in vitro. The process was inhibited by butylated hydroxyanisole and enhanced by vitamin C and linolenic acid, indicating a close relationship between cytotoxicity and the lipid peroxidizing ability of Fe3+ NTA. The cytotoxicity was suppressed by glucose and a temperature below 20 degrees C. Lipid peroxidation of Fe3+ NTA-treated cells was greater at 0 degree C than at 37 degrees C, contrary to results with Fe3+ NTA-treated plasma membranes of Ehrlich ascites tumor cell. These results suggested that metabolism and membrane fluidity are important factors in the expression of the Fe3+ NTA-induced cytotoxicity. H2O2 showed a lower cytotoxicity than did Fe3+ NTA but a greater lipid peroxidizing ability. H2O2 appeared to damage the cells less, and was quenched rapidly by cellular metabolism unlike Fe3+ NTA. In transferrin-free medium, Ehrlich ascites tumor cell readily incorporated Fe3+ NTA, and iron uptake was greater than NTA-uptake in Fe3+ NTA-treated cells, suggesting that Ehrlich ascites tumor cell incorporated iron from Fe3+NTA and metabolized it into an inert form such as ferritin.     

Downregulation of taurine uptake in multidrug resistant Ehrlich ascites tumor cells

Summary.  In daunorubicin resistant Ehrlich ascites tumor cells (DNR), the initial taurine uptake was reduced by 56% as compared to the parental, drug sensitive Ehrlich cells. Kinetic experiments indicated that taurine uptake in Ehrlich cells occurs via both high- and low-affinity transporters. The maximal rate constant for the initial taurine uptake was reduced by 45% (high-affinity system) and 49% (low affinity system) in the resistant subline whereas the affinity of the transporters to taurine was unchanged. By immunoblotting we identified 3 TauT protein bands in the 50–70 kDa region. A visible reduction in the intensity of the band with the lowest molecular weight was observed in resistant cells. Quantitative RT-PCR indicated a significant reduction in the amount of taurine transporter mRNA in the resistant cells. Drug resistance in DNR Ehrlich cells is associated with overexpression of the mdr1 gene product P-glycoprotein (P-gp). Using 5 progressively DNR resistant Ehrlich cell sublines with different P-gp expression pattern no correlation between taurine uptake and P-gp expression was found. Taurine uptake in MDR1 transfected NIH/3T3 mouse fibroblasts was in contrast to the findings in Ehrlich cells increased compared to the parental fibroblasts. It is concluded that the reduced taurine uptake in resistant Ehrlich cells reflects a down regulation of the taurine transporter at the mRNA and protein level and it is most probably not related to P-gp overexpression. Received October 22, 2001 Accepted November 26, 2001     

The effects of haem on translational control of protein synthesis in cell-free extracts from fed and lysine-derived Ehrlich ascites tumour cells

1. Addition of haem to cell-free extracts from Ehrlich ascites tumour cells stimulates protein synthesis only in extracts from cells previously incubated in nutritionally complete conditions. Extracts from amino-acid-deprived cells do not respond to haem. The stimulation of protein synthesis in fed cell extracts is due to increased initiation on endogenous mRNA mediated by an increase in the levels of 40-S-subunit X Met-tRNA initiation complexes. Extracts from starved cells exhibit a defect in 40-S initiation complex formation which cannot be overcome by haem. 2. Experiments to test for the presence of an inhibitor of initiation in Ehrlich cell extracts by monitoring effects on translation in haem-supplemented reticulocyte lysates have revealed that extracts from both fed and starved cells contain one or more inhibitory activities which shut off protein synthesis, dissagregate polysomes and reduce the level of 40-S initiation complexes in the lysate. Extracts from starved cells are more inhibitory for protein synthesis than those from fed cells. 3. Initiation factor eIF-2 is phosphorylated by an endogenous Ehrlich cell protein kinase in vitro, but this occurs to the same extent in extracts from fed and starved cells. 4. We propose a possible model for the role of eIF-2 in the control of protein synthesis by amino acid supply in Ehrlich cells.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.     

Dehydrogenases involved in the conversion of succinate to 4-hydroxybutanoate by Clostridium kluyveri.

A pathway of succinate fermentation to acetate and butanoate (butyrate) in Clostridium kluyveri has been supported by the results of 13C nuclear magnetic resonance studies of the metabolic end products of growth and the detection of dehydrogenase activities involved in the conversion of succinate to 4-hydroxybutanoate (succinic semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase). C. kluyveri fermented [1,4-13C]succinate primarily to [1-13C]acetate, [2-13C]acetate, and [1,4-13C]butanoate. Any pathway proposed for this metabolism must account for the reduction of a carboxyl group to a methyl group. Succinic semialdehyde dehydrogenase activity was demonstrated after separation of the crude extracts of cells grown on succinate and ethanol (succinate cells) by anaerobic nondenaturing polyacrylamide gel electrophoresis. 4-Hydroxybutanoate dehydrogenase activity in crude extracts of succinate cells was detected and characterized. Neither activity was found in cells grown on acetate and ethanol (acetate cells). Analysis of cell extracts from acetate cells and succinate cells by sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that several proteins were present in succinate cell extracts that were not present in acetate cell extracts. In addition to these changes in protein composition, less ethanol dehydrogenase and hydrogenase activity was present in the crude extracts from succinate cells than in the crude extracts from acetate cells. These data support the hypothesis that C. kluyveri uses succinate as an electron acceptor for the reducing equivalents generated from the ATP-producing oxidation of ethanol.     

Distribution of anti-RNA antibody-binding sites on the surfaces of Ehrlich ascites tumor cells

The distribution of anti-RNA antibody-binding sites on the surfaces of Ehrlich ascites tumor cells was studied by membrane immunofluorescence after binding anti-RNA antibody on the cell surfaces. Results showed that the cells formed caps after incubation with anti-RNA antibody at 4 degrees C and the elevation of their temperature to 37 degrees C. Pronase treatment of the cell surfaces completely inhibited reactivity with anti-RNA antibody. These results suggest that the RNAs on the surfaces of Ehrlich ascites tumor cells are linked to membrane protein membrane-bound cytoskeleton complexes.     

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength.

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Succinate transport by a ruminal selenomonad and its regulation by carbohydrate availability and osmotic strength

Washed cells of strain H18, a newly isolated ruminal selenomonad, decarboxylated succinate 25-fold faster than Selenomonas ruminantium HD4 (130 versus 5 nmol min-1 mg of protein-1, respectively). Batch cultures of strain H18 which were fermenting glucose did not utilize succinate, and glucose-limited continuous cultures were only able to decarboxylate significant amounts of succinate at slow (less than 0.1 h-1) dilution rates. Strain H18 grew more slowly on lactate than glucose (0.2 versus 0.4 h-1, respectively), and more than half of the lactate was initially converted to succinate. Succinate was only utilized after growth on lactate had ceased. Although nonenergized and glucose-energized cells had similar proton motive forces and ATP levels, glucose-energized cells were unable to transport succinate. Transport by nonenergized cells was decreased by small increases in osmotic strength, and it is possible that energy-dependent inhibition of succinate transport was related to changes in cell turgor. Since cells which were deenergized with 2-deoxyglucose or iodoacetate did not transport succinate, it appeared that glycogen metabolism was providing the driving force for succinate uptake. An artificial delta pH drove succinate transport in deenergized cells, but an artificial membrane potential (delta psi) could not serve as a driving force. Because succinate is nearly fully dissociated at pH 7.0 and the transport process was electroneutral, it appeared that succinate was taken up in symport with two protons. An Eadie-Hofstee plot indicated that the rate of uptake was unusually rapid at high substrate concentrations, but the low-velocity, high-affinity component could account for succinate utilization by stationary cultures.(ABSTRACT TRUNCATED AT 250 WORDS)