Depression of hepatic dolichol levels by cholesterol feeding

A study was conducted to determine whether repression of 3-hydroxy-3-methylglutaryl CoA reductase by a chronic high-cholesterol diet would deplete hepatic dolichol levels. Four-week-old male C57BL/6J mice were maintained on a control diet or a diet supplemented with 5% cholesterol. Animals from both groups were killed at various times and reductase activity and levels of free dolichol, dolichyl acyl ester, dolichyl phosphate, and ubiquinone were measured. The reductase activity was reduced by 90% within 1 week and remained depressed through 56 days. Initially, the levels of the free dolichol, acyl ester, phosphoryl ester, and ubiquinone were 7, 16, 5, and 80 micrograms/g liver, respectively. Early increases in the concentration of dolichyl phosphate and free dolichol were similar in both the cholesterol-fed and control groups. However, in the cholesterol-fed group the concentration of dolichyl acyl esters was only 50% of that in the control group by 7 days and it remained lower throughout the experiment. Total dolichol levels were lower by about 30%. Ubiquinone levels were transiently depressed at 7 days by 33% but returned to control levels by 4 weeks. After 56 days, the control values of dolichol and dolichyl phosphate remained constant whereas the dolichyl acyl ester levels continuously increased to a value of 133 micrograms/g of liver by 156 days. Subcellular fractionation of livers from 4-week-old mice indicated a lysosomal distribution of dolichol and dolichyl acyl ester and a lysosomal and microsomal distribution of dolichyl phosphate.     

Frog liver dolichols: separation and quantitative determination related to seasonality

1. The liver of female frogs shows a significantly higher dolichol content in May (134 micrograms/g) than in November (90 micrograms/g). The male frog liver (134 micrograms/g May; 119 micrograms/g November) does not show any significant change. 2. The rat liver does not show any significant change in dolichol content from May to November (30 micrograms/g). 3. The dolichyl-phosphate level was the same in male and female frog livers and in the rat liver. 4. Data is given of the distribution of dolichols according to the number of their isoprene residues.     

Divergence of larval resource acquisition for water conservation and starvation resistance in Drosophila melanogaster

Laboratory selection experiments have evidenced storage of energy metabolites in adult flies of desiccation and starvation resistant strains of D. melanogaster but resource acquisition during larval stages has received lesser attention. For wild populations of D. melanogaster, it is not clear whether larvae acquire similar or different energy metabolites for desiccation and starvation resistance. We tested the hypothesis whether larval acquisition of energy metabolites is consistent with divergence of desiccation and starvation resistance in darker and lighter isofemale lines of D. melanogaster. Our results are interesting in several respects. First, we found contrasting patterns of larval resource acquisition, i.e., accumulation of higher carbohydrates during 3rd instar larval stage of darker flies versus higher levels of triglycerides in 1st and 2nd larval instars of lighter flies. Second, 3rd instar larvae of darker flies showed ~40?h longer duration of development at 21°C; and greater accumulation of carbohydrates (trehalose and glycogen) in fed larvae as compared with larvae non-fed after 150?h of egg laying. Third, darker isofemale lines have shown significant increase in total water content (18%); hemolymph (86%) and dehydration tolerance (11%) as compared to lighter isofemale lines. Loss of hemolymph water under desiccation stress until death was significantly higher in darker as compared to lighter isofemale lines but tissue water loss was similar. Fourth, for larvae of darker flies, about 65% energy content is contributed by carbohydrates for conferring greater desiccation resistance while the larvae of lighter flies acquire 2/3 energy from lipids for sustaining starvation resistance; and such energy differences persist in the newly eclosed flies. Thus, larval stages of wild-caught darker and lighter flies have evolved independent physiological processes for the accumulation of energy metabolites to cope with desiccation or starvation stress.     

Effects of river red gum, Eucalyptus camaldulensis, litter on golden perch, Macquaria ambigua

Aquaria with added river red gum, Eucalyptus camaldulensis , litter became hypoxic, with decreased pH and contained up to 30 mg 1⁻¹ tannin and lignin. Survival of golden perch, Macquaria ambigua , larvae in aquaria treated with a simulated annual litter density of 450 g m⁻² for 72 h was 14·9% for 15-day-old larvae and 0% for 8-day-old larvae. A litter density of 1223 g m⁻² resulted in total mortality for both age groups of larvae. Aeration increased survival of larvae to a minimum of 68·8% in 1223 g m⁻² litter treatments compared to 89·8% in aerated controls and 86·8% in non-aerated controls. A kinetic behavioural assay was used to detect alarm responses in golden perch larvae and juveniles exposed to leachates from river red gum bark, leaves and wood. Eight-day-old larvae exposed to bark and wood leachates (0·001–10 g 1⁻¹) exhibited an initial period of hyperactivity, followed by a concentration-dependent decrease in spontaneous activity. Larvae exposed to leaf leachates displayed only a decrease in spontaneous activity. Four-month-old juveniles exposed to wood leachates were also initially hyperactive, then progressively developed mild hypoactivity at increasing leachate concentrations. Juveniles exposed to wood leachates at 20g 1⁻¹ for 30min suffered 97·5% mortality in 96 h. Wood leachates induced dose-dependent lamellar fusion, epithelial dissociation and necrosis in the gills. The presence of toxic leachates and low oxygen availability in flooded river red gum forests may make these habitats unsuitable as nursery areas for native fish.     

Plasmodium falciparum: inhibition of dolichol kinase by mefloquine

Dolichol kinase, a rate limiting enzyme for the supply with dolichyl monophosphate as glycosyl carrier lipid, was demonstrated in Plasmodium falciparum. The enzyme was found to be associated with the pellet fraction and to depend on cytidine triphosphate as phosphoryl donor. The apparent Km values of the plasmodial enzyme were determined to be 0.8 mM for cytidine triphosphate and 17 micrograms ml-1 for dolichol. In the presence of 0.5 mM mefloquine, the inhibition of enzyme activity was found to be about 50%.     

Hypoglycosylation due to dolichol metabolism defects

Dolichol phosphate is a lipid carrier embedded in the endoplasmic reticulum (ER) membrane essential for the synthesis of N-glycans, GPI-anchors and protein C- and O-mannosylation. The availability of dolichol phosphate on the cytosolic site of the ER is rate-limiting for N-glycosylation. The abundance of dolichol phosphate is influenced by its de novo synthesis and the recycling of dolichol phosphate from the luminal leaflet to the cytosolic leaflet of the ER. Enzymatic defects affecting the de novo synthesis and the recycling of dolichol phosphate result in glycosylation defects in yeast or cell culture models, and are expected to cause glycosylation disorders in humans termed congenital disorders of glycosylation (CDG). Currently only one disorder affecting the dolichol phosphate metabolism has been described. In CDG-Im, the final step of the de novo synthesis of dolichol phosphate catalyzed by the enzyme dolichol kinase is affected. The defect causes a severe phenotype with death in early infancy. The present review summarizes the biosynthesis of dolichol-phosphate and the recycling pathway with respect to possible defects of the dolichol phosphate metabolism causing glycosylation defects in humans.     

A defect in dolichol phosphate biosynthesis causes a new inherited disorder with death in early infancy

The following study describes the discovery of a new inherited metabolic disorder, dolichol kinase (DK1) deficiency. DK1 is responsible for the final step of the de novo biosynthesis of dolichol phosphate. Dolichol phosphate is involved in several glycosylation reactions, such as N-glycosylation, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, and C- and O-mannosylation. We identified four patients who were homozygous for one of two mutations (c.295T-->A [99Cys-->Ser] or c.1322A-->C [441Tyr-->Ser]) in the corresponding hDK1 gene. The residual activity of mutant DK1 was 2%-4% when compared with control cells. The mutated alleles failed to complement the temperature-sensitive phenotype of DK1-deficient yeast cells, whereas the wild-type allele restored the normal growth phenotype. Affected patients present with a very severe clinical phenotype, with death in early infancy. Two of the patients died from dilative cardiomyopathy.     

Extraction and quantitation of dolichol and dolichyl phosphate in soybean embryo tissue

A procedure for the quantitative extraction of both dolichol and dolichyl phosphate (Dol-P) in plant tissue (soybean embryos) into diethyl ether from an alkaline saponification mixture is described. A complete and quantitative separation of total dolichol and total Dol-P is then obtained based on their respective solubilities in diethyl ether and water. After separation dolichol and Dol-P can both be analyzed and quantitated directly by reverse-phase HPLC on C18 columns without additional purification. The two major homologs of dolichol and Dol-P are those with 17 and 18 isoprene units. The total dolichol and total Dol-P contents of dry embryos were 96.3 +/- 0.8 and 5.3 +/- 0.1 micrograms/g, respectively. The post-HPLC recoveries for dolichol and Dol-P were 101 +/- 2 and 84 +/- 3% respectively, using [1-14C]dolichol and Dol-P containing 20 isoprene units as recovery standards. Dol-P estimations could be carried out on material equivalent to as little as 65 mg embryo tissue.     

Dolichol: function, metabolism, and accumulation in human tissues.

Dolichol, a homologous series of alpha-saturated polyisoprenoid alcohols containing 14-24 isoprene units, was first isolated and characterized about 30 years ago. The phosphorylated form, dolichyl phosphate, is required for the biosynthesis of biologically important N-linked glycoproteins. Dolichol itself is synthesized by a common isoprenoid pathway from acetate and synthesis can be inhibited by some of the factors that inhibit cholesterol biosynthesis. It is metabolized very slowly and accumulates in tissues during aging and in certain lipid storage diseases. Dolichyl phosphate and cholesterol also accumulate in tissues during aging, but to a lesser extent than dolichol. Although dolichol and cholesterol have important metabolic functions, their accumulation in tissues can have deleterious effects.     

Absorption and distribution of [1-14C]dolichol intubated into rats

[1-14C]Dolichol was mixed in vitro with sunflower seed oil and intubated into rats. Radioactivity began to appear in the blood at 3 h and peaked after about 6 h. The absorbed radioactivity was rapidly cleared from the blood. At 7.5 h postintubation two thirds of the radioactivity in the serum was associated with chylomicrons and about one quarter with the high density lipoproteins. At 12 h the proportion of the radioactivity in the chylomicrons had fallen to one third and that in the high density lipoproteins had increased to one half of the total. Less than 0.02% of the dose was found in the tissues after 12 h. Liver and blood each contained about one third of the total, with smaller amounts in the lungs and spleen. The heart, testes, brain, and kidneys contained only traces of radioactivity. After 12 h most of the radioactivity in the tissues and feces was present as [1-14C]dolichol. The radioactive compounds in the urine (about 0.05% of the dose) were more polar than [1-14C]dolichol as determined by thin-layer chromatography.     

Chronic ethanol treatment: dolichol and retinol distribution in isolated rat liver cells

The aim of this study was to use chronic ethanol intoxication for 2 and 4 months as a means of studying the distribution of dolichol and retinol in isolated rat liver parenchymal cells, Kupffer cells, sinusoidal endothelial cells, and two subfractions of hepatic stellate cells: Ito 1 and Ito 2. Dolichol and retinol were studied in two batches of rats: on normal nutrition and after a load of vitamin A given 3 d before sacrifice. New observations reported are: (i) on normal nutrition, after 2 months of treatment, dolichol in HC seems to be the first target of chronic ethanol, while retinol is the first target in hepatic stellate cells; (ii) the various types of liver cells are differently affected by chronic ethanol, which highlights the importance of studying each type of sinusoidal cell; (iii) a load of vitamin A given when the damage has already occurred restores dolichol content in HC while retinol decreases; and, (iv) a link between dolichol and vitamin A metabolism might be supposed after the load of vitamin A: the percentage distribution of dolichol with 18 isoprene units (Dolichol -18) increases in all the control cells but decreases after chronic ethanol treatment. A different role of this dolichol and/or a different compartmentalization within the cell need to be further investigated.     

Sodium nitroprusside toxicity in Drosophila melanogaster: delayed pupation, reduced adult emergence, and induced oxidative/nitrosative stress in eclosed flies

The toxicity of sodium nitroprusside (SNP) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with SNP at concentrations of 0.01-1.5 mM. Food supplementation with SNP caused a developmental delay in flies and reduced adult eclosion. Biochemical analyses such as levels of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control and SNP-fed larvae. Larval exposure to SNP resulted in lower activities of aconitase and catalase in adult flies relative to the control cohort. However, larval treatment with SNP led to higher carbonyl protein content and higher activities of superoxide dismutase, glucose-6-phosphate dehydrogenase, thioredoxin reductase, and glutathione-S-transferase in flies. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by SNP. The study also suggests that the toxicity of SNP may arise not just from its direct effects, but also from its decomposition products such as nitric oxide and iron ions.     

The Effect of Oxygen on the Frequency of Somatic Recombination in DROSOPHILA MELANOGASTER

The influence of oxygen on the frequency of somatic recombination in the yellow singed system on the X chromosome of Drosophila melanogaster was studied under a variety of experimental conditions. Flies raised from egg to adult in atmospheres containing 70-90% oxygen were found to have significantly more mosaic spots on their abdominal tergites than were observed in flies which developed in air. First instar larvae X-rayed in from 0 to 100% oxygen demonstrated the existence of an oxygen effect for somatic recombination in the cells which form the abdominal hypoderm. The mosaic spot counts, beginning with the lowest numbers which were found in flies X-rayed in nitrogen, increased rapidly with rising oxygen tensions until the percentage in air was reached, then leveled off at the higher concentrations. Post-treatment with nitrogen of larvae X-rayed in air or oxygen created a substantially higher number of mosaic spots than were found when larvae, after being similarly irradiated, were instead placed into air or oxygen.     

Stimulation by dolichol phosphate-mannose of N-acetylglucosaminyl-lipid biosynthesis by membranes from class E Thy-1-negative mutant mouse lymphoma cells which are defective in dolichol phosphate-mannose biosynthesis

Dolichol phosphate-mannose has been shown previously to stimulate the biosynthesis of N-acetylglucosaminyl-diphosphate-dolichol (E. L. Kean (1985) J. Biol. Chem. 260, 12561-12571). Although the class E Thy-1-negative mutant mouse lymphoma cells are unable to synthesize dolichol phosphate-mannose, the addition of this compound exogenously to membranes from the mutant cells brought about a stimulation of N-acetylglucosaminyl-lipid synthesis similar to that obtained with membranes from wild type cells. The retention of this activity by the mutant cells supports the suggestion of a regulatory role for dolichol phosphate-mannose as an intrinsic property of the glucosaminyltransferase which catalyzes the initial reaction of the dolichol pathway.     

Low-level chemiluminescence from Drosophila melanogaster fed with chemical mutagens polycyclic aromatic hydrocarbon quinones and a carcinogenic bracken fern

The spontaneous photon emission (chemiluminescence) from Drosophila melanogaster fed chemical mutagens, polycyclic aromatic hydrocarbon quinones, and a carcinogenic bracken fern was studied. The fly chemiluminescence was evidently enhanced by mutagen or carcinogen administration and was increased proportionally to the administered amount of tested compound. Strong chemiluminescence was observed especially at the larval stage. Living larvae emitted stronger chemiluminescence than their homogenate. The chemiluminescence from Drosophila melanogaster fed polycyclic aromatic hydrocarbon quinones showed a linear relation with the mutation frequency in the Drosophila wing spot test. The chemiluminescence from flies fed a bracken fern decreased by the addition of free radical scavengers and active oxygen quenchers. The phosphatidylcholine hydroperoxide concentration in the flies was increased proportionally with the chemiluminescence intensity. It seems that the free radical formation is stimulated as shown by the enhanced chemiluminescence in mutagen- or carcinogen-dosed flies, and as a result, lipid peroxide accumulation accompanies mutation in Drosophila melanogaster.     

Rates of cholesterol, ubiquinone, dolichol and dolichyl-P biosynthesis in rat brain slices

Slices from the brain and liver of rats were prepared and upon incubation exhibited a continuous and high capacity for incorporation of radioactive precursors into proteins and lipids. Using [3H]mevalonate as precursor, the rates of biosynthesis of cholesterol, ubiquinone, dolichol and dolichyl-P in brain slices were determined and found to be 5.5, 0.25, 0.0093 and 0.0091 nmol/h/g, respectively. Dolichol and dolichyl-P accumulate to a limited extent, but almost all of these lipids in the brain originate from de novo synthesis. The calculated half-lives for cholesterol, ubiquinone, dolichol and dolichyl-P were 4076, 90, 1006 and 171 h, respectively. The results indicate that lipids formed via the mevalonate pathway in the brain have an active and independently regulated biosynthesis.     

Nondisjunction induced by ethanol in Drosophila melanogaster females.

The effect of ethanol on chromosomal segregation was investigated in Drosophila melanogaster females homozygous for a structurally normal X chromosome marked with the recessive mutation yellow (y/y). For chronic treatments the females were kept from eclosion in food supplemented with 10% or 15% (v/v) ethanol, mated 24 or 48 h later to wild-type males and brooded in freshly prepared ethanol food. For the acute treatments 24- or 48-h-old females were exposed for 60 min to a 75% (v/v) ethanol solution by means of soaked tissue paper placed at the bottom of regular culture vials and brooded daily after mating. The results obtained show that: (1) both treatments significantly increased the frequency of X-chromosome nondisjunction; (2) after acute treatment this effect declined in later broods; (3) the yield of malformed flies in the progeny of acutely treated females was significantly higher than control values and also declined in later broods; (4) ovary analysis showed that chronic ethanol treatments caused a cessation of egg production. The induction pattern of nondisjunction and malformed flies is interpreted as giving support to the assumption that these effects may result from a direct action of ethanol. Ethanol toxicity was assessed by exposing females of different ages to a 50% or a 75% (v/v) solution for 60 min and counting the surviving flies 24 h later. The surviving fraction decreased steeply from 1-day-old (100%) to 5-day-old females (1.8%). It is suggested that toxicity may have been due to the action of a metabolite of ethanol, probably acetaldehyde.     

The temporal pattern of vitellogenin synthesis in Drosophila grimshawi

The temporal pattern of protein production and, in particular, vitellogenin protein synthesis during the sexual maturation of Drosophila grimshawi females has been studied in vivo by briefly feeding the flies with 35S-methionine and 3H-amino acids. The overall level of incorporation was very low in young flies; it then progressively increased to reach a maximum with the onset of sexual maturity at 13-15 days. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses revealed three classes of proteins: those synthesized throughout the age spectrum, which constitute the majority of protein species; proteins synthesized primarily or only in young flies; and proteins synthesized only by the older flies. In this Drosophila species, the three vitellogenins (V1, V2, and V3) appeared to be synthesized in a two-phase pattern. In the first phase, small quantities of V1 and V2 were detected immunologically in the fat body and hemolymph of newly emerged and 1 day-old flies. These proteins did not accumulate in the hemolymph or the ovaries, apparently being unstable proteins. The second phase commenced in early vitellogenesis (7-9 days of age) with synthesis in the fat body of small quantities of V1 and V2, followed by V3 proteins. These proteins were secreted and accumulated in the hemolymph and 24 h later were found in the ovaries. Their quantities increased rapidly and a steady state of synthesis, release into the hemolymph, and uptake by the ovaries was reached by days 13-15. We have estimated that during the steady state of vitellogenin synthesis, a fly can synthesize in 24 h at least 152 micrograms of vitellogenins, which is more than 2% of its body weight, at an average rate of about 6.3 micrograms vitellogenins/h. About 2 micrograms of this are synthesized in the fat body, and about 4 micrograms in the ovaries. These findings are discussed in terms of their physiological implications and contrasted with the available data on Drosophila melanogaster.     

Studies on molecular species of choline glycerophospholipids of developing rat brain.

The chronological changes in molecular species of choline glycerophospholipids were studied for cerebra of 17-, 19- and 21-day-old rat fetuses, and 3-, 6-, 12-, 24- and 90-day-old rats. The molecular species found by gas chromatography-mass spectrometry and selected ion retrieval technique were phosphatidylcholines of '30 : 0, 32 : 0, 32 : 1, 34 : 0, 34 : 1, 34 : 2, 36 : 0, 36 : 1, 36 : 2, 36 : 3, and 36 : 4' where the larger number indicates the sum of chain lengths on positions C-1 and C-2; the smaller number is the total number of double bonds. Of these molecular species, '32 : 0' (mainly 16 : 0/16 : 0, dipalmitoyl glycerophosphorylcholine), '34 : 1' (mainly 16 : 0/18 : 1, palmitoyloleoyl glycerophosphorylcholine), '34 : 0' (16 : 0/18 : 0, palmitoylstearoyl glycerophosphorylcholine), '32 : 1' (mainly 16 : 0/16 : 1, palmitoylpalmitoleoyl glycerophosphorylcholine and '30 : 0' (14 : 0/16 : 0, myristoylpalmitoyl glycerophosphorylcholine) were main species. The '32 : 0' species increased to about 44% at around the 10th day and thereafter remained nearly constant. '34 : 1' and '34 : 0' decreased to about 17 and 6% at that time and then increased to about 30 and 14%, respectively. '30 : 0' increased from last stage of gestation to the 6th day and then decreased. '32 : 1' was about 16% for 17-day-old fetus and decreased grandually. '36 : 1' (18 : 0/18 : 1, stearoyloleoyl glycerophosphorylcholine) increased at the latter part of development.     

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.