The characterization and stereochemistry of biosynthesis of dolichols in rat liver

It was shown that rat liver contains a series of dolichols with chain lengths of from 17 (or possibly 16) to 21 isoprene residues, the main constituent of the mixture being dolichol-18. By using double-labelled radioactive mevalonates it was demonstrated that each of these dolichols possesses three biogenetically trans-isoprene residues and that the remaining residues are biogenetically cis, suggesting that these polyprenols are biosynthesized from all-trans-farnesyl pyrophosphate by the cis additions of isoprene residues, followed by saturation of the alpha-isoprene residue. The results obtained with these radioactive mevalonates also indicated that the activity of isopentenylpyrophosphate isomerase is low relative to the activity of prenyltransferase in rat liver.     

Uptake and modification of dietary polyprenols and dolichols in rat liver

Short and long dolichols and polyprenols in free form or esterified with fatty acids were incorporated into liposomes and administered to rats through a gastric tube. The free alcohols were taken up by the liver to different extents. While uptake in other organs was less, it also involved the fatty acid esters. The use of systems other than liposomes did not increase the efficiency of uptake. Most of the administered lipids were recovered in the lysosomes. Exogenous dolichols and polyprenols were both partly esterified in the liver and, to some extent, also phosphorylated; a portion of the polyprenols was also alpha-saturated. These results indicate that various polyisoprenes are taken up, to a small extent, from the diet by tissues under normal conditions and in liver these dietary lipids undergo terminal modifications.     

High-performance liquid chromatographic method for the determination of dolichols in tissues and plasma

High-performance liquid chromatographic methods for the determination of dolichols in tissues and plasma have been developed. The tissue concentration of dolichols was measured by high-performance liquid chromatography with uv detection and plasma levels of dolichols were determined fluorometrically after derivatization with anthracene-9-carboxylic acid. In both methods, 2,2-didecaprenylethanol was used as an internal standard. The method with fluorescence detection was sufficiently sensitive to measure the concentration of dolichols in human plasma.     

The uptake of dietary polyprenols and their modification to active dolichols by the rat liver

The uptake of dietary polyprenols was studied by administering, through a gastric tube, labeled alpha-saturated and alpha-unsaturated polyprenols, with 11 and 19 isoprene residues. The lipids appeared in all organs but mostly in the liver after 16 h where those with 11 isoprenes were in much higher concentration than the prenols with 19 isoprene residues; the distribution in the liver was studied in detail. About 45% of the polyprenols taken up were esterified with fatty acids. A part of the radioactivity (6-30%) appeared in the supernatant but mostly in water-soluble form. Among subcellular fractions, the highest uptake was found in the outer mitochondrial membranes. After 16 h, both 11- and 19-residue alpha-unsaturated injected prenols were present to a large extent as alpha-saturated compounds in liver homogenates and subcellular fractions. About 10-15% of the lipids were phosphorylated. The results suggest that a part of the dolichol phosphate pool, participating in glycosylation reactions, may derive from dietary unsaturated polyprenols which after uptake can be reduced and phosphorylated.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Birth seasonality and offspring production in threatened neotropical primates related to climate

Given the threatened status of many primate species, the impacts of global warming on primate reproduction and, consequently, population growth should be of concern. We examined relations between climatic variability and birth seasonality, offspring production, and infant sex ratios in two ateline primates, northern muriquis, and woolly monkeys. In both species, the annual birth season was delayed by dry conditions and El Niño years, and delayed birth seasons were linked to lower birth rates. Additionally, increased mean annual temperatures were associated with lower birth rates for northern muriquis. Offspring sex ratios varied with climatic conditions in both species, but in different ways: directly in woolly monkeys and indirectly in northern muriquis. Woolly monkeys displayed an increase in the proportion of males among offspring in association with El Niño events, whereas in northern muriquis, increases in the proportion of males among offspring were associated with delayed onset of the birth season, which itself was related, although weakly, to warm, dry conditions. These results illustrate that global warming, increased drought frequency, and changes in the frequency of El Niño events could limit primate reproductive output, threatening the persistence and recovery of ateline primate populations.     

Rat testicular dolichols: distribution and accumulation with age

Dolichols, linear isoprenoids essential in the biosynthesis of N-glycosylated glycoproteins, are abundant in testicular tissue. This study investigated the distribution of dolichols among testicular cell and subcellular fractions. In addition, the accumulation of dolichol within the rat testis as a function of age was investigated. Dolichol content expressed either as total dolichol/testicle or as dolichol/mg protein exhibited a marked and continuous increase between 14 and 60 days of age. The 4-, 6-, 9-, and 12-mo-old animals exhibited only minor increases in testicular dolichol content. Mean value for retired breeders was 279 ng dolichol/mg protein. Although previous studies have suggested that dolichol synthesis occurs primarily within the spermatogenic cell, elutriation-purified spermatogenic cell fractions showed very low concentrations of dolichol. Pachytene spermatocyte and round spermatid fractions contained 25.8 and 36.5 ng dolichol/mg protein, respectively. Washed epididymal sperm also had a very low dolichol content (18.8 ng dolichol/mg protein). Recovery studies during elutriation purification of spermatogenic cells showed that the majority of dolichol was contained within the Sertoli-rich tubular fragments. Microsomal fractions isolated from whole testis exhibited a small enrichment (1.6-fold) in dolichol content, whereas Golgi apparatus fractions exhibited a large (12-fold) enrichment over that of the initial homogenate. These studies suggest that, although dolichols may be synthesized within the spermatogenic cell, they accumulate within the Sertoli cell.     

Chemiluminescence quantitative immunohistochemical determination of MRP2 in liver biopsies.

Evaluation of protein expression in tissues and cells by electrophoretic and blotting techniques or by the quantification of the mRNA coding for the target protein is a common procedure in biochemistry research and clinical diagnoses. In this article, an alternative approach, based on an immunohistochemical procedure with chemiluminescent imaging detection, is described. The assay exploited the peculiar characteristics of the chemiluminescent detection of enzyme labels (high sensitivity and specificity, low background, easy quantification of the signal) for performing the direct, simple, and rapid quantitative evaluation of protein expression in tissues. When applied to the study of the levels of MRP2, a member of the human multidrug resistance-associated protein family, in samples obtained from formalin-fixed, paraffin-embedded liver biopsies, it allowed the reliable evaluation of the protein content of the tissue. Moreover, the analysis of clinical samples from patients with primary biliary cirrhosis under therapy with ursodeoxycholic acid gave results in line with those, previously reported in the literature, obtained with conventional protein expression analysis techniques.     

Separation and quantitative determination of dolichol and dolichyl phosphate in rat and trout liver

The dolichol concentrations in rat and trout liver were found respectively to be 50-59 and 16-21 micrograms/g using three experimental methods: densitometric scanning of thin-layer plates, colorimetric assay and HPLC analysis. By HPLC of benzoylated dolichols, the distribution of the dolichols according to the number of their isoprene residues, was determined in rat and trout liver. The major component was dolichol -18 in rat and dolichol -19 in trout liver. Dolichyl phosphate concentrations were found to be 6-7 micrograms/g of rat liver and 8-9 micrograms/g of trout liver by densitometric scanning of thin-layer plates.     

Planarian regeneration: quantitative differences in RNA and protein synthesis related to age

A comparative study of RNA and protein synthesis during regeneration of immature and adult planarians reveals fundamental differences in the regeneration process. Young planarians, which contain about 20 times more RNA/protein in their tissues than adults, actively synthesize RNA prior to any wound. A single stimulation of RNA synthesis is observed after 24 h following sectioning. The electrophoretic pattern of labelled RNA extracted either from intact or regenerating young planarians does not change significantly and shows, besides ribosomal RNA, an important fraction of RNA of heterogeneous molecular weights. This pattern is similar to that observed with extracts of RNA from regenerating adults but only after 24 h following sectioning (Martelly, I. and Le Moigne, A., Reprod. Nutr. Dev., 20 (1980) 1527–1537). Indeed, in adults, a preliminary phase of RNA metabolism is observed during the first day of regeneration. Young and adult planarians differ also in their time course of stimulation of protein synthesis after sectioning. While a lag time of more than 6 h is necessary in adults, protein synthesis is stimulated immediately after sectioning in the young. These differences in the pattern of macromolecular synthesis related to age are discussed in relation with the idea of cellular activation during the regeneration process.     

Alternative NMR method for quantitative determination of acyl positional distribution in triacylglycerols and related compounds

High-resolution 13C NMR spectroscopy has been used to analyze the positional distribution of fatty acids in model triacylglycerols. A novel method for quantitative determination of the positional distribution of unsaturated chains in triacylglycerols simultaneously with the ratio of saturated/unsaturated acyl chains has been proposed, utilizing the chemical shift differences of the aliphatic atoms C4, C5, and C6. The use of HSQC-TOCSY spectra allows unequivocal proof of the position of the unsaturated chain as well as complete assignment of the 13C NMR signals in tripalmitin.     

Frog liver glucose-6-phosphatase activity: seasonal variations of latency and kinetic parameters

The frog liver microsome glucose-6-phosphatase activity shows seasonal rhythm, being significatively higher in the winter than in summer. The latency and the sensibility to anionic detergent Deoxycholate is higher in the summer than in winter. During the summer a Km and epsilon Vmax decrease is observed.