[3H]Inositol incorporation into phosphoinositides of pig reticulocytes

Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP₂) of pig reticulocytes were extensively labelled when these cells were incubated with [³H]inositol. In marked contrast, a total lack of [³H]inositol labelling of phosphoinositides was observed in mature erythrocytes. Phosphoinositides of both reticulocytes and mature erythrocytes were labelled with ³²P but the labelling in reticulocytes was several-fold higher than in mature erythrocytes. Inclusion of Ca²⁺ (2 mM) + ionophore A23187 (2 μg/ml) during the labelling experiments substantially reduced the radioactivity incorporation into phosphoinositides of reticulocytes. When [³H]inositol-prelabelled reticulocytes were treated with Ca²⁺ + A23187 the levels of radioactive PI and PIP₂ did not change significantly. However, the PIP pool exhibited a remarkable sensitivity to Ca²⁺ as shown by a 75% increase in its radioactivity over the control. The ability to incorporate [³H]inositol into phosphoinositides remains transitorily intact in the reticulocyte stage. Thus, pig reticulocytes offer a suitable model in which to explore the physiological role of phosphoinositides in relation to cellular maturation process.     

Trypanosoma cruzi: incorporation of [3H]-palmitic acid and [3H]-galactose into components shed by trypomastigotes.

Trypomastigotes were metabolically labeled with [3H]-palmitic acid or [3H]-galactose and labeled components were detected in the culture medium. Thin layer chromatography of the shed material showed several lipids in the [3H]-palmitic acid labeled sample while the sugar was mainly incorporated into macromolecules. The material incorporated with the lipidic precursor was fractionated by DEAE-Sephadex (acetate form) and the amount of radioactivity was ten times higher in the acidic lipids than in the neutral lipids. When acidic lipids were further separated by Unisil, 73% of the radioactivity was recovered in the less polar fraction. Different patterns were obtained on comparison of the shed components with the lipids remaining in the parasite.     

Autoradiographic analysis of the effect of potassium orotate on the intensity of uridine-5 3H and proline-3H incorporation into wound fibroblasts]

The intensity of uridine-53H and proline-3H incorporation into fibroblast nuclei and cytoplasm as well as into intercellular spaces was studied in wound healing of mice given potassium orotate and in the control animals. It was shown that proline-3H incorporation was more intensive than that of uridine-53H. At the same time a significant increase in RNA synthesis in fibroblasts under the action of potassium orotate, and a less pronounced rise in proline-containing protein synthesis were noted in similar conditioelationships correlate with the fibroblast ultrastructural changes in the process of their differentiation.     

In vivo incorporation of [2-3H]-myo-inositol into frog opsin

The in vivo incorporation of [2-3H]-myo-inositol into frog retinal rod outer segment membranes was examined. About 25% of the recovered radioactivity was found to be protein-associated. Following acid hydrolysis of this material and extraction with hexane, all the radioactivity remained in the aqueous phase, indicating that the label was not in fatty acids. Following ion exchange column chromatography of the hydrolysate, the major radioactive compound comigrated on TLC with an internal standard of [U-14C]-myo-inositol. SDS polyacrylamide gel electrophoresis of unextracted membranes indicated that the majority of the label was associated with opsin. These results indicate that [2-3H]-myo-inositol was incorporated in vivo into opsin, presumably with retention of its chemical identity.     

Tissue-specific inhibition of [3H]thymidine incorporation into DNA by carcinogenic N-nitrosamines

The N-nitrosamines N-nitrosodimethylamine (DMN), N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were injected intraperitoneally 24 h before sacrifice in F344 rats and C57BL mice in doses of 297 mumoles/kg b.w. and 148 mumoles/kg b.w., respectively. 2 h before sacrifice, the animals were given an intraperitoneal injection of [3H]thymidine. The results showed that the examined N-nitrosamines inhibited the incorporation of [3H]thymidine into DNA in a few tissues of the rats and the mice. The results indicated that the N-nitrosamines exerted a tissue-specific inhibition of the [3H]thymidine incorporation in the tissues reported to be involved in the biotransformation of these substances. The observed inhibitory effects on the incorporation of [3H]thymidine by DMN, NNN and NNK were also correlated to a considerable extent to the reported sites of carcinogenicity. The present study indicates that measurements of [3H]thymidine incorporation into DNA in various tissues of experimental animals is a useful short-term bioassay to evaluate the potential tissue-specific carcinogenicity of the N-nitrosamines. The method may also be useful as a complement to other short-term in vivo tests in the screening of potential genotoxicity of several other chemicals.     

Autoradiographic analysis of the incorporation of uridine-H3 into hyperchromic motoneurons of mouse spinal cord]

Under conditions of local administration of uridine-H3 solution a study was made of the dynamics of the isotope incorporation into the RNA of the nuclei and the cytoplasm of the perikarion of the hyperchromic motor neurons of the ventro-lateral nucleus of mouse spinal cord. An increase in the content of hyperchromic neurocytes in the population under study was reached in mice as a result of swimming for four hours. A different character of the label regression curves for the hyperchromic and normochromic motor neurons was shown.     

3H]Inositol incorporation into phosphoinositides of pig reticulocytes

Phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) of pig reticulocytes were extensively labelled when these cells were incubated with [3H]inositol. In marked contrast, a total lack of [3H]inositol labelling of phosphoinositides was observed in mature erythrocytes. Phosphoinositides of both reticulocytes and mature erythrocytes were labelled with 32P but the labelling in reticulocytes was several-fold higher than in mature erythrocytes. Inclusion of Ca2+ (2 mM)+ ionophore A23187 (2 micrograms/ml) during the labelling experiments substantially reduced the radioactivity incorporation into phosphoinositides of reticulocytes. When [3H]inositol-prelabelled reticulocytes were treated with Ca2+ + A23187 the levels of radioactive PI and PIP2 did not change significantly. However, the PIP pool exhibited a remarkable sensitivity to Ca2+ as shown by a 75% increase in its radioactivity over the control. The ability to incorporate [3H]inositol into phosphoinositides remains transitorily intact in the reticulocyte stage. Thus, pig reticulocytes offer a suitable model in which to explore the physiological role of phosphoinositides in relation to cellular maturation process.     

High incorporation of [3H]inositol into phosphoinositides of human platelets during reversible electropermeabilisation

A new method for high incorporation of [3H]inositol into human platelets is described. The method involves incorporation of [3H]inositol during reversible electropermeabilisation by high voltage discharge, followed by resealing the cells during incubation at 37 degrees C. Between 10- and 20-fold increase of isotope uptake is achieved compared to control intact cells. Permeabilised resealed platelets maintain good responses to thrombin and collagen. Analysis of the incorporation of the label amongst the phosphoinositides shows 70% to be in PI, 20% in PIP, and 10% in PIP2. Stimulation with thrombin and analysis of the formation of IP1, IP2 and IP3 shows the labelling to occur in a hormone-sensitive pool. These studies indicate that reversible electropermeabilisation can be used to achieve good uptake of non-membrane penetrating substances such as inositol.     

Effects of FSH and LH on incorporation of [3H]thymidine into follicular DNA

To assess the roles of FSH and LH on follicular growth, after various experimental manipulations, hamster follicles were sorted into 10 stages and incubated for 4 h with [3H]thymidine. Stages 1-4 correspond to follicles with 1-4 layers of granulosa cells, respectively; Stage 5 = 5 or 6 layers of granulosa cells plus theca; Stage 6 = 7-8 layers of granulosa cells plus theca; Stage 7 = early formation of the antrum; Stages 8-10 = small, intermediate and large antral follicles, respectively. Phenobarbitone sodium injected at 13:00 h on pro-oestrus blocked the normal rise of blood FSH and LH concentrations at 15:00 h and prevented the increase of [3H]thymidine incorporation into follicles of Stages 1-9. The optimal treatment to reverse the effects of phenobarbitone was 1 microgram FSH and 2 micrograms LH injected i.p. at 13:00 h which restored DNA replication to follicles of Stages 2-10: FSH acted primarily on Stages 2-5 and LH on Stages 5-10. Injection of phenobarbitone at 13:00 h on prooestrus followed by 2.5 micrograms FSH at 22:00 h restored DNA synthesis by the next morning to follicles at Stages 1-8. In hamsters hypophysectomized at 09:00 h on the day of oestrus (Day 1), injection on Day 4 of 2.5 micrograms FSH restored DNA synthesis 6 h later to Stage 2-6 follicles. Unilateral ovariectomy on Day 3 resulted 6 h later in an acute rise in FSH and LH and change of follicles from Stage 4 to Stage 5 but, paradoxically, there was decreased synthesis of DNA in follicles of Stages 5-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]thymidine incorporation into whole liver as an alternative to [3H]thymidine incorporation into DNA as a parameter of cell proliferation in regenerating liver tissue in rats

OBJECTIVE: To monitor liver regeneration following partial hepatectomy, liver cell proliferation can be measured by assaying in vivo [3H]thymidine incorporation into liver cell DNA. We hypothesized that [3H]thymidine incorporation into whole liver tissue parallels [3H]thymidine incorporation into liver cell DNA, both in high proliferating and low proliferating liver. STUDY DESIGN: Liver cell proliferation in rats after partial hepatectomy or a sham operation was studied by measuring incorporation of [3H]thymidine into various fractions of liver tissue on days 1, 2, 3, 4 and 10 after surgery. RESULTS: [3H]thymidine incorporation into whole liver tissue and in the protein fraction correlated well with DNA-specific [3H]thymidine incorporation into regenerating (r > .80, P < .0001) and nonregenerating liver (r > .69, P < .005). [3H]thymidine incorporation into DNA was < 5% of the total amount of administered [3H]thymidine in both sham-operated and hepatectomized rats. Significant differences in [3H]thymidine incorporation into partially hepatectomized livers as compared to sham-operated rat livers were found on days 1 and 2 (whole liver tissue and protein fraction) or day 1 (DNA) after surgery. CONCLUSION: [3H]thymidine incorporation into whole liver tissue is a simple technique that can be used for the study of liver cell proliferation after partial hepatectomy in rats.     

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

Quantitative analysis of in-vitro incorporation of [3H]thymidine into hamster follicles during the oestrous cycle

Follicles were isolated from hamster ovaries at 09:00 h and 15:00 h on each of the 4 days of the oestrous cycle (Day 1 = oestrus; Day 4 = pro-oestrus) by microdissection and by a mixture of enzymes and classified into 10 stages with pre-calibrated pipettes (stage 1 = preantral follicles with 1 layer of granulosa cells; stage 10 = preovulatory antral follicles). The follicles at each stage were incubated for 4 h with [3H]thymidine with incorporation expressed per microgram follicular DNA or per follicle. A significant increase in thymidine per follicle occurred at 15:00 h on Days 1 and 3 of the cycle from stage 2 (bilaminar follicle) to stage 6 (7-8 layers granulosa cells plus theca). When expressed as thymidine per follicle or microgram DNA, there was a significant increase in incorporation for stages 1-4 (4 layers granulosa cells) on Day 4 at 15:00 h compared to 09:00 h, presumably as a consequence of the preovulatory increase in gonadotrophins. Follicles in stages 5 to 8 (preantral follicles with 5 or more layers of granulosa cells to small antral follicles), from which the next set of ovulatory follicles will be selected, did not show a significant peak in incorporation per microgram DNA until Day 1 at 09:00 and 15:00 h when the second increase in FSH is in progress. DNA synthesis was similarly sustained throughout Day 1 for stage 1-4 follicles. These results suggest that periovulatory changes in FSH and LH, directly or indirectly, are not only responsible for ovulation and the recruitment of the next set of follicles destined to ovulate but also stimulate DNA replication in smaller follicles which develop over the course of several cycles before they ovulate or become atretic.