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.     

Heavy metals and spermatozoan motility. II. Turbidity changes induced by divalent cations and adenosinetriphosphate in sea urchin sperm flagella

The initial binding of synthetic polyribonucleotides and different RNAs to Ehrlich ascites cells has been studied using cell electrophoresis. When cells were pre-incubated in the presence of polylysine significant differences in the binding of different polyribonucleotides were observed. For example, Poly (A):Poly (U) caused a greater change in electrophoretic mobility than Poly (I):Poly (C). The increased electrophoretically detectable change after treatment with Ehrlich ascites RNA compared with E. coli tRNA could be correlated with increased uptakes of labelled Ehrlich ascites RNA compared with tRNA. An interesting observation was that when Poly (A) was added before Poly (U), a greater effect on electrophoretic mobility was found compared with the effects when the order of addition was reversed. It is suggested that copolymer formation from the homopolymers can occur at the cell surface.     

Elevated polyamine levels in erythrocytes of SLC:ICR mice inoculated with ehrlich ascites carcinoma cells

Ehrlich ascites carcinoma cells (4×10⁵ cells/mouse) were inoculated intraperitoneally in 7-week-old SLC:ICR mice, and polyamine levels in peripheral erythrocytes and in ascites cells were determined periodically. Polyamine levels in peripheral erythrocytes increased linearly until 10 days after cell inoculation, while ascites cells showed exponential growth.The effect of carbazilquinone on cellular growth and polyamine levels in erythrocytes was also studied. When 1 or 2mg/kg of carbazilquinone was injected intraperitoneally on day 4 or on day 7, cellular growth was suppressed and the survival time of the mice was lengthened. The polyamine levels in erythrocytes were also markedly decreased 3 days after the carbazilquinone injection.These results suggest that the polyamine levels in peripheral erythrocytes are closely related to the cellular growth of Ehrlich ascites carcinoma cells.     

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.     

Pyruvate kinase isoenzymes in chromatin extracts of Ehrlich ascites tumour, Morris hepatoma 7777 and normal mouse and rat livers

A pyruvate kinase (EC 2.7.1.40) variant inhibited by L-cysteine has been found in Ehrlich ascites tumour and Morris hepatoma 7777, but not in normal mouse and rat livers used for comparison. Chromatin extracts of all materials studied contained three pyruvate kinase isoenzymes (alpha, beta, gamma) which showed the greatest electrophoretic mobility in normal mouse and rat livers. The isoenzyme mobility diminished in both tumour chromatin extracts, and the slow migrating gamma isoenzyme exhibited sensitivity to L-cysteine inhibition. This gamma isoenzyme sensitive to L-cysteine might be considered as a tumour marker. All tumour pyruvate kinase isoenzymes were insensitive to normal signal molecules, i.e., to ATP and fructose 1,6-diphosphate, which regulate liver pyruvate kinase activity. It was, however, noted that the binding of pyruvate kinase isoenzymes to DNA is connected with a diminution in their catalytic activity.     

Cytochalasin B and the sialic acids of Ehrlich ascites cells

The effect of cytochalasin B (CB) on the electrophoretic mobility and density of ionized sialic acid groups at the surface of Ehrlich ascites cells was examined together with a biochemical assay of the total sialic acid content of treated and control cells. Sialic acid assays indicated that CB-treated cells had a greater amount of total sialic acid and sialic acid sensitive to neuraminidase than control cells/cell. Equal amounts of sialic acid were removable by neuraminidase treatment from control cells and cells pretreated with neuraminidase and subsequently cultured with CB. The electrophoresis results showed a decrease in electrophoretic mobility in the presence of CB which could be reversed by growth in CB-free medium. Neuraminidase treatment did not make a significant additional reduction in the mobility of CB-treated cells. CB also prevented the recovery of electrophoretic mobility of neuraminidase treated cells. The results suggest that while CB does not inhibit sialic acid synthesis, it does alter the expression of ionized sialic acid groups at the electrokinetic surface. CB-containing culture media could be re-utilized several times suggesting that CB is not significantly bound or metabolized by Ehrlich ascites cells.     

Negatively charged RNase-susceptible molecules on the surface of ascites tumor cells

RNase-susceptible ionogenic groups on the cell surface membranes of two leukemic and two nonleukemic strains of ascites tumor cells were studied by cell electrophoresis, DEAE-Sephadex A-25 column and paper chromatography, and indirect membrane immunofluorescence. RNase treatment of the nonleukemic ascites tumor cells (Ehrlich ascites tumor and Sarcoma 180) produced a significant reduction in their electrophoretic mobilities. When the cells were labeled with [3H]uridine then incubated with RNase, there was a marked increased in the radioactive nucleotides present in the incubation medium as compared to the results of the experiment with RNase-untreated controls. Indirect membrane immunofluorescence studies of nonleukemic ascites tumor cells suggest that the sites that react with anti-RNA antibody are distributed diffusely on their surfaces. RNase treatment of these cells markedly reduced their ability to react with the antibody. It thus appears that RNAs are present on the surface membrane of nonleukemic ascites tumor cells and that RNase digests these RNAs, removing negatively charged nucleotides from their electrophoretic surfaces. This results in a reduction in mobility. In contrast, leukemic ascites cells (L1210 and C1498) incubated with RNase showed no significant change in mobility or in the amount of nucleotides released into the incubation medium. Moreover, no fluorescence was found on the surface of cells examined by indirect membrane immunofluorescence. This suggests that leukemic ascites cells are devoid of RNAs on their surface.     

Purification and properties of mouse thymus thymidylate synthase. Comparison of the enzyme from mammalian normal and tumour tissues

Mouse thymus thymidylate synthase has been purified to apparent electrophoretic homogeneity and compared with the enzyme from mouse tumour L1210 and Ehrlich ascites carcinoma cells. The enzyme is a dimer composed of 35,000 mol. wt monomers. Mouse thymus and tumour enzymes exhibit allosteric properties reflected by cooperative binding of both dUMP and 5-fluoro-dUMP. Activation energy for the reaction, catalyzed by thymidylate synthase from mouse tumour but not from mouse thymus, lowers at temperatures above 34 degrees C, reflecting a change of rate-limiting step in dTMP formation. MgATP at millimolar concentrations inhibits mouse thymus enzyme.     

Cytophotometric assays of ribonucleic acids in Ehrlich tumor cells]

The RNA content was measured in individual mouse Ehrlich ascites tumour cells by absorption cytophotometry after gallocyanin chrome alum staining. The cellular RNA content doubles during the preparation for mitosis and is twice as high as in an hyperdiploid line in an hypertetraploid cell lines.     

Kinetic estimation of the base sequence complexity of poly(A)-containing mRNA in Ehrlich-ascites-tumor cells and its abundance in nuclear RNA.

Poly(A)-containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27,000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from polysomal poly(A)-containing mRNA up to an rot of 3,000 M . s. It was found that all classes of the polysomal poly(A)-containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2% of the total nuclear RNA sequences were expressed as total polysomal poly(A)-containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)-containing mRNA sequence complexity.     

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound ⁴⁵Ca²⁺ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca²⁺ concentration. Crosslinking experiments with BS³ showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg²⁺-ATPase activity of myosin in the presence of Ca²⁺.     

Heterogeneity of alkaline ribonuclease in the mouse and Ehrlich ascites cells.

The specific activity of alkaline RNase II was l00 to 1800 times higher in mouse pancreas than in mouse liver, serum, ascites fluid, and Ehrlich ascites cell grown intraperitoneally. Ehrlich ascites cells grown in cell culture medium had a much lower alkaline RNase II activity than cells grown intraperitoneally. Chromatography on CM-52 cellulose of acid- and heat-treated preparations showned a considerable heterogeneity of the mouse enzymes. Depending on the source of the extract, two to six forms fo alkaline RNase were eluted. Pancreatic extract contained two RNase forms. These also seemed to be present as minor components in preparations from other sources except Ehrlich ascites cells grown in vitro. Ehrlich ascites cells grown in vivo contained forms of the RNase which were not present in other extracts. Possible reasons for this heterogeneity were investigated. In addition to their stability to acid and heat the different RNase forms were similar in that they were much more active at alkaline pH than at acidic pH, they did not require divalent metal ions for activity, and they degraded RNA 'endonucleolytically.' Also, native DNA, denatured DNA, and poly A were poor substrates compared with RNA. Some differences seemed to exist, however, with respect to their abilities to degrade poly U and poly C and their sensitivities to the endogenous RNase inhibitor.     

Production of oxygen free radicals by Ehrlich ascites tumour cells: effect of lipids

Phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and platelet activating factor (PAF) stimulated the generation of oxygen free radicals (nitro-blue tetrazolium reduction) in Ehrlich ascites tumour (EAT) cells. PAF was effective at an optimal concentration of 4 muM, but was inhibited by BN 52021, a specific PAF antagonist. Lyso-PAF was ineffective. Inclusion of different lipids during incubation prior to the addition of PAF, resulted in the activation/inhibition of free radical generation. Among the phospholipids at a concentration of 50 mug/ml, the order of activation was phosphatidylserine > phosphatidylglycerol > phosphoinositides > phosphatidylinositol > phosphatidylethanolamine. Phosphatidylcholine was not effective, while sphingolipids were inhibitory. In addition, Ehrlich ascites tumour cells grown in mice under marginal vitamin A deficiency, showed an augmented production of free radicals compared to control cells. This was suppressed by exogenous addition of vitamin A or superoxide dismutase. These results suggest that membrane lipids and dietary factors like vitamin A probably function as physiological modulators in regulating the free radical generation.     

STUDIES ON CELL DEFORMABILITY : I. Effect of Surface Charge

The deformability of the surface membranes of Sarcoma 37 and Ehrlich murine ascites tumor cells was assessed by the pressure required to suck a hemispherical bulge from these cells into a micropipette. It was shown that treatment with neuraminidase allowed the cells to be deformed with significantly less suction, and that enzymatic treatment also produced a significant reduction in surface charge as determined by measurement of cellular electrophoretic mobility. It is suggested that the increase in cellular deformability may be related to charge reduction, and that the charge at the cell periphery may affect not only the magnitude of the potential energy barriers hindering contact between cells, but also the ease with which cells can form low radius of curvature probes in order to help overcome these barriers.     

Cellular Electrophoretic Mobility and the Mitotic Cycle

The electrophoretic mobility of RPMI No. 41 cells grown in suspension, parasynchronized by double thymidine blocking and cold shock, is reported. No. 41 cells have a higher electrophoretic mobility during the mitotic peak phase than at other times in the mitotic cycle. Treatment of parasynchronous cells by neuraminidase reduces the mobility to the same value irrespective of the stage of the cells in the mitotic cycle. The higher electrophoretic mobility of cells in mitotic peak phase is probably due to a higher surface charge density at this time, possibly caused by a higher concentration of ionized neuraminic acid carboxyl groups at the hydrodynamic shear layer. The mobility of nonsynchronous rapidly and slowly growing cells differs; neuraminidase reduces their mobility by proportionately similar amounts. The results suggest that the differences in mobility between rapidly and slowly growing cells cannot be accounted for exclusively by differences in the amount of neuraminic acid groups at the shear layer.     

Electrokinetic determinations of enzymatic susceptibilities of cell surface-associated RNA

Earlier investigations suggested, using electrokinetic evidence, that RNA is present at the surfaces of some types of cultured and freshly isolated cells. In this report, further investigations of the nature of cell surface RNA of cultured Ehrlich ascites (EAT) cells are reported. These experiments were carried out by determining the changes in electrophoretic mobility of EAT cells after treatment with several highly purified nucleases, neuraminidase, and hyaluronidase. The results suggested that cell surface RNA is located at surface sites separate from those susceptible to neuraminidase and hyaluronidase, that alpha and omega termini of RNA are absent from the electrokinetic surface, and that the RNA present at the cell surface might exist predominantly in a double-stranded form. A model is proposed in which cell surface RNA strand termini are buried out of the electrokinetic surface, but where RNA extends from these buried termini into the electrokinetic surface in loops.     

Cell surface negativity and the binding of positively charged particles

The binding of positively charged, colloidal ferric oxide particles to the surfaces of Ehrlich ascites tumour cells, before and after incubation with neuraminidase and/or ribonuclease, has been studied by electron microscopy. An attempt has been made to correlate the amount of binding observed, with the electrophoretic mobilities of the cells under similar conditions to those under which they were exposed to the ferric oxide. Although a progressive loss of staining was observed with progressive loss of cellular net surface negativity, neither property was predictable from knowledge of the other. Some of the difficulties inherent in quantitative staining techniques of this type are discussed.     

Ribosomal RNA on the surfaces of nonleukemic mouse ascites tumor cells

RNAs on the cell surfaces of two nonleukemic and two leukemic strains of mouse ascites tumor cells were studied by fractionating the RNAs released from the cell surface by gentle pronase treatment after sucrose density gradient centrifugation, by indirect membrane immunofluorescence that used anti-RNA antibody and by cell electrophoresis. RNA was extracted from the cell supernatants of Ehrlich ascites tumor and sarcoma 180 cells (nonleukemic) that had been treated or not treated with pronase (1 microgram/ml, 37 degrees C, 20 min) followed by sucrose density gradient centrifugation. It was clearly demonstrated that the amounts of ribosomal RNA (18S and 28S) released after pronase treatment were approximately 80% greater than that of nonpronase-treated cells. Ehrlich ascites tumor cells that had been treated with actinomycin D (100 micrograms/kg body weight of mouse, 16 h) in vivo released an amount of ribosomal RNA after pronase treatment only 20% greater than the value for untreated cells. Actinomycin D treatment greatly reduced both the cell surface negative charge and the cell surface immunofluorescence when rabbit anti-RNA antibody was used. Under the same experimental conditions with actinomycin D, only ribosomal RNA synthesis showed preferential inhibition, not the syntheses of poly A-containing messenger RNA, 4S or other small-size RNAs. In contrast, L1210 and C1498 cells (leukemic) showed no change in the amounts of ribosomal RNA released after pronase treatment. L1210 cells also showed no change in the surface negative charge after being treated with actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dephosphorylation of the small heat shock protein hsp25 by calcium/calmodulin-dependent (type 2B) protein phosphatase.

The dephosphorylation of the mouse small heat shock protein hsp25 within an extract obtained from Ehrlich ascites tumor cells is inhibited by the calcium chelator EGTA and at concentrations of microcystin-LR which are characteristic for inhibition of calcium/calmodulin-dependent (2B type) protein phosphatases. Furthermore, the dephosphorylation of hsp25 in the cell-free system derived from Ehrlich ascites tumor could be increased specifically by addition of the calcium/calmodulin-dependent (2B type) protein phosphatase calcineurin. Dephosphorylation of the heat shock protein hsp25 is also obtained in an in vitro system containing phosphorylated recombinant hsp25, 1 mM Ca2+, calmodulin, and calcineurin specifying hsp25 as the direct substrate for this enzyme. The expression of two isoforms of the catalytic subunit of the mouse calcium/calmodulin-dependent (2B type) protein phosphatases in Ehrlich ascites tumor cells is demonstrated by polymerase chain reaction using specific oligonucleotide primers to the catalytic and calmodulin-binding domain, respectively. Northern blot analysis using the amplified fragments as probes shows that the mRNA of one isoform of the mouse calcium/calmodulin-dependent protein phosphatase is of medium abundance in EAT cells. These data suggest a calcium/calmodulin-dependent dephosphorylation of the small stress protein in EAT cells also in vivo. Since it is known that heat shock increases the intracellular calcium level and that thermotolerance is influenced by calcium chelators, ionophores, and anti-calmodulin drugs, the changes in the degree of hsp25 phosphorylation induced by thermal stress resulting in an altered thermoresistance could be explained at least partially by the calcium/calmodulin-dependent dephosphorylation through protein phosphatases 2B.