Biological properties of a DHA-containing structured phospholipid (AceDoPC) to target the brain.

1-acetyl,2-docosahexaenoyl-glycerophosphocholine (AceDoPC) has been made to prevent docosahexaenoyl (DHA) to move to the sn-1 position as it rapidly does when present in 1-lyso,2-docosahexaenoyl-GPC (lysoPC-DHA), an efficient DHA transporter to the brain. When incubated with human blood, AceDoPC behaves closer to lysoPC-DHA than PC-DHA in terms of binding to plasma albumin and lipoproteins, and DHA incorporation into platelets and red cells. In addition, AceDoPC prevents more efficiently the deleterious effects of the experimental stroke in rats than does unesterified DHA. Also, AceDoPC inhibits platelet-activating factor-induced human blood platelet aggregation. Overall, AceDoPC might act as an efficient DHA transporter to the brain, and as a neuro-protective agent by itself.

DHA metabolism: targeting the brain and lipoxygenation.

Docosahexaenoic acid (DHA), the end-product of the metabolism of omega-3 family fatty acids, is the main polyunsaturated fatty acid of the brain, but its accumulation is incompletely understood. This paper reviews how it could accumulate through specific uptake of DHA-containing lysophosphatidylcholine (LysoPC-DHA). DHA migrates very easily from the sn-2 position of LysoPC, which could be considered as the physiological form of polyunsaturated LysoPC, to the sn-1 position, which is much more stable. An approach preventing migration by acetylating the sn-1 position, while retaining the main physico-chemical properties of the carrier, is described. Also, the double lipoxygenation and bond-isomerization of DHA into 10(S),17(S)-docosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid, named PDX, by soybean lipoxygenase is described. As in other E,Z,E conjugated trienes, PDX is shown to inhibit human blood platelet aggregation at submicromolar concentrations.

Mild phenotype in a 15-year-old boy with Pallister-Killian syndrome.

Pallister-Killian syndrome is a rare disorder characterized by multiple congenital anomalies, coarse face, pigmentary skin changes, seizures, severe mental retardation, and the presence of an extra metacentric chromosome i(12p) confined to skin fibroblasts only. Here, we report on an unusual case of i(12p) in a 15-year-old boy presenting with mild mental retardation, minor facial features (long face, prognathism, short neck), normal weight, length, and OFC parameters as well as hyperpigmented streaks. The boy attended normal school until the age of 14 years. Because of hyperpigmented stripes, chromosome analysis was performed on skin fibroblasts. This study showed that 37% of the cells had an additional isochromosome for the short arm of chromosome 12. This observation illustrates the phenotypic variability of i(12p) and emphasizes the importance of skin fibroblasts chromosome analysis in patients with pigmentary skin changes.

A novel automated strategy for screening cryptic telomeric rearrangements in children with idiopathic mental retardation.

Cryptic unbalanced subtelomeric rearrangements are known to cause a significant proportion of idiopathic mental retardation in childhood. Because of the limited sensitivity of routine analyses, the cytogenetic detection of such rearrangements requires molecular techniques, namely FISH and comparative genomic hybridisation (CGH). An alternative approach consists in using genetic markers to detect segmental aneusomy. Here, we describe a new strategy based upon automated fluorescent genotyping to search for non mendelian segregation of telomeric microsatellites. A total of 29 individuals belonging to 24 unrelated families were screened and three abnormal patterns of segregation were detected (two rearrangements and one parental disomy). This study gives strong support to the view that cryptic telomeric rearrangements significantly contribute to idiopathic mental retardation and demonstrates that fluorescent genotyping is a very sensitive and cost-effective method to detect deletions, duplications and uniparental disomies.

Ebstein anomaly associated with rearrangements of chromosomal region 11q.

Ebstein anomaly (EA) is a relatively uncommon congenital heart defect and it is very rarely associated with a chromosomal anomaly. We report two distinct rearrangements of the chromosomal region 11q arm in two unrelated patients with Ebstein anomaly, renal malformation, minor anomalies, and the Pierre Robin sequence. The first patient had an interstitial deletion of chromosome 11 [46,XY,del(11)(11q21q23), and the other had a tertiary trisomy of chromosome 11qter (47,XX,+der(22)t(11;22)(q23;q11.2) Its association with either a chromosome 11q deletion or a duplication in some individuals suggests that a rearrangement of the 11q region is likely to cause a shift of the individuals' underlying liability to develop EA above a certain threshold.

Regulation of PDE-4 cAMP phosphodiesterases by phosphatidic acid.

Phosphatidic acid (PA) has been previously shown to activate specifically some of the isoforms of type 4 cylic nucleotide phosphodiesterases (PDE-4) in an acellular system. In the present work, we have investigated the mechanism of PA-activating effect by using a recombinant PA-sensitive isoform, PDE-4D3. The enzyme was specifically activated by acidic phospholipids, but not by zwitterionic phospholipids or anionic detergents. The importance of the role of PA acidic groups in the activation process was confirmed by studying the influence of pH and ionic strength on activation. Crosslinking experiments suggested that PA might influence the ability of PDE-4D3 to form dimers. Binding studies performed with radiolabeled PA showed that PA binds to a PDE-4D3 preparation in a saturable manner. Specifically bound PA was displaced by anionic, but not by zwitterionic phospholipids. With a preparation of PDE-4B2, a PDE-4 isoform insensitive to PA activation, PA binding was only displaced by high concentrations of unlabeled PA, suggesting that high-affinity PA binding sites are only present on PDE-4D3. These data support the hypothesis that PA-activating effect depends on direct binding of the effector on specific sites carried by the PDE-4D3 protein.

[3-dimensional (3D) echography in obstetrics. Advantages and limits]. / Echographie en 3 dimensions (3D) en Obstétrique. Avantages et limites.

Three-dimensional ultrasound (3D) offers new options in imaging modes: such as simultaneous rotation and translation of the three perpendicular planes displayed, surface rendering, or transparent mode providing an imaging of structures with high echogenicity (bones, skull). This new imaging mode is extending the field of conventional two-dimensional ultrasound, but for now it has to be evaluated.

Specific effects of n-3 fatty acids and 8-bromo-cGMP on the cyclic nucleotide phosphodiesterase activity in neonatal rat cardiac myocytes.

The authors have previously resolved four forms of cyclic nucleotide phosphodiesterase (PDE) in neonatal rat cultured cardiomyocytes by use of high-performance liquid chromatography (HPLC) and have shown that the response of the cGMP-stimulated PDE to the effector cGMP was markedly reduced as compared to that of the corresponding isoform present in the heart ventricle of adult rat (70% v 350%). When neonatal rat ventricular myocytes were grown in a medium enriched in docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA), an increase of the basal level of cAMP and mainly cGMP was observed. The cGMP-PDE specific activity in the unfractionated cytosol of DHA-, EPA- or 8-bromo-cGMP-enriched cardiomyocytes was lower than that observed in control cells. Whereas the cAMP-PDE specific activity remained unchanged whatever the treatment used. At the same time, the response of the cGMP-stimulated PDE to cGMP was substantially increased in n-3 fatty acid-enriched cardiomyocytes and reached the same level as in the whole ventricle (340%). The treatment of neonatal cardiomyocytes with 8-bromo-cGMP also re-established the sensitivity of this cGMP-stimulated isozyme to cGMP (320%). These results suggest a common mechanism for polyunsaturated fatty acids and 8-bromo-cGMP in the regulation of the cGMP-stimulated PDE activity in cardiomyocytes from new-born rats.

Selective inhibition of rat heart cAMP phosphodiesterases by lipophilic C-methyl-2-phenyl-4H-1-benzopyran-4-ones (C-methylflavones).

A series of eight methoxylated C-methyl-2-phenyl-4H-1-benzopyran-4-ones 3, 6, 10-15 was evaluated as inhibitors of rat heart cytosolic cyclic nucleotide phosphodiesterase (PDE). The 2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-3,8-dimethyl-4H-1-benzopyran-4-one (3) and the 2-(4-methoxyphenyl)-5,7-dimethoxy-3,8-dimethyl-4H-1-benzopyran-4-one (10) have never been previously described. Inhibition was performed on the whole cytosolic preparation and on the four PDE isoforms after HPLC purification. The flavones 3, 6, 10, 13 and 14 were selective and potent inhibitors of the isoforms, namely ROI (rolipram-sensitive) and CGI (cGMP-sensitive) PDEs specifically hydrolyzing cAMP. The di-C-methylflavones 3 and 13 have been shown to be potent inhibitors of these two isoforms, with IC50 values in the micromolar range.

Developmental differences in distribution of cyclic nucleotide phosphodiesterase isoforms in cardiomyocytes and the ventricular tissue from newborn and adult rats.

We characterized cyclic nucleotide phosphodiesterase (PDE) activities in neonatal rat cardiomyocytes and in whole ventricle from newborn and adult rats, to determine whether cyclic nucleotide hydrolytic activities might be influenced by the developmental stage of the animal or by cell isolation and culture procedures. Using anion-exchange high-performance liquid chromatography (HPLC), we obtained four soluble PDE activities from adult rat ventricle. Peak 1, a cyclic GMP-specific PDE was stimulated by Ca2+/calmodulin; peak 2 hydrolyzed cyclic AMP and cyclic GMP, the addition of cyclic GMP stimulating the hydrolysis of cyclic AMP. Peaks 3 and 4 selectively hydrolyzed cyclic AMP, peak 3 being very sensitive to inhibition by rolipram and peak 4 consisting of two different enzyme activities, one inhibited by cyclic GMP (peak 4b) and the other inhibited by rolipram (peak 4a). In cardiomyocytes and whole ventricle from newborn rats, the response of the two cyclic GMP-sensitive PDE isoforms (cyclic GMP-stimulated and cyclic GMP-inhibited PDE) to the effector cyclic GMP was markedly reduced as compared with that of the corresponding isoforms (peaks 2 and 4b) present in the heart ventricle of adult rats. The other peaks were analogous to peaks 1, 3, and 4a of the whole ventricle. The profile of PDE activities in nonmyocardial cells did not differ from that of myocytes. Therefore, the lack of cyclic GMP effects was not due to culture conditions. Differences in PDE activities observed between cardiomyocytes from newborn rats and ventricle from adult rats might be age dependent.

Inhibition of the different phosphodiesterase isoforms of rat heart cytosol by free fatty acids.

The sensitivity of the various phosphodiesterase (PDE) isoforms present in the cytosolic compartment of rat heart to the main fatty acids of the saturated, n-3 and n-6 families was assessed. High-performance liquid chromatography (HPLC) on a Mono Q ion-exchange column resolved four separate cyclic nucleotide phosphodiesterase activities: a calmodulin-activated fraction, a cyclic GMP-stimulated fraction, a cyclic AMP-specific rolipram-sensitive fraction, and a cyclic GMP-inhibited fraction. Polyunsaturated fatty acids (PUFA) were more potent inhibitors than saturated fatty acids whatever the considered PDE isoform. Although all PDE isoforms were affected, the cyclic GMP-stimulated isoform was the most sensitive to the inhibitory effect of PUFAs. The possible influences of free fatty acids (FFA) on cardiac contractility through PDE inhibition are discussed.

Effect of two flavonoid compounds on central nervous system. Analgesic activity.

The psychopharmacological profile of quercetin (Q) and penta-O-ethylquercetin (PQ) showed for both compounds a sedative effect on central nervous system in mice. In this set of pharmacological tests (hypothermia, spontaneous motility, psychomotor activity) penta-O-ethylquercetin always exerted a more pronounced effect than quercetin, probably due to the difference of lipophilicity. In analgesia experiments such as acetic acid-induced writhings, penta-O-ethylquercetin showed a significant dose-related effect whereas quercetin was inactive. As pretreatment with naloxone failed to antagonize the analgesic activity of penta-O-ethylquercetin, it was suggested that penta-O-ethylquercetin acted mainly peripherally.

Cardiovascular action of penta-O-ethylquercetin in the rat.

Intravenous (i.v.) administration of penta-O-ethylquercetin (PQ) dissolved in glycerolformal (GF) induced a rapid and transient fall in arterial blood pressure of rats. The mechanism appears rather complex. Beside its inhibitory properties toward cardiac cyclic AMP phosphodiesterase, the drug seems to lower the adrenergic tone, thus acting both at the central (stimulation of the cardiomoderator medullary center) and peripheral (partial blockade of postsynaptic alpha-adrenergic receptors, decrease in noradrenaline liberation) levels. Repeated i.v. administration of variable dosages of PQ caused a decrease in the hypotensive effect of PQ/GF which might be related to a tachyphylaxy phenomenon. In marked contrast, continuous i.v. infusion of PQ/GF progressively increased arterial blood pressure, which very likely involves a quite different mechanism of action.

Inhibition of the different cyclic nucleotide phosphodiesterase isoforms separated from rat brain by flavonoid compounds.

A series of synthetic pentasubstituted analogs of quercetin were evaluated for their ability to inhibit the various phosphodiesterase isoforms resolved from rat brain cytosol by isoelectric focusing. All the tested compounds were more potent in inhibiting the calcium plus calmodulin-independent isoforms than the dependent ones. Out of the two calcium-independent cyclic AMP-specific isoforms present in brain preparations, the Rolipram-sensitive enzyme proved to be the most sensitive to flavonoid inhibition. In contrast with the antidepressant compound Rolipram which is totally devoid of anticalmodulin property, these flavonoid derivatives exhibited anticalmodulin activity as illustrated by their higher inhibitory potency toward the calmodulin-dependent isoform in the presence of calcium plus calmodulin than in the presence of EGTA and by the ability of [3H] penta-O-ethylquercetin to bind to calmodulin in a calcium-dependent way.

Flavonoid modulation of protein kinase C activation.

The flavonoid quercetin exhibited a biphasic effect on calcium and phospholipid-dependent protein kinase (protein kinase C) activity from rat brain and pig thyroid. At a low concentration (10(-7) M) quercetin stimulated the enzyme activity whereas at higher concentrations quercetin was inhibitory. By contrast the synthetic penta-0-ethylquercetin stimulated protein kinase C activity in a dose-dependent manner. When fresly dispersed pig thyroid cells were treated with penta-0-ethylquercetin or 12-0-tetradecanoylphorbol 13-acetate (TPA), a 50% decrease of the cytosolic protein kinase C activity was observed. These results suggest that the lipophilicity as well as other structural determinants may be crucial for the ability of flavonoids to regulate (inhibit or activate) the enzyme activity.

Activation of the cyclic nucleotide phosphodiesterase from rat heart cytosol by phospholipase C.

Phospholipase C (clostridium perfringens) significantly increased the cyclic nucleotide phosphodiesterase activity of a crude 105 000 g supernatant from rat heart. This activation only concerned the basal activity of cyclic GMP phosphodiesterase determined with 0.25 microM cyclic GMP as substrate, in the presence of EGTA, whereas stimulation was found to be independent of EGTA when phosphodiesterase activity was measured with 0.25 microM cyclic AMP. Similar qualitative results were found for the three cytosolic forms of phosphodiesterase separated from rat heart supernatant by isoelectric focusing. Supplementary experiments provided evidence that the activation of the cyclic AMP-specific phosphodiesterase was attributable to Phospholipase C activity and not to contaminating protease(s). In contrast, the stimulation of the cyclic GMP phosphodiesterase activity appeared to be largely dependent on the proteolytic activity of commercial Phospholipase C. Phosphatidic acid also significantly increased the cyclic AMP phosphodiesterase activity of the rat heart cytosol. These results suggest that the activation of cardiac cyclic AMP phosphodiesterase may be related to changes in phospholipid metabolism, notably the accumulation of phosphatidate, and relevant to physiological regulatory processes.

Pentasubstituted quercetin analogues as selective inhibitors of particulate 3':5'-cyclic-AMP phosphodiesterase from rat brain.

Some penta-O-substituted analogues of quercetin were synthesized and tested for the inhibition of cytosolic and particulate rat brain cyclic AMP and cyclic GMP phosphodiesterase activities. Ten of these compounds are potent and highly selective inhibitors of cAMP hydrolysis with respect to cGMP hydrolysis. They inhibit more potently the particulate enzyme than the cytosolic preparation. The highest selectivity was observed with penta-O-ethylquercetin and analogue 6d, which proved to be more selective and more potent inhibitors than the reference compound Ro 20-1724. Some structure-activity relationships are discussed.

Selective inhibition of separated forms of cyclic nucleotide phosphodiesterase from rat heart by some pentasubstituted quercetin analogs.

Synthetic analogs of quercetin were evaluated as inhibitors of cyclic nucleotide phosphodiesterase in rat heart preparations. Three main enzymatic forms of cyclic nucleotide phosphodiesterase can be resolved from rat heart cytosol by isoelectric focusing. Inhibition studies were performed with the whole cytosolic and particulate preparations and with the cytosolic separated enzymatic forms. All the compounds studied proved more potent as inhibitors of the particular preparation than as inhibitors of the cytosolic fraction. With the exception of water-soluble derivatives and quercetin, they showed a better potency to inhibit cyclic AMP than cyclic GMP phosphodiesterase activity of the cytosolic and particulate preparations; likewise, among the three separated enzymatic forms, the cyclic AMP specific form of pI 5.55-6 is the most inhibited by these flavonoid compounds. In all cases, the highest selectivity was observed with pentaethyl quercetin (2).