High production of cold-tolerant chitinases on shrimp wastes in bench-top bioreactor by the Antarctic fungus Lecanicillium muscarium CCFEE 5003: Bioprocess optimization and characterization of two main enzymes

The Antarctic fungus Lecanicillium muscarium CCFEE-5003 was preliminary cultivated in shaken flasks to check its chitinase production on rough shrimp and crab wastes. Production on shrimp shells was much higher than that on crab shells (104.6 ± 9.3 and 48.6 ± 3.1 U/L, respectively). For possible industrial applications, bioprocess optimization was studied on shrimp shells in bioreactor using RSM to state best conditions of pH and substrate concentration. Optimization improved the production by 137% (243.6 ± 17.3). Two chitinolytic enzymes (CHI1 and CHI2) were purified and characterized. CHI1 (MW ca. 61 kDa) showed optima at pH 5.5 and 45 °C while CHI2 (MW ca. 25 kDa) optima were at pH 4.5 and 40 °C. Both enzymes maintained high activity levels at 5 °C and were inhibited by Fe⁺⁺, Hg⁺⁺ and Cu⁺⁺. CHI2 was strongly allosamidin-sensitive. Both proteins were N-acetyl-hexosaminidases (E.C. 3.2.1.52) but showed different roles in chitin hydrolysis: CHI1 could be defined as “chitobiase” while CHI2 revealed a main “eso-chitinase” activity.     

Modulation of Lipid Kinase PI4KIIα Activity and Lipid Raft Association of Presenilin 1 Underlies γ-Secretase Inhibition by Ginsenoside (20S)-Rg3

Amyloid β-peptide (Aβ) pathology is an invariant feature of Alzheimer disease, preceding any detectable clinical symptoms by more than a decade. To this end, we seek to identify agents that can reduce Aβ levels in the brain via novel mechanisms. We found that (20S)-Rg3, a triterpene natural compound known as ginsenoside, reduced Aβ levels in cultured primary neurons and in the brains of a mouse model of Alzheimer disease. The (20S)-Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts where catalytic components of the γ-secretase complex are enriched. The Aβ-lowering activity of (20S)-Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIα (PI4KIIα), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol 4,5-bisphosphate synthesis. PI4KIIα overexpression recapitulated the effects of (20S)-Rg3, whereas reduced expression of PI4KIIα abolished the Aβ-reducing activity of (20S)-Rg3 in neurons. Our results substantiate an important role for PI4KIIα and phosphoinositide modulation in γ-secretase activity and Aβ biogenesis.     

Mechanisms of Nitrosylation and Denitrosylation of Cytoplasmic Glyceraldehyde-3-phosphate Dehydrogenase from Arabidopsis thaliana

Nitrosylation is a reversible post-translational modification of protein cysteines playing a major role in cellular regulation and signaling in many organisms, including plants where it has been implicated in the regulation of immunity and cell death. The extent of nitrosylation of a given cysteine residue is governed by the equilibrium between nitrosylation and denitrosylation reactions. The mechanisms of these reactions remain poorly studied in plants. In this study, we have employed glycolytic GAPDH from Arabidopsis thaliana as a tool to investigate the molecular mechanisms of nitrosylation and denitrosylation using a combination of approaches, including activity assays, the biotin switch technique, site-directed mutagenesis, and mass spectrometry. Arabidopsis GAPDH activity was reversibly inhibited by nitrosylation of catalytic Cys-149 mediated either chemically with a strong NO donor or by trans-nitrosylation with GSNO. GSNO was found to trigger both GAPDH nitrosylation and glutathionylation, although nitrosylation was widely prominent. Arabidopsis GAPDH was found to be denitrosylated by GSH but not by plant cytoplasmic thioredoxins. GSH fully converted nitrosylated GAPDH to the reduced, active enzyme, without forming any glutathionylated GAPDH. Thus, we found that nitrosylation of GAPDH is not a step toward formation of the more stable glutathionylated enzyme. GSH-dependent denitrosylation of GAPC1 was found to be linked to the [GSH]/[GSNO] ratio and to be independent of the [GSH]/[GSSG] ratio. The possible importance of these biochemical properties for the regulation of Arabidopsis GAPDH functions in vivo is discussed.     

Efficiency of flagging and dragging for tick collection

It is acknowledged that data from field studies on tick ecology might be biased by collection methods, but actually comparative studies are still limited. Herein we assessed whether the efficiency of flagging and dragging varies according to tick developmental stage, species, season and habitat. Ticks were collected in three sites bordered by an oak forest. The abundance of ticks collected by each collection method varied according to tick species, developmental stage, season, and habitat. Flagging was in general more efficient in collecting adult ticks, especially in spring and winter. Females were more frequently collected by flagging in the meadow and grassland habitats and males in the man-made trail. Flagging collected significantly more adults of Dermacentor marginatus, Hyalomma marginatum, Haemaphysalis inermis and Ixodes ricinus. Flagging was more efficient in collecting D. marginatus and I. ricinus in spring, and H. inermis and I. ricinus females in both spring and winter. In summer and autumn tick abundances were generally similar, with the exception of D. marginatus female in autumn. Flagging was more efficient in collecting D. marginatus adults in the meadow habitat and in the man-made trail, and I. ricinus adults in the meadow and grassland habitats. Dragging was more efficient in grassland for R. turanicus. Our results suggest that variations in terms of collection method performance are associated to factors linked to tick behaviour, habitat characteristics, and climate. Field studies employing these collection methods should take this into account to avoid misleading conclusions about tick population dynamics and tick-borne pathogen transmission risk.     

A new small mammal assemblage from the M013 Terre Rosse fissure filling (Gargano,South-Eastern Italy)

The taxonomic study of the small mammal assemblage from fissure M013, sampled by a team of the University of Torino during the 2005–2009 excavations in the Dell’Erba Quarry (Apricena, Foggia) is presented. The assemblage includes the Echinosoricinae Apulogalerix cf. pusillus and the Crocidosoricinae ?Lartetium cf. dehmi; the Myomiminae species Stertomys simplex and Stertomys lyrifer; a new genus and species of Cricetodontinae and a single specimen of Hattomys cf. nazarii. Muridae include Mikrotia cf. parva, Mikrotia sp. 1 and a new genus and species of Murinae, phylogenetically related to Mikrotia. The occurrence of the new Murinae, the new Cricetodontinae, the two species of glirids and the Crocidosoricinae, as well as the absence of Apodemus and Prolagus, indicates M013 as the oldest Gargano's faunal assemblage known to date, despite the occurrence of Hattomys cf. nazarii, Mikrotia cf. parva and Mikrotia sp. 1, which most probably results from infiltrations from younger fissure fillings. The M013 assemblage is an absolute novelty for the Abruzzo-Apulian Palaeobioprovince, opening new perspectives for the timing and mode of dispersal of the forerunners of the Gargano fauna.     

Discovery,synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.