Molecular and biochemical characterization of three GH62 α-l-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum

Penicillium funiculosum is an industrial fungus exploited for its capacity to secrete a wide array of glycosyl hydrolases (GHs) and glycosyl transferases (GTs). These enzymes are part of an enzymatic cocktail that is commercialized under the name RovabioExcel^®, which is used as feed additive in animal nutrition. The genome sequence of this filamentous fungus has revealed a remarkable richness in several accessory enzymes, and notably in α-l-arabinofuranosidases (α-l-AFases) that participate in the hydrolysis of arabinoxylans (AX) in corn/wheat fibers used in poultry feed. Here, we report on the molecular and biochemical characterization of three GH62 family α-l-AFases encoding genes in this filamentous fungus. Amino acids sequences showed strong similarities (>65%) between them, as well with GH62 enzymes from other filamentous fungi. Interestingly, one of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1) of 37 amino acids length, which was shown to help the protein to bind to microcrystalline cellulose. Also, this PfABF62c showed optimal pH and temperature of 2.8 and 50 °C, respectively, whereas optimal activity for PfABF62a and PfABF62b were measured at 40 °C and at pH ranging between 2.6 and 4.5. Arabinan and arabinoxylan, but no other sugars or polymers were found to augment the thermal transition of the three enzymes by 3–5 °C as measured by differential scanning fluorimetry. Finally, enzymatic hydrolysis fingerprints of heteroxylans allowed concluding that the mode of action of the GH62 enzymes from this fungal species was to remove arabinofuranosyl residues linked in position O-2 and O-3 of substituted xylose units in arabinoxylan chains.     

Nicotine reduces mortality of developing rats exposed to high-altitude hypoxia and partially suppresses the duration of cortical epileptic afterdischarges

Nicotine has been repeatedly reported as substance possessing neuroprotective properties. This study focused on the possible beneficial effects of nicotine against the high-altitude hypoxia (9000 m for one hour). 15 min prior to hypoxia exposition rats (12- and 35-day-old) were treated with nicotine. Next day electrodes have been implanted and the effects of nicotine and hypoxia (or both factors) on duration of afterdischarges (ADs) were tested. Administration of nicotine declined the hypoxia-induced mortality in 35-day-old animals. Nicotine pretreatment had no effect on ADs duration in 12-day-old pups, therefore brought about suppression of ADs in 35-day-old animals. Taken together, our data show that nicotine exhibits an anticonvulsant effect that is age-dependent. The mechanisms of nicotine neuroprotective properties include probably the influence of calcium homeostasis, increase synthesis of variety of growth factors, inhibition of the caspase cascades and antioxidant capability of nicotine.     

Effects of human cultural neuronal and mesenchymal stem cells on the rat learning and brain state after acute hypoxia

The aim of the study was to compare the effects of neurotransplantation of cultural neural stem cells (NSC) and mesenchymal stem cells (MSC) on the rat behaviour and brain state after acute hypoxia. It was shown that development of two-way avoidance defensive conditioning in a shuttle box improved in rats-recipients with NSC, but not MSC as compared to control. Both the transplants of NSC and transplants of MSC exert neuroprotective influence on the rat brain. NSC both in vitro (before transplantation) and in vivo (on day 27 after transplantation) gave rise to all neural cell types: stem/progenitor cells, precursors of neurons and glia, neurons and glial cells. MSC population in vitro and in vivo (on day 10 after transplantation) consisted of fibroblast-like cells which were eliminated by day 20 after transplantation and were surrounded by reactive glia. We suggest that effects of NSC may be connected with their good survival and potential to differentiate into neurons and with trophic influence on the brain of recipient, whereas MSC only have possible positive trophic effect at early stages after transplantation.     

Testosterone-mediated sex differences in the face shape during adolescence: subjective impressions and objective features

Sex identification of a face is essential for social cognition. Still, perceptual cues indicating the sex of a face, and mechanisms underlying their development, remain poorly understood. Previously, our group described objective age- and sex-related differences in faces of healthy male and female adolescents (12–18 years of age), as derived from magnetic resonance images (MRIs) of the adolescents' heads. In this study, we presented these adolescent faces to 60 female raters to determine which facial features most reliably predicted subjective sex identification. Identification accuracy correlated highly with specific MRI-derived facial features (e.g. broader forehead, chin, jaw, and nose). Facial features that most reliably cued male identity were associated with plasma levels of testosterone (above and beyond age). Perceptible sex differences in face shape are thus associated with specific facial features whose emergence may be, in part, driven by testosterone.     

Inhibition of malaria parasite blood stages by tyrocidines, membrane-active cyclic peptide antibiotics from Bacillus brevis

Tyrothricin, a complex mixture of antibiotic peptides from Bacillus brevis, was reported in 1944 to have antimalarial activity rivalling that of quinine in chickens infected with Plasmodium gallinaceum. We have isolated the major components of tyrothricin, cyclic decapeptides collectively known as the tyrocidines, and tested them against the human malaria parasite Plasmodium falciparum using standard in vitro assays. Although the tyrocidines differ from each other by conservative amino acid substitutions in only three positions, their observed 50% parasite inhibitory concentrations (IC(50)) spanned three orders of magnitude (0.58 to 360 nM). Activity correlated strictly with increased apparent hydrophobicity and reduced total side-chain surface area and the presence of ornithine and phenylalanine in key positions. In contrast, mammalian cell toxicity and haemolytic activities of the respective peptides were considerably less variable (2.6 to 28 microM). Gramicidin S, a structurally analogous antimicrobial peptide, was less active (IC(50)=1.3 microM) and selective than the tyrocidines. It exerted its parasite inhibition by rapid and selective lysis of infected erythrocytes as judged by fluorescence and light microscopy. The tyrocidines, however, did not cause an overt lysis of infected erythrocytes, but an inhibition of parasite development and life-cycle progression.     

Emotional Reactions of Mothers Facing Premature Births: Study of 100 Mother-Infant Dyads 32 Gestational Weeks

Objectives

This current study has been conducted to clarify the relationship between the mother''s post-traumatic reaction triggered by premature birth and the mother-infant interactions. In this article, the precocious maternal feelings are described.

Methods

A multicenter prospective study was performed in three French hospitals. 100 dyads with 100 very premature infants and their mothers were recruited. Mothers completed, at two different times self-questionnaires of depression/anxiety, trauma and social support. The quality of interactions in the dyads was evaluated.

Results

Thirty-nine percent of the mothers obtained a score at HADS suggesting a high risk of depression at the first visit and approximately one-third at visit two. Seventy-five percent of the mothers were at risk of suffering from an anxiety disorder at visit one and half remained so at visit two. A “depressed” score at visits one and two correlated with a hospitalization for a threatened premature labor. We noted a high risk of trauma for 35% of the mothers and high interactional synchrony was observed for approximately two-thirds of the dyads. The mothers'' psychological reactions such as depression and anxiety or postnatal depression correlate strongly with the presence of an initial trauma. At visit one and visit two, a high score of satisfaction concerning social support correlates negatively with presence of a trauma. A maternal risk of trauma is more frequent with a C-section delivery.

Conclusions

Mothers'' psychological reactions such as depression and anxiety correlate greatly with the presence of an initial trauma. The maternal traumatic reaction linked to premature birth does not correlate with the term at birth, but rather with the weight of the baby. Social support perceived by the mother is correlated with the absence of maternal trauma before returning home, and also seems to inhibit from depressive symptoms from the time of the infant''s premature birth.     

Discovery of Novel Inhibitors of a Disintegrin and Metalloprotease 17 (ADAM17) Using Glycosylated and Non-glycosylated Substrates

A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3–19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118–122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.