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.     

Purification and properties of RNA polymerase I from Ehrlich ascites cells

DNA-dependent RNA polymerase I (or A) (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) was purified from Ehrlich ascites cells after solubilization from isolated nuclei. The purification was accomplished by a procedure involving initial precipitation with ammonium sulfate, following by chromatographies on DEAE-Sephadex and phosphocellulose ion exchange resins and gel filtration on Sepharose 6B. A chromatographically homogeneous enzyme was obtained which was purified about 2300-fold relative to nuclear extracts. The specific activity of the most purified enzyme fraction was 230 nmol of [3H]UTP incorporated into RNA per mg of protein in 10 min at 37 degrees C, which is similar to those reported for the highly purified RNA polymerase I from mouse myeloma and calf thymus. The elution position on Sepharose 6B gel filtration indicated a molecular weight of approx. 580 000. Analysis of the purified enzyme by polyacrylamide gel electrophoresis under nondenaturing conditions revealed only one protein band. Certain heterogeneity in the RNA polymerase I fractions was found in the early chromatographic steps, but not in the most purified fractions.     

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.     

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.     

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.     

An ultrastructural study of heterokaryons derived from Theileria parva-infected bovine lymphoblasts and Ehrlich ascites tumour cells

An ultrastructural study was made of Sendai virus induced heterokaryons derived from Theileria parva-infected lymphoblasts and Ehrlich ascites tumour cells. When fusion occurred, parasites were successfully integrated into the cytoplasm of the resulting heterokaryons where they appeared as morphologically normal macroschizonts. Homokaryon formation was also noted. This occurred frequently between Ehrlich ascites tumour cells and rarely with lymphoblasts. A small proportion of heterokaryons contained altered forms of T parva showing nuclear components, vesicular structures and paired organelles similar to the microshizont stage of the parasite.     

Switching the amino acid specificity of an aminoacyl-tRNA synthetase

The accuracy of protein synthesis essentially rests on aminoacyl-tRNA synthetases that ensure the correct attachment of an amino acid to the cognate tRNA molecule. The selection of the amino acid substrate involves a recognition stage generally followed by a proofreading reaction. Therefore, to change the amino acid specificity of a synthetase in the aminoacylation reaction, it is necessary to alleviate the molecular barriers which contribute its editing function. In an attempt to accommodate a noncognate amino acid into the active site of a synthetase, we chose a pair of closely related enzymes. The current hypothesis designates glutaminyl-tRNA synthetase (GlnRS) as a late component of the protein synthesis machinery, emerging in the eukaryotic lineage by duplication of the gene for glutamyl-tRNA synthetase (GluRS). By introducing GluRS-specific features into the Rossmann dinucleotide-binding domain of human GlnRS, we constructed a mutant GlnRS which preferentially aminoacylates tRNA with glutamate instead of glutamine. Our data suggest that not only the transition state for aminoacyl-AMP formation but also the proofreading site of GlnRS are affected by that mutation.     

Aminoacylation of tRNA in the evolution of an aminoacyl-tRNA synthetase

Aminoacyl-tRNA synthetases catalyze aminoacylation of tRNAs by joining an amino acid to its cognate tRNA. The selection of the cognate tRNA is jointly determined by separate structural domains that examine different regions of the tRNA. The cysteine-tRNA synthetase of Escherichia coli has domains that select for tRNAs containing U73, the GCA anticodon, and a specific tertiary structure at the corner of the tRNA L shape. The E. coli enzyme does not efficiently recognize the yeast or human tRNACys, indicating the evolution of determinants for tRNA aminoacylation from E. coli to yeast to human and the coevolution of synthetase domains that interact with these determinants. By successively modifying the yeast and human tRNACys to ones that are efficiently aminoacylated by the E. coli enzyme, we have identified determinants of the tRNA that are important for aminoacylation but that have diverged in the course of evolution. These determinants provide clues to the divergence of synthetase domains. We propose that the domain for selecting U73 is conserved in evolution. In contrast, we propose that the domain for selecting the corner of the tRNA L shape diverged early, after the separation between E. coli and yeast, while that for selecting the GCA-containing anticodon loop diverged late, after the separation between yeast and human.     

Differential response of human melanoma and Ehrlich ascites cells in vitro to the ribosome-inactivating protein luffin

OBJECTIVE: To analyze the short- and medium-term results of the Burch-like urethropexy with bone anchors in the treatment of genuine stress urinary incontinence. METHODS: We performed the conventional Burch technique which was modified with the use of 4 bone anchors for bony fixation. Forty-four female patients with genuine stress urinary incontinence were operated on from November, 1995 to November, 1997. RESULTS: The patients had a bladder catheter indwelling for 4 to 9 days and only 3 of them required intermittent catheterization during two months. All patients recovered spontaneous micturition. The postoperative urinary continence was 93% at a mean follow-up of 11 months. CONCLUSIONS: Although our initial results seem encouraging, a continuous and objective follow-up is warranted to assess the long-term efficacy of this technique.     

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.     

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.     

Lipid components of aminoacyl-tRNA synthetase complex in eukaryotes and their function

This review includes data about lipids composition of eukariotic high molecular complex of aminoacyl-tRNA synthetases under normal conditions and under the effect of ionizing radiation. The role of different lipids in formation of the structure of high molecular complex and functioning is discussed. The role of different groups of phospholipids, prostaglandins in providing the normal work of high molecular complex and some synthetases in control and pathological state is considered. A conclusion has been made that different groups of lipids composed the high molecular complex aminoacyl-tRNA synthetases. Some of them play structural role and other (prostaglandins, some phospholipids) are the regulatory components of the complex.