Design, synthesis and activity as acid ceramidase inhibitors of 2-oxooctanoyl and N-oleoylethanolamine analogues

The synthesis of novel N-acylethanolamines and their use as inhibitors of the aCDase is reported here. The compounds are either 2-oxooctanamides or oleamides of sphingosine analogs featuring a 3-hydroxy-4,5-hexadecenyl tail replaced by ether or thioether moieties. It appears that, within the 2-oxooctanamide family, the C3-OH group of the sphingosine molecule is required for inhibition both in vitro and in cultured cells. Furthermore, although the (E)-4 double bond is not essential for inhibitory activity, the (E) configuration is required, since the analogue with a (Z)-4 unsaturation was not inhibitory. None of the oleamides inhibited the aCDase in vitro. Conversely, with the exception of N-oleoylethanolamine and its analogs with S-decyl and S-hexadecyl substituents, all the synthesized oleamides inhibited the aCDase in cultured cells, although with a relatively low potency. We conclude that novel aCDase inhibitors can evolve from N-acylation of sphingoid bases with electron deficient-acyl groups. In contrast, chemical modification of the N-oleoylsphingosine backbone does not seem to offer an appropriate strategy to obtain aCDase inhibitors.     

Oxidative DNA damage and antioxidant defenses in the European common lizard (Lacerta vivipara) in supercooled and frozen states

The European common lizard (Lacerta vivipara) tolerates long periods at sub-zero temperatures, either in the supercooled or the frozen state. Both physiological conditions limit oxygen availability to tissues, compelling lizards to cope with potential oxidative stress during the transition from ischemic/anoxic conditions to reperfusion with aerated blood during recovery. To determine whether antioxidant defenses are implicated in the survival of lizards when facing sub-zero temperatures, we monitored the activities of antioxidant enzymes and oxidative stress either during supercooling or during freezing exposures (20 h at -2.5 degrees C) and 24 h after thawing in two organs of lizards--muscle and liver. Supercooling induced a significant increase in the total SOD and GPx activity in muscle (by 67 and 157%, respectively), but freezing had almost no effect on enzyme activity, either in muscle or in liver. By contrast, thawed lizards exhibited higher GPx activity in both organs (a 133% increase in muscle and 59% increase in liver) and a significant decrease in liver catalase activity (a 47% difference between control and thawed lizards). These data show that supercooling (but not freezing) triggers activation of the antioxidant system and this may be in anticipation of the overgeneration of oxyradicals when the temperature increases (while thawing or at the end of supercooling). Oxidative stress was assessed from the content of 8-oxodGuo and the different DNA adducts resulting from lipid peroxidation, but it was unaltered whatever the physiological state of the lizards, thus demonstrating the efficiency of the antioxidant system that has been developed by this species. Overall, antioxidant defenses appear to be part of the adaptive machinery for reptilian tolerance to sub-zero temperatures.     

Antimicrobial peptides from diverse families isolated from the skin of the Asian frog, Rana grahami

Seven peptides with antimicrobial activity were isolated in pure form from an extract of the skin of the Yunnanfu Kunming frog Rana grahami Boulenger, 1917. The peptides were identified as belonging to the nigrocin-2 (three peptides), brevinin-1 (one peptide), brevinin-2 (three peptides), and esculentin-1 (one peptide) families. Nigrocin-2GRb (GLFGKILGVGKKVLCGLSGMC) containing three lysine residues, represented the peptide with highest potency against microorganisms (MIC = 3 microM against Escherichia coli, 12.5 microM against Staphylococcus aureus and 50 microM against Candida albicans) and the greatest hemolytic activity against human erythrocytes (LD50 = 40 microM). In contrast, nigrocin-2GRa (GLLSGILGAGKHIVCGLSGLC) and nigrocin-2GRc (GLLSGILGAGKNIVCGLSGLC), with only a single lysine residue, showed weak antimicrobial and hemolytic activity. Phylogenetic relationships among Eurasian ranid frogs are less well understood than those of North American ranids but the primary structures of the R. grahami antimicrobial peptides suggest a close relationship of this species with the Japanese pond frogs R. nigromaculata and R. porosa brevipoda.     

GLUT8 is dispensable for embryonic development but influences hippocampal neurogenesis and heart function

GLUT8 is a glucose transporter isoform expressed at high levels in testis; at intermediate levels in the brain, including the hippocampus; and at lower levels in the heart and several other tissues. GLUT8 is located in an intracellular compartment and does not appear to translocate to the cell surface, except in blastocysts, where insulin has been reported to induce its surface expression. Here, we generated mice with inactivation of the glut8 gene. We showed that expression of GLUT8 was not required for normal embryonic development and that glut8-/- mice had normal postnatal development, glucose homeostasis, and response to mild stress. Adult glut8-/- mice showed increased proliferation of hippocampal cells but no defect in memory acquisition and retention. Absence of GLUT8 from the heart did not alter heart size and morphology but led to an increase in P-wave duration, which was not associated with abnormal Nav1.5 Na+ channel or connexin expression. Thus, absence of GLUT8 expression in the mouse caused complex but mild physiological alterations.     

Peroxisome proliferator-activated receptor gamma regulates E-cadherin expression and inhibits growth and invasion of prostate cancer

Peroxisome proliferator-activated receptor gamma (PPARgamma) might not be permissive to ligand activation in prostate cancer cells. Association of PPARgamma with repressing factors or posttranslational modifications in PPARgamma protein could explain the lack of effect of PPARgamma ligands in a recent randomized clinical trial. Using cells and prostate cancer xenograft mouse models, we demonstrate in this study that a combination treatment using the PPARgamma agonist pioglitazone and the histone deacetylase inhibitor valproic acid is more efficient at inhibiting prostate tumor growth than each individual therapy. We show that the combination treatment impairs the bone-invasive potential of prostate cancer cells in mice. In addition, we demonstrate that expression of E-cadherin, a protein involved in the control of cell migration and invasion, is highly up-regulated in the presence of valproic acid and pioglitazone. We show that E-cadherin expression responds only to the combination treatment and not to single PPARgamma agonists, defining a new class of PPARgamma target genes. These results open up new therapeutic perspectives in the treatment of prostate cancer.     

Expression of slow myosin heavy chain during muscle regeneration is not always dependent on muscle innervation and calcineurin phosphatase activity

In the literature, there is an ambiguity as to the respective roles played by calcineurin phosphatase activity (CPA) and muscle innervation in the reestablishment of the slow-twitch muscle phenotype after muscle regeneration in different species. In this study, we wanted to determine the role of calcineurin and muscle innervation on the appearance and maintenance of the slow phenotype during mouse muscle regeneration. The pattern of myosin expression and CPA was analyzed in adult (n=15), regenerating (n=45) and denervated-regenerating (n=32) slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. Moreover, in a second group of denervated-regenerating mice (n=9), the animals were treated with a calcineurin inhibitor. Regeneration was induced by injection of cardiotoxin and in the denervated-regenerating group, denervation was carried out by cutting the sciatic nerve before the administration of cardiotoxin. In innervated-regenerating soleus muscle, CPA increased continuously after 10 days postinjury and by 21 days, there was a 3.5-fold increase in CPA compared with adult basal level, whereas in innervated-regenerating EDL muscle, CPA remained unchanged. Moreover, our results show that in denervated-regenerating muscles, the MyHC profile was identical in spite of the functional differences inherent in these muscles. In long-term denervated-regenerating muscles, a slow muscle phenotype was reexpressed both in the presence or absence of calcineurin inhibitor. Our results show that although in innervated-regenerating mouse muscle, the appearance of a slow phenotype is correlated with a peak of CPA, in denervated-regenerating muscles, a slow phenotype is triggered and maintained in a calcineurin- and nerve-independent manner.     

Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes

Site-directed mutagenesis followed by functional characterization is a widely used approach to obtain information on the structure-function relationship of proteins. Due to time and cost considerations, the number of amino acids studied is frequently reduced. To address the need for convenient parallel production of numerous point mutants of a protein, we developed an automated method to perform classical site-directed mutagenesis, protein purification, and characterization in a high-throughput manner. The process consists of a succession of six fully automated protocols that can be adapted to any automated liquid handling systems. Our procedure allows construction, validation, and characterization of hundreds of site-directed mutants of a given protein in just 4 days. The method is especially adapted to projects aiming at the study of unique or multiple mutants without the need to construct and screen large libraries of random mutants. The usefulness of the technique is illustrated by the construction and characterization of tens of single mutants of the penicillin-binding protein 2x (PBP2x) from Streptococcus pneumoniae. Moreover, seven mutations of PBP2x were obtained simultaneously in a single experiment with efficiency close to 90%.