Bis(monoacylglycero)phosphate, a peculiar phospholipid to control the fate of cholesterol: Implications in pathology.

Bis(monoacylglycero)phosphate (BMP) is a structural isomer of phosphatidylglycerol that exhibits an unusual sn1:sn1' stereoconfiguration, based on the position of the phosphate moiety on its two glycerol units. Early works have underlined the high concentration of BMP in the lysosomal compartment, especially during some lysosomal storage disorders and drug-induced phospholipidosis. Despite numerous studies, both biosynthetic and degradative pathways of BMP remained not completely elucidated. More recently, BMP has been localized in the internal membranes of late endosomes where it forms specialized lipid domains. Its involvement in both dynamics and lipid/protein sorting functions of late endosomes has started to be documented, especially in the control of cellular cholesterol distribution. BMP also plays an important role in the late endosomal/lysosomal degradative pathway. Another peculiarity of BMP is to be naturally enriched in docosahexaenoic acid and/or to specifically incorporate this fatty acid compared to other polyunsaturated fatty acids, which may confer specific biophysical and functional properties to this phospholipid. This review summarizes and updates our knowledge on BMP with an emphasis on its possible implication in human health and diseases, especially in relation to cholesterol homeostasis.

Quantification of splice variants using real-time PCR.

A reliable and robust method for measuring the expression of alternatively spliced transcripts is an important step in investigating the significance of each variant. So far, accurate quantification of splice variants has been laborious and difficult due to the intrinsic limitations of conventional methods. The many advantages of real-time PCR have made this technique attractive to study its application in quantification of splice isoforms. We use skipping of exon 37 in the NF1 gene as a model to compare and evaluate the different strategies for quantitating splice variants using real-time PCR. An overview of three different possibilities for detecting alternative transcripts is given. We propose the use of a boundary-spanning primer to quantify isoforms that differ greatly in abundance. We describe here a novel method for creating a reliable standard curve using one plasmid containing both alternative transcripts. In addition, we validate the use of an absolute standard curve based on a dilution series of fluorometrically quantified PCR products.

Dual regulation of glutathione peroxidase by docosahexaenoic acid in endothelial cells depending on concentration and vascular bed origin.

Docosahexaenoic acid (DHA) has been reported to elicit oxidative stress, which in turn can induce antioxidant enzymes. Glutathione peroxidase (GPx) has received particular attention in this respect, as this enzyme is specifically required for the degradation of lipid hydroperoxides. Because we previously found that DHA could protect against oxidative stress when used in low amounts, we have compared the effect of a low (10 microM) versus high (100 microM) concentration of DHA on oxidant/antioxidant balance in bovine retinal and bovine aortic endothelial cells (BREC and BAEC). At 100 microM, DHA elicited a marked oxidative stress, as evidenced by high malondialdehyde levels and decreased plasmalogen phosphatidylethanolamine in both cells, and for BAEC only, a decrease of alpha-tocopherol. At 10 microM, DHA induced a slight increase of malondialdehyde in both cells, but did not affect alpha-tocopherol levels, which is indicative of a mild oxidative stress. In BREC, 10 microM DHA slightly but significantly decreased cytosolic GPx (cGPx) activity whereas 100 microM had no effect. In contrast, in BAEC, DHA 10 microM did not affect cGPx activity, whereas 100 microM increased it. The decreased cGPx activity in BREC was associated with a lower level of protein, suggesting a transcriptional and/or posttranscriptional effect. Phospholipid hydroperoxide GPx (PHGPx) activity was not modified by DHA at either concentration in BREC, whereas it was increased in BAEC when using 100 microM. Our results confirm that large amounts of DHA lead to oxidative stress, but do no support an antioxidant action of DHA at low concentration, in endothelium. Nevertheless, we showed that DHA can exert opposite effects on GPx regulation in endothelial cells, with regard to its concentration and to vascular bed origin.

Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects.

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders and is caused by mutations in the NF1 gene. Mutation detection is complex due to the large size of the NF1 gene, the presence of pseudogenes and the great variety of possible lesions. Although there is no evidence for locus heterogeneity in NF1, mutation detection rates rarely exceed 50%. We studied 67 unrelated NF1 patients fulfilling the NIH diagnostic criteria, 29 familial and 38 sporadic cases, using a cascade of complementary techniques. We performed a protein truncation test starting from puromycin-treated EBV cell lines and, if no mutation was found, continued with heteroduplex, FISH, Southern blot and cytogenetic analysis. We identified the germline mutation in 64 of 67 patients and 32 of the mutations are novel. This is the highest mutation detection rate reported in a study of typical NF1 patients. All mutations were studied at the genomic and RNA level. The mutational spectrum consisted of 25 nonsense, 12 frameshift, 19 splice mutations, six missense and/or small in-frame deletions, one deletion of the entire NF1 gene, and a translocation t(14;17)(q32;q11.2). Our data suggest that exons 10a-10c and 37 are mutation-rich regions and that together with some recurrent mutations they may account for almost 30% of the mutations in classical NF1 patients. We found a high frequency of unusual splice mutations outside of the AG/GT 5 cent and 3 cent splice sites. As some of these mutations form stable transcripts, it remains possible that a truncated neurofibromin is formed.

Biosynthesis and inactivation of N-arachidonoylethanolamine (anandamide) and N-docosahexaenoylethanolamine in bovine retina.

N-Arachidonoylethanolamine (anandamide; AEA) and 2-arachidonoylglycerol (2-AG), the two proposed endogenous agonists of cannabinoid receptors, and the putative AEA biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), were identified in bovine retina by means of gas chromatography-electron impact mass spectrometry (GC-EIMS). This technique also allowed us to identify N-docosahexanoylethanolamine (DHEA) and 2-docosahexanoylglycerol (2-DHG), two derivatives of docosahexaenoic acid (DHA), one of the most abundant fatty acids esterified in retina phospholipids and necessary for optimal retinal function. N-Docosahexaenoylphosphatidylethanolamine (NDHPE), the potential biosynthetic precursor for DHEA, was also found. The fatty acid composition of the sn-1 and sn-2 positions of bovine retina's most abundant phospholipid classes, also determined here, were in agreement with a phospholipid-dependent mechanism for 2-AG, 2-DHG, AEA, and DHEA biosynthesis, as very high levels of polyunsaturated fatty acids, including DHA, were found on the sn-2 position of phosphatidylcholine (PC) and -ethanolamine (PE), and measurable amounts of di-docosahexanoyl-PC and -PE, two potential biosynthetic precursors of NDHPE, were detected. Accordingly, we found that isolated particulate fractions from bovine retina could release AEA and DHEA in a time-dependent fashion. Finally, a fatty acid amide hydrolase (FAAH)-like activity with subcellular distribution and pH dependency similar to those reported for the brain enzyme was also detected in bovine retina. This activity was inhibited by FAAH inhibitors, phenylmethylsulfonyl fluoride and arachidonoyltrifluoromethylketone, and appeared to recognize DHEA with a lower efficiency than AEA. These data indicate that AEA and its congeners may play a physiological role in the mammalian eye.

Regulation of n-3 and n-6 fatty acid metabolism in retinal and cerebral microvascular endothelial cells by high glucose.

The present study was undertaken to determine whether polyunsaturated fatty acid metabolism is affected by high glucose levels in cerebral and retinal microvascular endothelial cells. The metabolism of [3-14C]22:5n-3 and [1-14C]18:2n-6 was studied in cells previously cultured for 5 days in normal (5 mM) or high (30 mM) glucose medium. After incubation of retinal endothelial cells with [3-14C]22:5n-3 in the high glucose condition, the formation of labeled 24:6n-3 and 22:6n-3 was increased, and that of labeled 24:5n-3 was decreased, compared with the normal glucose condition. The changes were found for fatty acids esterified in cellular lipids and those released into the medium. After incubation with [1-14C]18:2n-6, levels of all elongation/desaturation products were increased at the expense of the precursor in retinal endothelial cells cultured in high glucose medium. The changes were primarily found for esterified fatty acids, with the release of n-6 fatty acids being minor in both glucose concentrations. By contrast, high glucose levels did not affect the metabolism of [3-14C]22:5n-3 and [1-14C]18:2n-6 in cerebral endothelial cells. The changes in metabolic activity of retinal endothelial cells were not reflected in the fatty acid composition. The present data suggest that high glucose can increase the desaturation process in retinal but not cerebral endothelial cells. This may produce some lipid abnormalities in retinal microvasculature and contribute to altered vascular function observed in diabetic retinopathy.

Effect of diet on the fatty acid and molecular species composition of dog retina phospholipids.

Dogs were born to mothers fed commercial diets low or enriched in n-3 fatty acids and raised on those diets until they were about 50 d old. Retinas were removed, lipids were extracted, and total phospholipids were analyzed for fatty acid and molecular species composition. Animals from the low n-3 group had significantly lower retinal levels of 22:6n-3 and higher levels of n-6 fatty acids, especially 20:4n-6 and 22:5n-6. There was no difference in the retinal levels of 18:2n-6, and only small differences were found in saturated and monounsaturated fatty acids. The most dramatic differences in molecular species occurred in 22:6n-3-22:6n-3 (4.7 vs. 0.8%) and 18:0-22:6n-3 (27.6 vs. 14.4%); total molecular species containing 22:6n-3 were significantly lower in the low n-3 group (45.5 vs. 24.0%). Molecular species containing 20:4n-6 and 22:5n-6 were greater in the low n-3 animals (13.0 vs. 25.7%), as were molecular species containing only saturated and monounsaturated fatty acids (40.8 vs. 35.4%). These results show that modest differences in the amount of n-3 fatty acids in the diets of dogs can have profound effects on the fatty acid and molecular species composition of their retinas.

Polyunsaturated fatty acid metabolism in retinal and cerebral microvascular endothelial cells.

Docosahexaenoic acid (22:6n-3), an n-3 essential fatty acid derived from elongation and desaturation of linolenic acid (18:3n-3), is found in abundant proportion in the brain and the retina. It is generally assumed that the liver is the major source of 22:6n-3 for these organs, although some retinal and cerebral cells, such as retinal pigment epithelium (Wang and Anderson, 1993. Biochemistry. 32:13703-13709) and brain astrocytes (Moore et al. 1991. J. Neurochem. 56:518-524) have the ability to produce 22:6n-3. The aim of the present study was to determine whether retinal and cerebral microvascular endothelium could synthesize 22:6n-3. After incubation of both cultured bovine retinal and rat cerebral endothelial cells with [3-14C] 22:5n-3 in presence of serum, radioactivity was primarily recovered in 20:5n-3, indicating active retroconversion reactions in both tissues. However, 22:6n-3, 24:5n-3, and 24:6n-3 were also labeled. All of these metabolites were released in the medium as free fatty acids. Retinal endothelial cells preferentially released labeled 24-carbon metabolites, whereas cerebral endothelial cells released relatively more 20:5n-3 and 22:6n-3. With heat-inactivated serum or no serum, both endothelial cell preparations showed relatively higher retroconversion levels. However, in serum-deprived cells, the elongation/desaturation pattern was affected in retinal cells only, with an accumulation of 24:5n-3 relative to a decrease of 24:6n-3 and 22:6n-3. Fatty acid composition analyses revealed a decrease in long-chain polyunsaturated n-6 and n-3 fatty acids in retinal cells maintained in inactivated serum compared to normal serum, while no change was found in cerebral cells. Taken together, these results suggest that 1) the synthesis of 22:6n-3 by both retinal and cerebral endothelial cells is independent of a delta4-desaturase; 2) retinal and cerebral endothelia could be a source of 22:6n-3 for the retina and the brain, respectively; and 3) retinal endothelial delta6-desaturase, which converts 24:5n-3 to 24:6n-3, could be stimulated by serum components.

Modulation of norepinephrine-stimulated cyclic AMP accumulation in rat pinealocytes by n-3 fatty acids.

This work showed that docosahexaenoic (22:6n-3) and eicosapentaenoic (20:5n-3) acid supplementation for 48 h have opposite effects on the norepinephrine-stimulated cyclic AMP accumulation in rat pinealocytes. We found that 22:6n-3 supplementation of pineal cells, done by increasing specifically 22:6n-3 in phospholipid and triacylglycerol pools, led to inhibition of norepinephrine-stimulated cyclic AMP production whereas 20:5n-3 supplementation, by increasing 20:5n-3, and 22:5n-3 and 22:6n-3 in the same pools, stimulated it. In contrast, direct treatment of pinealocytes with each fatty acid (50 microM) did not affect cyclic AMP production in the presence of (0.1-10 microM) norepinephrine. The results indicate that, using pharmacological agents such as forskolin or prazosin: (a) neither basal nor forskolin-stimulated cyclic AMP levels were modified in fatty acid-supplemented cells compared to control cells; (b) in the presence of 1 microM prazosin, the activation by 20:5n-3 was still effective whereas no additional inhibition of norepinephrine stimulation was observed in 22:6n-3-supplemented cells. Taken together our results suggest that 22:6n-3 or 20:5n-3 supplementation modulates specifically the alpha 1- or beta-adrenoceptors in the rat pineal gland.