Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line

Summary The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3, under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2′-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanism(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5°C. (b) The concentration limits for BrdU incorporation are 5 to 100 μM; baseline frequency is about 11 SCE/metaphase (constant up to 20 μM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 μM) cause a decrease of SCE rates below that found at 100 μM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division. This work was supported by a grant from the Medizinisch-wissenschaftlicher Fond des Bürgermeisters der Bundeshauptstadt Wien.     

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line

The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3 , under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2'-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanisms(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5 degrees C. (b) The concentration limits for BrdU incorporation are 5 to 100 microM; baseline frequency is about 11 SCE/metaphase (constant up to 20 microM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 microM) cause a decrease of SCE rates below that found at 100 microM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division.     

Effect of noradrenaline on rest-dependent and excitation-dependent exchange of calcium in the ventricular muscle of guinea-pig heart

The effect of noradrenaline (NA) on the "contractile calcium" was investigated in the ventricular muscle of guinea-pig heart. It was found that NA in doses increasing the steady-state contractile force by 50-100% increased the beat-to-beat exchange of 45Ca (Ca1 fraction) by about 50%, and significantly increased Ca2 fraction which is lost from the cells at rest and re-accumulated during subsequent stimulation. It has been proposed elsewhere that this fraction may control the force of contractile response to the triggering Ca1 fraction. It is concluded that NA increases beat-to-beat Ca exchange and affects intracellular Ca distribution.     

Subcellular localization of LGN during mitosis: evidence for its cortical localization in mitotic cell culture systems and its requirement for normal cell cycle progression

Mammalian LGN/AGS3 proteins and their Drosophila Pins orthologue are cytoplasmic regulators of G-protein signaling. In Drosophila, Pins localizes to the lateral cortex of polarized epithelial cells and to the apical cortex of neuroblasts where it plays important roles in their asymmetric division. Using overexpression studies in different cell line systems, we demonstrate here that, like Drosophila Pins, LGN can exhibit enriched localization at the cell cortex, depending on the cell cycle and the culture system used. We find that in WISH, PC12, and NRK but not COS cells, LGN is largely directed to the cell cortex during mitosis. Overexpression of truncated protein domains further identified the Galpha-binding C-terminal portion of LGN as a sufficient domain for cortical localization in cell culture. In mitotic COS cells that normally do not exhibit cortical LGN localization, LGN is redirected to the cell cortex upon overexpression of Galpha subunits of heterotrimeric G-proteins. The results also show that the cortical localization of LGN is dependent on microfilaments and that interfering with LGN function in cultured cell lines causes early disruption to cell cycle progression.     

Effect of disinfectants on variola virus in cell culture.

Twenty kinds of disinfectants were examined for ability to inactivate variola virus. Cytopathic effect and plaque formation on monolayer cultures of an established monkey kidney cell line were used as indicators of virus inactivation. A micromethod using microplate cultures, and not requiring a CO2 incubator, was adopted. The procedures were straightforward, showing good reproducibility. Among the compounds tested, several were found to be superior because of the minimum concentrations required for complete inactivation of virus. The purified viruses were shown to be more sensitive to the compounds than were the crude samples. The virus inactivation kinetics curves were determined by plaque counting. The usefulness of this method for quantitative analysis of disinfecting effect is suggested.     

Effect of disinfectants on variola virus in cell culture.

Twenty kinds of disinfectants were examined for ability to inactivate variola virus. Cytopathic effect and plaque formation on monolayer cultures of an established monkey kidney cell line were used as indicators of virus inactivation. A micromethod using microplate cultures, and not requiring a CO2 incubator, was adopted. The procedures were straightforward, showing good reproducibility. Among the compounds tested, several were found to be superior because of the minimum concentrations required for complete inactivation of virus. The purified viruses were shown to be more sensitive to the compounds than were the crude samples. The virus inactivation kinetics curves were determined by plaque counting. The usefulness of this method for quantitative analysis of disinfecting effect is suggested.     

Mitochondrial calcium uptake in the perfused contracting rat heart and the influence of epinephrine on calcium exchange

Perfused rat hearts were exposed to solutions containing ⁴⁵Ca²⁺ with and without epinephrine. They were subjected to differential centrifugation and the distribution of Ca and ⁴⁵Ca in mitochondria and microsomes was determined. It was found that the mitochondria contain most of the calcium of the intact heart and that the exchange of mitochondrial calcium with extracellular calcium was extremely rapid. This process was accelerated in hearts stimulated by epinephrine.     

Kinetic analysis of calcium movements in cell culture

Summary The distribution of intracellular calcium was determined in isolated kidney cells by kinetic analyses of⁴⁵Ca fluxes. Isotopic desaturation curves reveal an intracellular calcium compartment with a very slow time constant. The size of this calcium compartment is markedly increased by raising the extracellular calcium, by increasing the extracellular phosphate and may contain up to 99% of the intracellular exchangeable calcium. Accumulation of calcium in this pool is completely abolished by two specific inhibitors of mitochondrial calcium uptake, Antimycin A and Warfarin^®. These results suggest that this compartment represents a pool of calcium in the cell mitochondria. The sudden removal of phosphate from the medium immediately stimulates calcium efflux from the cell. Conversely, an increase in medium phosphate immediately inhibits calcium efflux. Both effects are rapidly reversible. Finally, calcium efflux from the cells is stimulated after the cells are exposed to low temperature suggesting that calcium transport out of the cell may be regulated by the cytoplasmic calcium activity. These experiments are consistent with the view that mitochondria play an important role in the control and regulation of cytoplasmic calcium activity and of calcium transport.     

Ultrastructural localization of calcium deposits during in vitro culture of pig oocytes

Calcium deposits were localized using the combined oxalate-pyroantimonate technique in follicle-enclosed oocytes fixed in situ. These deposits can be observed within vacuoles, mitochondria, and on the surface of yolk granules as well as in the caryoplasm, but are absent from the endoplasmic reticulum. Isolation of the oocyte from the follicle resulted in the immediate depletion of these calcium deposits. Replenishment of these deposits started during the first 8 hr of in vitro culture of the oocyte and they were gradually replenished to the levels observed before the liberation of oocytes during in vitro maturation to the stage of metaphase II.     

Effect of temperature on calcium exchange in digitonin-treated rat ventricular myocytes

The effect of a change in temperature on net mitochondrial Ca2+ exchange has been investigated in a suspension of adult rat ventricular myocytes. Temperature was varied between 42 degrees C and 15 degrees C. Hypothermia reduced the initial rate of respiration-dependent Ca2+ uptake and reduced the Na+-sensitivity of Ca2+ efflux. The net result of these alterations is that at low temperatures, the Ca2+ level at which a steady-state between mitochondria and sarcoplasm is maintained, will be raised.     

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration

Membrane currents and changes in intracellular calcium ion concentration ([Ca²⁺]_i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca²⁺-indicator, fura-2, and changes in [Ca²⁺]_i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs⁺, Ni²⁺), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni²⁺-sensitive current elicited by ramp repolarization from +90 mV to –80 mV. Ramps were sufficiently rapid that little change in [Ca²⁺]_i occured during the ramp. The (constant) [Ca²⁺]_i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni²⁺-sensitive ramp current varied linearly with 1n([Ca²⁺])_i. The I-V relations at different [Ca²⁺]_i over the range –60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca²⁺]_i had changed. The relationship between [Ca²⁺]_i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca²⁺-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca²⁺ to the exchanger during the [Ca²⁺]_i-transient and the effects of membrane voltage on the exchanger.     

Effect of drugs on Trypanosoma cruzi and on its interaction with heart muscle cell "in vitro"

Megazol, nifurtimox, benznidazol and allopurinol were investigated, by light and electron microscopy, for their action on T. cruzi. Both the direct effect upon amastigote and trypomastigote forms and the effect upon the interaction of heart muscle cells (HMC) with bloodstream trypomastigotes were studied. The proliferation of amastigotes in Warren medium was inhibited in a dose-dependent manner by megazol, nifurtimox and benznidazol. Treatment of amastigotes (25-50 microM/24 h) and trypomastigotes (25 microM/24h) led to several ultrastructural alterations in the parasites. These three drugs also had a potent effect on the treatment of infected heart muscle cells when added at the beginning of the interaction or after one or three days of infection. The interiorized parasites showed a similar pattern of ultrastructural alterations as observed by the direct effect on the amastigotes. The primary heart muscle cell culture proved to be a suitable model for the study of drugs on intracellular parasites. Likewise, the amastigote proliferation in axenic medium was shown to be an adequate assay for an initial trial of drugs. These parameters seem very reliable to us for a systematic investigation of the mechanism of action of new drugs.     

Effect of calcium, phosphate and nitrogen on cell growth and biosynthesis of cell wall polysaccharides by Silene vulgaris cell culture

Medium nutrients such as calcium, phosphorus, nitrogen and nitrate to ammonium ratio have significant influence on the growth, biosynthetic and biochemical characteristics of polysaccharides produced by Silene vulgaris (M.) G. cell culture. Cell growth and production of polysaccharides was limited by an absence of any of these components in the medium. Optimal growth of the callus and production of arabinogalactan were achieved at 1.5-4.5 microM calcium while the optimal production of pectin named silenan was observed at 3.0-4.5 microM. The phosphate contents in the medium in the range of 0.63-3.75 microM were favorable for callus growth. Production of silenan was maximal at 1.25-3.75 microM phosphate. Optimal growth of the callus was achieved at 30-90 microM nitrogen. Maximal production of silenan was observed at 60 microM nitrogen while the optimal production of arabinogalactan was at 90 microM nitrogen (at ratio of NH(4)(+):NO(3)(-) as 1:2). A presence both of nitrate and ammonium is needed for the silenan biosynthesis (the NH(4)(+):NO(3)(-) ratio as 1:1 and 1:2). Yields and volumetric production of arabinogalactan were maximal at deletion of ammonium from the nutrient medium (ratio 0:1). Absence of calcium or nitrogen in the medium leads to a decrease of the galacturonic acid residues in silenan. The galactose residues contents in arabinogalactan were decreased in the absence of nitrogen and calcium in the medium.     

Changes in intercellular exchange intensity in an L cell culture]

Intercellular junctions permeable to fluorescein Na were studied with the aid of intracellular glass microelectrodes in the cultures of transformed mouse-embryo cells (L-strain). The degree of coupling was correlated with the cell culture density. In addition, the degree of coupling increased after short-time incubated of the cells with 1mM lanthanum. Such an incubation did not influence other features of cells: the value of the membrane potential, the character of growth or the inclusion of the vital dye neutral red. Some other factors stimulated cellular aggregation (concanavalin A, protamine, decreasing the pH) but did not influence the degree of coupling.     

Excitation-contraction coupling in heart. XIX. Effect of hypoxia on calcium transport by subcellular particles in the isolated perfused rat heart.

To examine the role of changes in calcium transport by subcellular particles in the pathogenesis of contractile failure due to oxygen lack, both mitochondrial and microsomal fractions were obtained from the isolated hypoxic rat hearts and their calcium binding and uptake abilities were determined by the Millipore filtration technique. The contractile force decreased by about 40, 60 and 70% of the control within 5, 10 and 30 min respectively, of perfusing the heart with hypoxic medium containing glucose. In hearts perfused for 10 min with hypoxic medium containing glucose, calcium binding and uptake by the microsomal fraction decreased significantly. However, mitochondrial calcium binding, but not uptake, decreased significantly on perfusing the hearts with hypoxic medium containing glucose for 20 to 30 min when the microsomal calcium transport was markedly depressed. Reduction in contractile force, calcium binding and uptake by the microsomal fraction as well as calcium binding by mitochondria of hearts made hypoxic for 30 min recovered towards normal upon reperfusion with control medium for 15 min. On the other hand, omitting glucose from the hypoxic medium significantly decreased calcium binding by mitochondrial and microsomal fractions within 10 min of perfusion in comparison to the control and accelerated the effects of hypoxia upon contractile force and microsomal calcium uptake. In contrast to the hypoxic hearts, the mitochondrial calcium uptake decreased significantly and the magnitude of depression in the microsomal calcium binding was appreciably greater in hearts made to fail to a comparable degree upon perfusion with substrate-free medium. The observed defects in calcium transporting properties of microsomal and mitochondrial membranes appear secondary to the contactile failure in hypoxic hearts.