In vivo incorporation of cytidine-monophosphate-ciliatine into rat liver lipids.

1. In vivo this investigation was carried out in order to compare the incorporation into rat lipids of free [1,2-minus 14C]-ciliatine and CMP-[1,2-minus 14C]-ciliatine which is the precursor in phosphonolipid biosynthesis. 2. The incorporation of the radioactivity from CMP-[1,2-minus 14C]-ciliatine took place more rapidly than that from free [1,2-minus 14C]-ciliatine in both liver and kidney. The amount of radioactivity from the CMP-[1,2-minus 14C]-ciliatine incorporated into total liver lipids was about 5 times higher than that incorporated into total liver lipids of rat two hrs after injecting free-[1,2-minus 14C]-ciliatine. 3. The amount of [1,2-minus 14C]-ciliatine incorporated into total liver lipids was 15 and 21 times higher than that incorporated into total kidney lipids of rat two and four hrs after injecting free [1,2-minus 14C]-ciliatine. 4. If the main pathway for the phosphonolipid biosynthesis is via CMP-ciliatine, the rate of phosphonolipid formation from CMP-ciliatine must therefore be higher than that from free-ciliatine. The results obtained here indicate therefore that the main pathway for phosphonolipid biosynthesis is a pathway involving CMP-ciliatine. 5. An unknow compound was detected in the water soluble fraction of the acid hydrolyzate of liver phosphonolipids. This material migrated with the N-trimethyl-derivative of ciliatine on the thin-layer chromatogram. The result shows that there is therefore a possibility of methylation of exogenous ciliatine to the phosphonate analogue of choline in the mammalian body.     

Incorporation of [35S]sulphate into glycosaminoglycans by mineralized tissues in vivo.

To investigate the metabolism of proteoglycans in young growing rats, calvaria, incisors, femoral diaphysis and metaphysis were labelled in vivo for 0.5-72 h with [35S]sulphate. At each time point the specific radioactivity, expressed as c.p.m. of [35S]sulphate/micrograms of uronic acid, of papain-resistant macromolecules in each tissue was determined. The identity of the glycosaminoglycans was established by the use of specific enzymic and chemical methods of degradation. Incorporation of the label into each tissue was maximal at 12 h; it then declined to 50-75% of that value by 72 h. Chondroitin sulphate was the predominant glycosaminoglycan in each tissue, representing 80-96% of the total; heparan sulphate comprised 2-14% of the total; in general, radioactive material sensitive to keratanase comprised less than 1% of the total. The relative amount of labelled chondroitin sulphate increased, whereas that of heparan sulphate decreased, with increasing time of incorporation. These data show that 25-50% of the newly synthesized glycosaminoglycans are lost from mineralizing tissues, during the time in which the newly secreted organic matrix becomes mineralized.     

Dehydroepiandrosterone sulphate in rat brain: incorporation from blood and metabolism in vivo

The incorporation of [7-³H]dehydroepiandrosterone[³⁵S]sulphate into brain tissue elements from the circulatory system and its metabolic fate in the brain were studied in developing rats. Approximately 0.037 % of [³H] and 0.023% of [³⁵S] were incorporated into the brain within 15 min after the intracardiac injection of the labelled steroid. More than one-half of the incorporated [³H] was recovered as free steroid, whereas the rest was recovered as sulphate. The ³H/³⁵S ratio in the sulphate fraction suggested that the sulphate entered the brain with the sulphate linkage intact. Upon intracerebral injection of the double-labelled steroid, approximately 6 per cent of the radioactivity was recovered in the brain at 30 min after the injection and 1 per cent was recovered at 1 h after the injection. Of the remaining radioactivity recovered from the brain, 5 per cent was found in the free steroid fraction, probably formed by hydrolysis of the sulphate; 90 per cent was in the sulphate ester fraction; and the rest was in the fraction of more polar compounds. To identify the metabolites, [4-¹⁴C]dehydroepiandrosterone sulphate was injected into the rat brain. Significant amounts of radioactivity were found in androstenediol sulphate, which was isolated from the brain. This compound was apparently derived from dehydroepiandrosterone sulphate by reduction of the 17-keto group to a 17β-hydroxyl group without prior hydrolysis. There was suggestive evidence that free androstenediol was also formed in the brain in this experiment.     

"In vivo" (35S)methionine interaction with rat liver tRNA

As part of a study to characterize the methionine role in tumorigenesis, we report that methionine sulfur interacts with rat liver tRNA "in vivo" (35S) radioactivity remained associated to the nucleic acid after a number of treatments, including tRNA deacylation. Similar data were obtained after administration of (methyl-3H) methionine, while no comparable tRNA labelling was detected when the aminoacid labelled in the aliphatic chain was given. Hplc analysis of (35S) tRNA enzymic hydrolysate showed two unidentified UV-absorbing radioactive peaks. NMR spectra of these two peaks did not reveal any thiomethyl group.     

The incorporation in vitro of sulphate ions into synaptic-membrane glycoproteins.

The very-low-density-lipoprotein secretion rate of isolated hepatocytes obtained from rats fed a high-fat diet was half that of cells from control animals. In fat-fed rats, the initial cellular uptake of [l-14C]oleate in vitro was decreased by 25%, its esterification to triacylglycerols and phospholipids by 50% and its incorporation into very-low-density-lipoprotein triacylglycerols by 70%. Exogenous oleate was not the main precursor of very-low-density lipoproteins in these animals. Lipogenesis, a minor source of very-low-density lipoproteins with the control diet in our experimental conditions, was inhibited by 84% after fat-feeding. A short-term inhibition of lipogenesis in vitro did not result in a decrease in very-low-density-lipoprotein secretion rate. The results suggest that fat-feeding decreased availability of exogenous as well as endogenous fatty acids for synthesis of very-low-density lipoproteins.