Composition of fatty acids triacylglycerols and unsaponifiable matter in Calophyllum calaba L. oil from Guadeloupe

The composition of the kernel oils of two Calophyllum species (Calophyllum calaba L. and Calophyllum inophyllum L.) was investigated. The physico-chemical properties and fatty acid composition of the kernel oils were examined. In two species, oleic acid C18:1 (39.1-50%) is the dominating fatty acid followed by linoleic acid C18:2 (21.7-31.1%) as the second major fatty acid. Stearic C18:0 (13.4-14.3%) and palmitic C16:0 (11-13.7%) acids are the major saturates. The oils contains an appreciable amount of unsaturated fatty acids (70.8-73.10%). Most of the fatty acids are present as triacylglycerol (76.7-84%), twenty one triacylglycerols are detected with predominantly unsaturated triacylglycerols. The total unsaponifiable content, its general composition and the identity of the components of the sterol and tocopherol fractions are presented. In both species, analysis of the unsaponifiable fractions revealed the preponderance of phytosterols, mainly stigmasterol (35.8-45.1%) and beta-sitosterol (41.1-43.1%). Among the eight tocopherols and tocotrienols present in two species, variations exist; alpha-tocopherol (183 mg/kg) is the main tocopherol in Calophyllum calaba L. and Delta-tocotrienol (236 mg/kg) is the dominant tocotrienol in Calophyllum inophyllum L.     

Pharmacological characterisation of (E)-N-(3-iodoprop-2-enyl)-2beta-carbomethoxy-3beta-(4'-methylphenyl)nortropane (PE2I) binding to the rat neuronal dopamine transporter expressed in COS cells.

The interaction of (E)-N-(3-iodoprop-2-enyl)-2beta-Carbomethoxy-3beta-(4'-methylphenyl) nortropane (PE2I) with the rat neuronal dopamine transporter (DAT) was studied in transfected COS cells by measuring its ability to inhibit DA uptake and by measuring its affinity in radioligand binding experiments. Saturable [3H]DA uptake was measured in COS cells transiently transfected with the cDNA sequence encoding the rat DAT. Pharmacological characterisation of this uptake revealed functional properties with a V(max) value of 45.05+/-2.62 pmol/mg protein per min and a K(m) value of 2.86+/-0.28 microM. The specific [3H]DA uptake was fully inhibited by 1 microM PE2I. Concentration response curves revealed the high potency of PE2I in inhibiting DA uptake (pEC(50) value of 8.70+/-0.33), 25 times higher than that observed for the reference DAT inhibitor, GBR 12935. On crude homogenates from transfected COS cells, PE2I displaced the specific binding of [3H]GBR 12935 with a pK(i) value of 7.73+/-0.13. Accordingly, [125I]PE2I was found to specifically recognise a single binding site population which is almost completely displaced by GBR 12935 and nomifensine. Saturation experiments revealed the high affinity of [125I]PE2I (K(D) value of 3.8+/-0.63 nM) that correlates with the high potency of PE2I in inhibiting the [3H]DA uptake. This contrasts with the results obtained with GBR 12935 for which a discrepancy was found between its high affinity in binding assays (K(D) value of 0.43+/-0.04 nM) and its rather low potency in functional assays (pEC(50) value of 7.30+/-0.05). A relatively high level of [3H]GBR 12935 binding was detected in non transfected COS cells. Such nomifensine resistant binding is attributed to the interaction of GBR 12935 with cytochrome P-450 as it was displaced by cis-(Z)-flupentixol (an inhibitor of cytochrome P-450). Such interaction was not observed using PE2I. Taken together, these data demonstrate that PE2I was a highly potent inhibitor of cloned DAT compared with GBR 12935 and provided a useful tool for further investigations in cells transfected with cDNA encoding the DAT.     

Molecular and Morphological Identification of Mealybug Species (Hemiptera: Pseudococcidae) in Brazilian Vineyards

Mealybugs (Hemiptera: Pseudococcidae) are pests constraining the international trade of Brazilian table grapes. They damage grapes by transmitting viruses and toxins, causing defoliation, chlorosis, and vigor losses and favoring the development of sooty mold. Difficulties in mealybug identification remain an obstacle to the adequate management of these pests. In this study, our primary aim was to identify the principal mealybug species infesting the major table grape-producing regions in Brazil, by morphological and molecular characterization. Our secondary aim was to develop a rapid identification kit based on species-specific Polymerase Chain Reactions, to facilitate the routine identification of the most common pest species. We surveyed 40 sites infested with mealybugs and identified 17 species: Dysmicoccus brevipes (Cockerell), Dysmicoccus sylvarum Williams and Granara de Willink, Dysmicoccus texensis (Tinsley), Ferrisia cristinae Kaydan and Gullan, Ferrisia meridionalis Williams, Ferrisia terani Williams and Granara de Willink, Phenacoccus baccharidis Williams, Phenacoccus parvus Morrison, Phenacoccus solenopsis Tinsley, Planococcus citri (Risso), Pseudococcus viburni (Signoret), Pseudococcus cryptus Hempel, four taxa closely related each of to Pseudococcus viburni, Pseudococcus sociabilis Hambleton, Pseudococcus maritimus (Ehrhorn) and Pseudococcus meridionalis Prado, and one specimen from the genus Pseudococcus Westwood. The PCR method developed effectively identified five mealybug species of economic interest on grape in Brazil: D. brevipes, Pl. citri, Ps. viburni, Ph. solenopsis and Planococcus ficus (Signoret). Nevertheless, it is not possible to assure that this procedure is reliable for taxa that have not been sampled already and might be very closely related to the target species.     

Group I metabotropic glutamate receptors activate the p70S6 kinase via both mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK 1/2) signaling pathways in rat striatal and hippocampal synaptoneurosomes

Group I metabotropic glutamate receptors (mGluRs) have been demonstrated to play a role in synaptic plasticity via a rapamycin-sensitive mRNA translation signaling pathway. Various growth factors can stimulate this pathway, leading to the phosphorylation and activation of mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that modulates the activity of several translation regulatory factors, such as p70S6 kinase. However, little is known about the cellular and molecular mechanisms that bring the plastic changes of synaptic transmission after stimulation of group I mGluRs. Here, we investigated the role of the mTOR-p70S6K and the ERK1/2-p70S6K pathways in rat striatal and hippocampal synaptoneurosomes after group I mGluR stimulation. Our findings show that (S)-3,5-dihydroxyphenylglycine (DHPG) increases significantly the activation of mTOR and p70S6K (Thr389, controlled by mTOR) in both brain areas. The mTOR activation is dose-dependent and requires the stimulation of mGluR1 subtype receptors as for the p70S6K activation observed in striatum and hippocampus. In addition, the p70S6K (Thr421/Ser424) activation via the ERK1/2 activation is increased and involved also mGluR1 receptors. These results demonstrate that group I mGluRs are coupled to mTOR-p70S6K and ERK1/2-p70S6K pathways in striatal and hippocampal synaptoneurosomes. The translational factor p70S6K could be involved in the group I mGluRs-modulated synaptic efficacy.     

Interaction of Double-stranded RNA-dependent Protein Kinase (PKR) with the Death Receptor Signaling Pathway in Amyloid �� (A��)-treated Cells and in APPSLPS1 Knock-in Mice

For 10 years, research has focused on signaling pathways controlling translation to explain neuronal death in Alzheimer Disease (AD). Previous studies demonstrated in different cellular and animal models and AD patients that translation is down-regulated by the activation of double-stranded RNA-dependent protein kinase (PKR). Among downstream factors of PKR, the Fas-associated protein with a death domain (FADD) and subsequent activated caspase-8 are responsible for PKR-induced apoptosis in recombinant virus-infected cells. However, no studies have reported the role of PKR in death receptor signaling in AD. The aim of this project is to determine physical and functional interactions of PKR with FADD in amyloid-β peptide (Aβ) neurotoxicity and in APP_SLPS1 KI transgenic mice. In SH-SY5Y cells, results showed that Aβ42 induced a large increase in phosphorylated PKR and FADD levels and a physical interaction between PKR and FADD in the nucleus, also observed in the cortex of APP_SLPS1 KI mice. However, PKR gene silencing or treatment with a specific PKR inhibitor significantly prevented the increase in pT⁴⁵¹-PKR and pS¹⁹⁴-FADD levels in SH-SY5Y nuclei and completely inhibited activities of caspase-3 and -8. The contribution of PKR in neurodegeneration through the death receptor signaling pathway may support the development of therapeutics targeting PKR to limit neuronal death in AD.     

Mutations in IMPG1 Cause Vitelliform Macular Dystrophies

Vitelliform macular dystrophies (VMD) are inherited retinal dystrophies characterized by yellow, round deposits visible upon fundus examination and encountered in individuals with juvenile Best macular dystrophy (BMD) or adult-onset vitelliform macular dystrophy (AVMD). Although many BMD and some AVMD cases harbor mutations in BEST1 or PRPH2, the underlying genetic cause remains unknown for many affected individuals. In a large family with autosomal-dominant VMD, gene mapping and whole-exome sequencing led to the identification of a c.713T>G (p.Leu238Arg) IMPG1 mutation, which was subsequently found in two other families with autosomal-dominant VMD and the same phenotype. IMPG1 encodes the SPACR protein, a component of the rod and cone photoreceptor extracellular matrix domains. Structural modeling indicates that the p.Leu238Arg substitution destabilizes the conserved SEA1 domain of SPACR. Screening of 144 probands who had various forms of macular dystrophy revealed three other IMPG1 mutations. Two individuals from one family affected by autosomal-recessive VMD were homozygous for the splice-site mutation c.807+1G>T, and two from another family were compound heterozygous for the mutations c.461T>C (p.Leu154Pro) and c.1519C>T (p.Arg507^∗). Most cases had a normal or moderately decreased electrooculogram Arden ratio. We conclude that IMPG1 mutations cause both autosomal-dominant and -recessive forms of VMD, thus indicating that impairment of the interphotoreceptor matrix might be a general cause of VMD.     

Sulfatide and GM1 ganglioside modulate the high-affinity dopamine uptake in rat striatal synaptosomes: evidence for the involvement of their ionic charges

The present study was undertaken to examine the effects of the anionic glycolipids GM1 ganglioside and sulfatide on the high-affinity dopamine (DA) uptake in rat striatal synaptosomes.After 1h of incubation, GM1 stably bound to synaptosomes and modified the activity of the neuronal dopamine transporter (DAT). With 1.2 and 12 microM GM1, V(max) decreased by 13 and 23%, respectively, reflecting a slight reduction of the number of functional uptake sites and K(m) was lowered by 21 and 33%, thus showing an increase of the affinity. Treatment of synaptosomes with 1.2 microM of sulfatide, which possesses an anionic sulfated group, led to a similar decrease of V(max) (19%) than GM1, but to a significantly higher reduction of K(m) (35%). In fact, sulfatide associated to synaptosomes in a 3.5-fold higher extent than GM1. Conversely, when GM1 and sulfatide were replaced by GM1 alcohol and galactosylceramide, respectively, no modification of the DA uptake occurred, although these neutral glycolipids incorporated into the synaptosomes to the same extent as the related anionic compounds.Altogether, these results demonstrate the key role of negative charges linked to the oligosaccharide chains of glycolipids in the modulation of DA transport across the synaptosomal membrane.     

The oxindole/imidazole derivative C16 reduces in vivo brain PKR activation

Inhibition of double-stranded RNA-dependent protein kinase (PKR) represents an interesting strategy for neuroprotection. However, inhibiting this kinase which triggers the apoptotic process could favour in counterpart cell proliferation and tumorigenesis. Here, we use an in vivo model of 7-day-old rat displaying a high activation of brain PKR to investigate the effects of a new PKR inhibitor identified as an oxindole/imidazole derivative (C16). We show for the first time that acute systemic injection of C16 specifically inhibits the apoptotic PKR/eIF2alpha signaling pathway without stimulating the proliferative mTOR/p70S6K signaling mechanism.     

Inflammatory Stress on Autophagy in Peripheral Blood Mononuclear Cells from Patients with Alzheimer's Disease during 24 Months of Follow-Up

Recent findings indicate that microglia in Alzheimer’s disease (AD) is senescent whereas peripheral blood mononuclear cells (PBMCs) could infiltrate the brain to phagocyte amyloid deposits. However, the molecular mechanisms involved in the amyloid peptide clearance remain unknown. Autophagy is a physiological degradation of proteins and organelles and can be controlled by pro-inflammatory cytokines. The purpose of this study was to evaluate the impact of inflammation on autophagy in PBMCs from AD patients at baseline, 12 and 24 months of follow-up. Furthermore, PBMCs from healthy patients were also included and treated with 20 μM amyloid peptide 1–42 to mimic AD environment. For each patient, PBMCs were stimulated with the mitogenic factor, phytohaemagglutin (PHA), and treated with either 1 μM C16 as an anti-inflammatory drug or its vehicle. Autophagic markers (Beclin-1, p62/sequestosome 1 and microtubule-associated protein-light chain 3: LC3) were quantified by western blot and cytokines (Interleukin (IL)-1β, Tumor necrosis Factor (TNF)-α and IL-6) by Luminex X-MAP^® technology. Beclin-1 and TNF-α levels were inversely correlated in AD PBMCs at 12 months post-inclusion. In addition, Beclin-1 and p62 increased in the low inflammatory environment induced by C16. Only LC3-I levels were inversely correlated with cognitive decline at baseline. For the first time, this study describes longitudinal changes in autophagic markers in PBMCs of AD patients under an inflammatory environment. Inflammation would induce autophagy in the PBMCs of AD patients while an anti-inflammatory environment could inhibit their autophagic response. However, this positive response could be altered in a highly aggressive environment.