Structural basis for substrate specificity in group I nucleoside hydrolases

Enzymes with nucleoside hydrolase activity (NHs) belonging to homology group I either are markedly specific for pyrimidine nucleoside substrates or hydrolyze with comparable efficiencies the N-glycosidic bond in all common nucleosides. The biochemical and structural basis for these differences in substrate specificity is still unknown. Here we characterize the binding interactions between the slowly hydrolyzed substrate inosine and the Escherichia coli pyrimidine-specific NH YeiK using cryotrapping and X-ray crystallography. Guided by the structural features of the Michaelis complex, we show the synergic effect of two specific point mutations in YeiK that increase the catalytic efficiency toward purine nucleosides to values comparable to those of natural nonspecific NHs. We demonstrate that the integrity of an active-site catalytic triad comprised of two hydroxylated amino acids and one histidine residue is a requirement for the highly efficient hydrolysis of inosine by group I NHs. Instead, cleavage of the YeiK-preferred substrate uridine is not affected by mutations at the same locations, suggesting a different fine chemical mechanism for the hydrolysis of the two nucleoside substrates. Our study provides for the first time direct evidence that distinct subsets of amino acid residues are involved in the hydrolysis of purine or pyrimidine nucleosides in group I NHs.     

Relationships among site characteristics,taxonomical structure and functional trait distribution of arthropods in forest,urban and agricultural soils of Southern Italy

Urbanization and agriculture cause alterations in soil biodiversity. As arthropods quickly respond to soil changes, they have been recognized as good indicators of soil quality. This study aimed to evaluate if (1) soil physico-chemical properties act as environmental filters of the functional trait distribution of soil arthropod community; (2) the abundance of certain functional traits is linked to different land uses; (3) the taxonomical and functional approaches provide complementary information about the evaluation of biological soil quality. The study was performed at forest, agricultural and urban sites in downtown and surroundings of Naples. The soils were characterized for texture, pH, organic matter content, water content, total Cd, Cr, Cu, Ni, Pb and Zn concentrations. The forest soils showed the highest organic matter and water contents, and the lowest metal concentrations; the agricultural soils showed the lowest organic matter and water contents and high Cd and Cu concentrations, whereas the urban soils showed high Pb and Zn concentrations. The forest soils showed the highest arthropod density, taxa richness and diversity. The distribution of the functional traits in the arthropod communities of the different land uses highlighted similarity only for the preference to occupy soil and for the percentage contribution of detritivores. The abundance of arthropods living above litter and omnivores were positively correlated to Pb and Zn concentrations, whereas predators were positively correlated to Cu concentrations. The findings suggest that the arthropod identification at order level could be enough to detect differences in functional traits among forest, urban, and agricultural soils.     

Exploitation of underground bee nests by three sympatric consumers in Loango National Park,Gabon

Honey represents a highly nutritious resource for animals, but is difficult to obtain given bees' defensive strategies. We investigated exploitation of the underground nests of stingless bees (Meliplebeia lendliana) by three sympatric consumers in Loango National Park, Gabon: the central African chimpanzee (Pan troglodytes troglodytes), forest elephant (Loxodonta cyclotis) and honey badger (Mellivora capensis). Given the differences in their respective morphological traits and sensory abilities, we hypothesized that chimpanzees would be more limited in digging out the bee nests, compared to the other two competitors, and would show behavioral strategies to overcome such constraints. Our dataset comprised camera trap footage recorded over 60 mo at 100 different bee nests. Chimpanzees visited the nests more often than the other consumers, showing a frequency of extraction success comparable to that observed in honey badgers, the most efficient digger. Both chimpanzees and honey badgers increased their extractive attempts across the dry season, whereas elephants did not. The soil hardness was greater during the dry season than the wet season and, possibly in order to compensate for this, chimpanzees showed a tendency toward digging at nests found in relatively softer soil. They also seemed to be inhibited by indirect cues left by other consumers, possibly as a risk‐avoidance strategy. Overall, chimpanzees and honey badgers extracted the underground nests of stingless bees with similar frequencies, whilst forest elephants did so only occasionally. Moreover, chimpanzees can use tools and other behavioral strategies to overcome the physical limitations that may constrain their exploitation of this resource.     

Effect of Harvest Time and Frequency on Biomass Quality and Biomethane Potential of Common Reed (<Emphasis Type="Italic">Phragmites australis</Emphasis>) Under Paludiculture Conditions

This study examined the effect of harvest time (from May to September) and dry matter partitioning on biomethane potential and methane yield per unit area of Phragmites australis cultivation under paludiculture conditions. The experimental site is part of a larger experimental platform (San Niccolò, Pisa) located within the Massaciuccoli Lake Basin in Central Italy (Tuscany, IT). The study also took into account the double cut strategy by evaluating the regrowth from June to September. Biomethane potentials ranged from 384 to 315 and from 412 to 283 NL CH₄ kg VS^?1 (normal liters of methane per kg of volatile solids) for leaves and stems, respectively. About digestion kinetics, maximum daily production rate (R _max) was significantly affected by harvest time and not by plant partitioning. Along the harvest season, biomethane yield per unit area was mostly driven by the biomass yield showing an increasing trend from May (1659 Nm³ ha^?1) to September (3817 Nm³ ha^?1). The highest value was obtained with the double harvest option (4383 Nm³ ha^?1), although it was not statistically different from the single harvest carried out in September. Owing to its remarkably lower yields, P. australis cannot be considered along the same lines as crops conventionally used for biogas production, but it may represent an interesting option for paludiculture cropping systems by coupling peatland restoration with bioenergy production. September harvest management seemed the most feasible option, although further investigation on crop lifespan is needed.     

Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA)

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused in the majority of cases by deletions of the DMD gene and are readily detectable in affected males by multiplex polymerase chain reaction (PCR). However, different approaches must be used for the identification of female carriers, in which deletions are not detectable by PCR, because of the presence of a normal X chromosome. In this study, we used the multiple ligation probe amplification (MLPA) tool for the identification of female carriers of DMD deletions or duplications in 12 families with a single affected male, 10 of which were previously diagnosed as carriers of a DMD rearrangement, and the remaining two as having an unknown disease-causing mutation. In all the investigated affected males, MLPA analysis confirmed the presence of a DMD rearrangement, and in six of them allowed the refinement of the breakpoints. In 12 female relatives of the affected patients, MLPA analysis showed a DMD deletion or duplication, confirming their carrier status. Two of these were the mother and the sister of a patient whose disease-causing mutation was not known. MLPA analysis thus proved to be an useful tool for the analysis of both affected males and females carriers of DMD rearrangements in cases in which the disease-causing mutation in the affected male was not known, providing useful information for the genetic counselling of the family.Valentina Gatta and Oronzo Scarciolla contributed equally to this work.     

2-aryl-8-chloro-1,2,4-triazolo[1,5-a]quinoxalin-4-amines as highly potent A1 and A3 adenosine receptor antagonists

Some 2-aryl-8-chloro-1,2,4-triazolo[1,5-a]quinoxaline derivatives 2-18, obtained by introducing different substituents on either the 4-amino moiety (acyl or carbamoyl groups) or the 2-phenyl ring (4-OCH3) of previously reported 8-chloro-2-phenyl-1,2,4-triazolo[1,5-a]quinoxalin-4-amine (1), have been synthesized and tested in radioligand binding assays at bovine A1 and A(2A) and at cloned human A1 and A3 adenosine receptors. The rationally designed 8-chloro-2-(4-methoxy-phenyl)-1,2,4-triazolo[1,5-a]quinoxalin-4-acetylamine (14) can be considered one of the most potent and hA3 versus hA1 selective AR antagonists reported till now. The structure-activity relationships of compounds 2-18 are in agreement with those of previously reported 2-aryl-1,2,4-triazolo[4,3-a]quinoxalines (series A) and 2-arylpyrazolo[3,4-c]quinolines (series B), thus suggesting a similar AR binding mode. In fact, the importance for the A3 receptor-ligand interaction of both a strong acidic NH proton donor and a C=O proton acceptor at position-4, able to engage hydrogen-bonding interactions with specific sites on the A3 AR, has been confirmed. Using our recently published hA3 receptor model, to better elucidate our experimental results, we decided to theoretically depict the putative TM binding motif of the herein reported 1,2,4-triazolo[1,5-a]quinoxaline derivatives on human A3 receptor. Structure-activity relationships have been explained analyzing the three-dimensional structure of the antagonist-receptor models obtained by molecular docking simulation.