The FMRP/GRK4 mRNA interaction uncovers a new mode of binding of the Fragile X mental retardation protein in cerebellum

Fragile X syndrome (FXS), the most common form of inherited intellectual disability, is caused by the silencing of the FMR1 gene encoding an RNA-binding protein (FMRP) mainly involved in translational control. We characterized the interaction between FMRP and the mRNA of GRK4, a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase super-family, both in vitro and in vivo. While the mRNA level of GRK4 is unchanged in the absence or in the presence of FMRP in different regions of the brain, GRK4 protein level is increased in Fmr1-null cerebellum, suggesting that FMRP negatively modulates the expression of GRK4 at the translational level in this brain region. The C-terminal region of FMRP interacts with a domain of GRK4 mRNA, that we called G4RIF, that is folded in four stem loops. The SL1 stem loop of G4RIF is protected by FMRP and is part of the S1/S2 sub-domain that directs translation repression of a reporter mRNA by FMRP. These data confirm the role of the G4RIF/FMRP complex in translational regulation. Considering the role of GRK4 in GABAB receptors desensitization, our results suggest that an increased GRK4 levels in FXS might contribute to cerebellum-dependent phenotypes through a deregulated desensitization of GABAB receptors.     

Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1_BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1_BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1_BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1_BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs.     

Potential role of NK cells in future anti-tumor immunotherapies

Natural Killer (NK) cells are no longer considered as relatively unimportant bystander cells having the capacity to kill certain tumor and virus-infected cells in a mysterious way. During the last decade a significant progress has been made in understanding biology of NK cells in particular their mechanisms of recognition and killing of target cells. This progress has led to novel knowledge-based clinical applications of NK cells as immunotherapeutic tools in various disease settings, especially in bone marrow transplantation for leukemia patients. The potential of NK cell therapy for eradicating solid tumors has not been fully exploited. In this mini-review, we examine the rationale behind these therapies and discuss the problems confronting researchers in their usage as therapeutic agents.     

Photosynthetic activity and leaf antioxidative responses of Atriplex portulacoides subjected to extreme salinity

Responses of Atriplex portulacoides upon 40-day-long exposure to salinity (0?C1,000?mM NaCl) were investigated. Mother plants originated from a sabkha located in a semi-arid region of Tunisia. The plant relative growth rate and leaf expansion increased significantly at 200?mM NaCl but decreased at higher salinities. Interestingly, the plants survived salinity as high as 1,000?mM NaCl without displaying salt-induced toxicity symptoms. Despite significant increase in leaf Na⁺ and Cl^? concentrations upon salt treatment, no significant effect on leaf relative water content was registered. Chlorophyll contents and the gas exchange parameters showed a significant stimulation at the optimal salinity (200?mM NaCl) followed by a decline at higher salinities. Extreme salinity hardly impacted the maximal efficiency of photosystem II photochemistry (F _v/F _m), but a marked decrease in the relative quantum yield of photosystem II (??_PSII) was observed, along with a significant increase in non-photochemical quenching (NPQ). Leaf malondialdehyde and carotenoid contents were generally unaffected following salt exposure, whereas those of anthocyanins, polyphenols, and proline increased significantly, being maximal at 1,000?mM NaCl. Leaf superoxide dismutase (EC 1.15.1.1), ascorbate peroxidase (EC 1.11.1.11), and glutathione reductase (EC 1.6.4.2) activities were significantly stimulated by salinity, whereas catalase (EC 1.11.1.6) activity was maximal in the 0?C400?mM NaCl range. As a whole, protecting the photosynthetic machinery from salt-induced photodamage together with the sustained antioxidant activity may account for the performance of A. portulacoides under high salinity.     

On the disposition of phospholipids in freshly isolated myelin sheath preparations from bovine brain

The extent of availability of the four major classes of phospholipids in freshly isolated bovine brain myelin has been studied using phospholipase C (Clostridium perfringens), phospholipase A (bee venom), sphingomyelase (Staphalococcus aureus) and trinitrobenzenesulphonate. The results have been compared with those of a previous study (Gwarsha et al., 1984). Taken together, the data suggest that 47–67% of the phosphatidylcholine, 45–66% of the ethanolamine-containing phosphoglycerides, 52–55% of the spingomyelin and 10–32% of the phosphatidylserine + phosphatidylinositol in the fresh myelin is available to these probes and hence may be located at the external surface of the membrane bilayer.     

Correlation between synthesis variation of 2-alkylquinolones and the antifungal activity of a Burkholderia cepacia strain collection

Twenty epiphytic and rhizospheric bacterial strains harbouring strong antifungal activities were isolated from the Tunisian environment. This group of bacteria was identified as Burkholderia cepacia genomovar I using 16S rDNA and recA fragment gene sequence analyses for two selected strains and RFLP technique for the eighteen other ones. This identification did not show variability between isolates despite the significant differences in the antifungal activities of their culture supernatant and the organic crude extract against Aspergillus niger and other phytopathogenic fungi. Chromatographic and mass spectrometric analyses of these extracts allowed us to confirm the difference between strains of the group. Their metabolic production showed differences in term of contents and quantities of secreted molecules, particularly those which were identified to be involved in the antifungal activities. Two metabolites, named Bc-255 and Bc-257 secreted by the entire group at different amounts, have been purified and tested separately against A. niger. Bc-255 showed an activity twice as high as those shown by Bc-257. The structural characterization of these two compounds by mass spectrometry and nuclear magnetic resonance spectroscopy allowed their identification as two analogous 2-alkylquinolones with only one difference at the alkyl chain.     

Pituitary adenylate cyclase-activating polypeptide protects astroglial cells against oxidative stress-induced apoptosis

Oxidative stress, associated with a variety of disorders including neurodegenerative diseases, results from accumulation of reactive oxygen species (ROS). Oxidative stress is not only responsible for neuron apoptosis, but can also provoke astroglial cell death. Numerous studies indicate that pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuron survival, but nothing is known regarding the action of PACAP on astroglial cell survival. Thus, the purpose of the present study was to investigate the potential glioprotective effect of PACAP on H(2)O(2)-induced astrocyte death. Pre-treatment of cultured rat astrocytes with nanomolar concentrations of PACAP prevented cell death provoked by H(2)O(2) (300 μM), whereas vasoactive intestinal polypeptide was devoid of protective activity. The effect of PACAP on astroglial cell survival was abolished by the type 1 PACAP receptor antagonist, PACAP6-38. The protective action of PACAP was blocked by the protein kinase A inhibitor H89, the protein kinase C inhibitor chelerythrine and the mitogen-activated protein (MAP)-kinase kinase (MEK) inhibitor U0126. PACAP stimulated glutathione formation, and blocked H(2)O(2)-evoked ROS accumulation and glutathione content reduction. In addition, PACAP prevented the decrease of mitochondrial activity and caspase 3 activation induced by H(2)O(2). Taken together, these data indicate for the first time that PACAP, acting through type 1 PACAP receptor, exerts a potent protective effect against oxidative stress-induced astrocyte death. The anti-apoptotic activity of PACAP on astrocytes is mediated through the protein kinase A, protein kinase C and MAPK transduction pathways, and can be accounted for by inhibition of ROS-induced mitochondrial dysfunctions and caspase 3 activation.     

Salt tolerance analysis of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> NOK2 accession under saline conditions and potassium supply

The response to salt treatment and K⁺ provision of two Arabidopsis thaliana accessions grown for 17 days in the presence of 50 mM NaCl was investigated. Leaf and root dry weight deposition was restricted by salt, more in Col accession than in NOK2 accession. In both accessions, the growth inhibition induced by salinity was associated with a decrease in total leaf surface area, which resulted from diminished leaf number, but not from restriction of individual leaf surface area. Comparing the effects of salt on dry matter production and total leaf surface area revealed large difference between Col and NOK2 for net assimilation rate (the amount of whole plant biomass produced per unit leaf surface area), which was augmented by salt and K⁺ in NOK2 but not in Col. This result, which suggested a better capacity of NOK2 to preserve its photosynthetic machinery against salt stress, was in agreement with the effect of NaCl on photosynthetic pigments. Indeed, salt significantly reduced chlorophyll and carotenoid content in Col leaves but had no impact on NOK2 leaf pigment content. Since K⁺ provision had only marginal effects on these responses to salt stress, leaf mineral unbalance was unlikely. Guaiacol peroxidase activity was augmented by salt treatment in leaves and roots of both accessions. Salinity decreased the catalase activity in Col leaves and in roots, and increased this activity in NOK2 organs. In conclusion, when aggressed by salt, NOK2 was able (1) to produce more leaves than Col, and (2) to efficiently protect its photosynthetic apparatus, perhaps by developing more efficient antioxidative defense through increased catalase and peroxidase activities. Consequently, the overall photosynthetic activity was higher and more robust to salt aggression in NOK2 than in Col.     

Environmental <Emphasis Type="Italic">Burkholderia cepacia</Emphasis> Strain Cs5 Acting by Two Analogous Alkyl-Quinolones and a Didecyl-Phthalate Against a Broad Spectrum of Phytopathogens Fungi

An environmental Burkholderia cepacia strain named Cs5 was isolated and identified first using API biochemical identification system and then with 16S rDNA and recA sequence homology search. This bacterium exhibited a broad spectrum of fungicidal activities against Alternaria alternata, Aspergillus niger, Fusarium culmorum, F. graminearum, F. oxysporum and Rhizoctonia solani. In the liquid conditions, the MIC of A. niger and R. solani were reached with, respectively, 1.25–2% of the Cs5 liquid culture supernatant. However, in the solid conditions, the same inhibition was caused in the presence of 3% of the Cs5 supernatant. The exhibition of these two fungi at low concentrations of supernatant Cs5 caused various morphological changes of their mycelia which were observed by confocal microscopy. Three antifungal compounds, named Cs5-255, Cs5-257 and Cs5-446, were purified from the Cs5 culture. The structural analysis of these molecules showed that Cs5-255 and Cs5-257 are analogous and belonged to the alkyl-quinolone family, while Cs5-446 was a didecyl-phthalate, isolated for the first time from a bacterium.