Structure-activity relationships of neuropeptide FF: role of C-terminal regions

A structure-activity study was carried out to determine the importance of the C-terminal amino acids of the octapeptide Neuropeptide FF (NPFF) in binding and agonistic activity. Affinities of NPFF analogues were tested toward NPFF receptors of the rat spinal cord and the human NPFF2 receptors transfected in CHO cells. The activities of these analogues were evaluated by their ability to both inhibit adenylate cyclase in NPFF2 receptor transfected CHO cells and to reverse the effect of nociceptin on acutely dissociated rat dorsal raphe neurons. The substitutions of Phenylalanine8 by a tyrosine, phenylglycine or homophenylalanine were deleterious for high affinity. Similarly, the replacement of Arginine7 by a lysine or D.Arginine induces a loss in affinity. The pharmacological characterization showed that the presence of the amidated Phe8 and Arg7 residues are also extremely critical for activation of anti-opioid effects on dorsal raphe neurons. The sequence of the C-terminal dipeptide seems also to be responsible for the high affinity and the activity on human NPFF2 receptors. The results support the view that a code messaging the molecular interaction toward NPFF-receptors is expressed in the C-terminal region of these peptides but the N-terminal segment is important to gain very high affinity.     

Myristoylation-dependent N-terminal cleavage of the myristoylated alanine-rich C kinase substrate (MARCKS) by cellular extracts

The myristoylated alanine-rich C kinase substrate (MARCKS) has been proposed to regulate the plasticity of the actin cytoskeleton at its site of attachment to membranes. In macrophages, MARCKS is implicated in various cellular events including motility, adhesion and phagocytosis. In this report we show that macrophage extracts contain a protease which specifically cleaves human MARCKS, expressed in a cell-free system or in E. coli, between Lys-6 and Thr-7. Cleavage of MARCKS decreases its affinity for macrophage membranes by ca. one order of magnitude, highlighting the contribution of the myristoyl moiety of MARCKS to membrane binding. Importantly, cleavage requires myristoylation of MARCKS. Furthermore, MARCKS-related protein (MRP), the second member of the MARCKS family, is not digested. Since Thr-7 is lacking in MRP this suggests that Thr-7 at the P1 position is important for the recognition of lipid-modified substrates. A different product is observed when MARCKS is incubated with a calf brain cytosolic extract. This product can be remyristoylated in the presence of myristoyl-CoA and N-myristoyl transferase, demonstrating that cycles of myristoylation/demyristoylation of MARCKS can be achieved in vitro. Although the physiological relevance of these enzymes still needs to be demonstrated, our results reveal the presence of a new class of cleaving enzymes recognizing lipid-modified protein substrates.     

Insights into the unfolding pathway and identification of thermally sensitive regions of phytase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> by molecular dynamics simulations

Thermal stability is of great importance in the application of commercial phytases. Phytase A (PhyA) is a monomeric protein comprising twelve α-helices and ten β-sheets. Comparative molecular dynamics (MD) simulations (at 310, 350, 400, and 500 K) revealed that the thermal stability of PhyA from Aspergillus niger (A. niger) is associated with its conformational rigidity. The most thermally sensitive regions were identified as loops 8 (residues 83–106), 10 (161–174), 14 (224–230), 17 (306–331), and 24 (442–444), which are present on the surface of the protein. It was observed that solvent-exposed loops denature before or show higher flexibility than buried residues. We observed that PhyA begins to unfold at loops 8 and 14, which further extends to loop 24 at the C-terminus. The intense movement of loop 8 causes the helix H2 and beta-sheet B3 to fluctuate at high temperature. The high flexibility of the H2, H10, and H12 helices at high temperature resulted in complete denaturation. The high mobility of loop 14 easily transfers to the adjacent helices H7, H8, and H9, which fluctuate and partially unfold at high temperature (500 K). It was also observed that the salt bridges Asp110–Lys149, Asp205–Lys277, Asp335–Arg136, Asp416–Arg420, and Glu387–Arg400 are important influences on the structural stability but not the thermostability, as the lengths of these salt bridges did not increase with rising temperature. The salt bridges Glu125–Arg163, Asp299–Arg136, Asp266–Arg219, Asp339–Lys278, Asp335–Arg136, and Asp424–Arg428 are all important for thermostability, as the lengths of these bridges increased dramatically with increasing temperature. Here, for the first time, we have computationally identified the thermolabile regions of PhyA, and this information could be used to engineer novel thermostable phytases. Numerous homologous phytases of fungal as well as bacterial origin are known, and these homologs show high sequence similarity. Our findings could prove useful in attempts to increase the thermostability of homologous phytases via protein engineering.     

Discrepancy between WISC-III and WISC-IV Cognitive Profile in Autism Spectrum: What Does It Reveal about Autistic Cognition?

The cognitive profile and measured intellectual level vary according to assessment tools in children on the autism spectrum, much more so than in typically developing children. The recent inclusion of intellectual functioning in the diagnostic process for autism spectrum disorders leads to the crucial question on how to assess intelligence in autism, especially as some tests and subtests seem more sensitive to certain neurodevelopmental conditions. Our first aim was to examine the cognitive profile on the current version of the most widely used test, the Wechsler Intelligence Scales for Children (WISC-IV), for a homogenous subgroup of children on the autism spectrum, i.e. corresponding to DSM-IV diagnosis of “autism”. The second aim was to compare cognitive profiles obtained on the third edition versus 4^th edition of WISC, in order to verify whether the WISC-IV yields a more distinctive cognitive profile in autistic children. The third aim was to examine the impact of the WISC-IV on the cognitive profile of another subgroup, children with Asperger’s Syndrome. 51 autistic, 15 Asperger and 42 typically developing children completed the WISC-IV and were individually matched to children who completed the WISC-III. Divergent WISC-IV profiles were observed despite no significant intelligence quotient difference between groups. Autistic children scored significantly higher on the Perceptual Reasoning Index than on the Verbal Comprehension Index, a discrepancy that nearly tripled in comparison to WISC-III results. Asperger children scored higher on the VCI than on other indexes, with the lowest score found on the Processing Speed Index. WISC-IV cognitive profiles were consistent with, but more pronounced than WISC-III profiles. Cognitive profiles are a valuable diagnostic tool for differential diagnosis, keeping in mind that children on the autism spectrum might be more sensitive to the choice of subtests used to assess intelligence.     

Analyses archéométriques dans la grotte ornée Mayenne-Sciences (Thorigné-en-Charnie, Mayenne)

Since 1998, a multidisciplinary team works on the study of the representations in the cave Mayenne-Sciences (Thorigné-en-Charnie, Mayenne). Particularly, there are trials for U-TH (TIMH) datations of the speleothems recovering the drawings and fossils of the bats; this would help to know at what date the decorated cave was closed. The studies already allowed to know better the black drawings, executed with a wooden charcoal crayon. Thus, it was possible to make removals which gave two dates from the Gravettian phase; this feeds again the discussion about the chronocultural position of this cave, on of the most septentrional caves we know.     

Lipotropha dorci n. sp. [Neogregarinida], parasite des larves deDorcus parallelipipedus L. [Coleopt. scarab.]

Résumé Des larves deDorcus parallelipipedus L. provenant de Saint-Hilaire-de-Brethmas (Gard) dans la région méditerranéenne de France et récoltées dans des troncs décomposés de chataigniers, se sont révélées intensément parasitées par des Grégarines appartenant à l'ordre desNeogregarinida. Nous étudions leur localisation dans le tissu adipeux, leur cycle, l'action pathogène sur l'h?te et discutons leur place systématique.

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Summary Lipotropha dorci n. sp. (Neogregarinida) is a parasite developing inDorcus parallelepipedus. The parasite is situated only in the fat body which can be destroyed completely by its activity. The life cycle ofLipotropha includes only one type of schizogony with 3–4 μ long merozo?ts, the spherical uninucleate gametocytes are gathered in pairs and form 23 μ long gametokysts. These give rise to 11 to 12 μ long sporocysts with a thick, non ornamented envelope, gathered round rests of cytoplasma. The size of these different stages enables to distinguishLipotropha dorci from the already described species:Lipotropha macrospora Keilin andLipotropha microspora Keilin.

     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.