Dynamics of a 3H-lysine pulse label in the culturing of L cells

Using autoradiography, 3H-lysine, 3H-thymidine, and 3H-tryptophane puls labeled L-cells were examined as long as through four passages. Our studies demonstrate that no renewal of 3H-lysine labels takes place in chromosomes and nuclei. Unlike, the cytoplasmic labels of 3H-lysine and chromosomal, nuclear and cytoplasmic labels of 3H-tryptophane showed an intensive renewal. A question of renewal of lysine-rich histones is discussed.     

Rhythm of protein concentration and incorporation of 3H-lysine into hepatocytes in vitro]

"In vitro" incubated slices of the rat liver, by the method of interferometry combined with radioautography, have demonstrated hour's rhythmic fluctuations in protein concentration and 3H-lysine incorporation into proteins. The biochemical method has demonstrated fluctuations in protein synthesis rate in the same cells with an average period of about one hour. The possibility to apply the interferometric method in combination with radioautography for revealing quantitative changes of protein in slices of organic cultures is demonstrated.     

Simultaneous photometry of the DNA and silver-grain count in liver cells labelled with 3H-thymidine or 3H-leucine

A modified method was proposed for reflected light simultaneous measurement of DNA content and of the silver grain number in the nucleus or cytoplasm of the same cell. Specimens-smears of isolated liver cells incorporated 3H-thymidine and 3H-leucine were prepared on coverslips and after processing were mounted on the slide glasses with smeared side facing downwards to avoid the influence of grains on DNA content measurements. To decrease the background, label measurements were carried out in polarized light. It was shown that the intensity of 3H-leucine incorporation in hepatocytes increases proportionally with cell ploidy degree.     

Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts

Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with ³H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.     

Incorporation of 3H-leucine, 3H-lysine, and 3H-tryptophan during the cell cycle of Chinese hamster ovary cells: a flow cytometric analysis

Exponentially growing Chinese hamster ovary cells were pulse labeled with 3H-leucine, 3H-lysine, or 3H-tryptophan, fixed, and stained by either the acriflavine-Feulgen procedure or with fluorescein isothiocyanate (FITC). Protein content as determined by FITC fluorescence was representative of protein content determined biochemically by the method of Lowry. Utilizing a fluorescence-activated cell sorter, 3H-labeled cells were sorted according to their DNA or protein content and the incorporation of 3H-leucine, 3H-lysine, or 3H-tryptophan determined by liquid scintillation counting. The rates of 3H-leucine, 3H-lysine, and 3H-tryptophan incorporation increased with respect to increasing DNA content (G1, mid-S, G2+M). The rate of 3H-lysine incorporation increased continuously with increasing protein content, whereas the rates of 3H-leucine and 3H-tryptophan incorporation were constant initially with an increase in incorporation near mid-cycle followed by a slight decrease. Matrix algebra modeling of the increase in protein content suggests that 3H-lysine incorporation is consistent with a sigmoidal increase in protein content, however, 3H-leucine and 3H-tryptophan incorporation do not follow either the exponential, linear, or sigmoidal models. Matrix algebra simulation of the FITC protein distribution indicates that while the rate of protein accumulation is not linear, the exponential and sigmoidal models fit the experimental data equally well.     

Autoradiographic and liquid scintillation assessment of beta-amino-propionitrile-induced inhibition of collagen maturation using 3H-proline and 3H-lysine

BAPN (0.1 mg/day) was injected into chick embryos for 4 days starting on the 7th day of incubation. On the 11th day, the embryos were administered either 3H-proline or 3H-lysine. 36 h later, the incorporation of each isotope by the periosteal osteogenic cells as well as into bone matrix was investigated by autoradiography. The incorporation of the two isotopes into whole bones was assessed by liquid scintillation counting. 3H-proline incorporation into the cellular or matrical compartments was unaffected by treatment. As compared to the controls, 3H-lysine label in BAPN-treated embryonic bones was significantly higher in the cellular compartment but was reduced over the bone matrix. The data provide the first direct morphological evidence that BAPN probably induces certain changes in the maturation of collagen involving lysyl residues which result in an inhibition of cross-linkage formation in collagen.     

Effects of [3H]UdR on the cell-cycle progression of L1210 cells

Tritium-labelled uridine [( 3H]UdR) perturbs progression of L1210 cells through the mitotic cycle. The main effect manifests as a slowdown or arrest of a portion of cells in G2 and is already observed 2 hr after addition of 0.5-5.0 microCi/ml of [3H]UdR into cultures. At 2.5-5.0 microCi/ml of [3H]UdR a slowdown of cell progression through S is also apparent. Additionally, there is an increase in the number of cells with DNA values higher than 4C in cultures growing in the presence of [3H]UdR for 8-24 hr. A pulse of [3H]UdR of 2 hr duration labels predominantly (95%) cellular RNA. The first cell-cycle effects (G2 slowdown) are observed when the amount of the incorporated [3H]UdR is such that, on average there are fewer than thirty-six [3H] decays per cell which corresponds to approximately 12-19 rads of radiation. The S-phase slowdown is seen at a dose of incorporated [3H]UdR twice as high as that inducing G2 effects. The specific localization of [3H]UdR in nucleoli, peripheral nucleoplasm and in cytoplasm, as well as differences in the kinetics of the incorporation in relation to phases of the cell cycle are discussed in the light of the differences between the effects of [3H]UdR and [3H]thymidine. Mathematical modelling of the cell-cycle effects of [3H]UdR is provided.     

The extraction and purification of DNA labelled with [methyl-3H]thymidine in aquatic bacterial production studies

DNA from natural populations of aquatic bacteria labelled withtritiated thymidine can be accurately and easily separated,on membrane filters, from other labelled macromolecules.     

Initiation and growth of microtubules from mitotic centers in lysed mammalian cells

Metaphase PtK1 cells, lysed into polymerization-competent microtubule protein, maintain a spindle which will gain or lose birefringence depending on the concentration of disassembled tubulin subunits used in the lysis medium. Concentrations of tubulin subunits greater than the equilibrium monomer value promote a rate and extent of birefringence increase that is proportional to the subunit concentration. Increase in spindle birefringence can be correlated with an increase in tubule number, though the relationship is not strictly linear. Increase in spindle tubule number is due to an vivo-like initiation of tubules at the mitotic centers, as well as tubulin addition onto pre-existing spindle fragments. Colcemid-treated prometaphase cells lysed into polymerization-competent tubulin develop large asters in the region of the centrioles and short tubules at kinetochores, making it unlikely that all microtubule formation in lysed cell preparations is dependent on tubulin addition to short tubule fragments. Asters can also form in colcemid-treated prometaphase cells lysed in tubulin that is incapable of spontaneous tubule initiation, suggesting that the centriolar region serves a tubule-initiator function in our lysed cell preparations. The ability of the centriole to initiate microtubule assembly is a time-dependent process-a ripening effect takes place between prophase and late prometaphase. Ripening is expressed by an increase in the number and length of tubules found associated with the centriolar region.     

Chemoattractant and guanosine 5''-[gamma-thio]triphosphate induce the accumulation of inositol 1,4,5-trisphosphate in Dictyostelium cells that are labelled with [3H]inositol by electroporation.

The analysis of the inositol cycle in Dictyostelium discoideum cells is complicated by the limited uptake of [3H]inositol (0.2% of the applied radioactivity in 6 h), and by the conversion of [3H]inositol into water-soluble inositol metabolites that are eluted near the position of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] on anion-exchange h.p.l.c. columns. The uptake was improved to 2.5% by electroporation of cells in the presence of [3H]inositol; electroporation was optimal at two 210 microseconds pulses of 7 kV. Cells remained viable and responsive to chemotactic signals after electroporation. The intracellular [3H]inositol was rapidly metabolized to phosphatidylinositol and more slowly to phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. More than 85% of the radioactivity in the water-soluble extract that was eluted on Dowex columns as Ins(1,4,5)P3 did not co-elute with authentic [32P]Ins(1,4,5)P3 on h.p.l.c. columns. Chromatography of the extract by ion-pair reversed-phase h.p.l.c. provided a good separation of the polar inositol polyphosphates. Cellular [3H]Ins(1,4,5)P3 was identified by (a) co-elution with authentic [32P]Ins(1,4,5)P3 and (b) degradation by a partially purified Ins(1,4,5)P3 5-phosphatase from rat brain. The chemoattractant cyclic AMP and the non-hydrolysable analogue guanosine 5'-[gamma-thio]triphosphate induced a transient accumulation of radioactivity in Ins(1,4,5)P3; we did not detect radioactivity in inositol 1,3,4-trisphosphate or inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In vitro, Ins(1,4,5)P3 was metabolized to inositol 1,4- and 4,5-bisphosphate, but not to Ins(1,3,4,5)P4 or another tetrakisphosphate isomer. We conclude that Dictyostelium has a receptor- and G-protein-stimulated inositol cycle which is basically identical with that in mammalian cells, but the metabolism of Ins(1,4,5)P3 is probably different.