Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

The DRD2 TaqIA polymorphism associated with changed midbrain volumes in healthy individuals

The human DRD2 gene is located on chromosome 11q22-q23 and contains one specific functional polymorphism called TaqIA, which characteristically presents two alleles referred to as A1 and A2. Evidence indicates that the A1 allele impacts brain dopaminergic function and may confer an increased risk of developing Parkinson's disease. However, possible morphological changes underlying such genetic variant remain to be clarified. The aim of this study was to provide an in vivo demonstration of changes in brain structures associated with the TaqIA polymorphism of the DRD2 gene. Optimized voxel-based morphometry (VBM) was applied to high-resolution MR brain images of 70 healthy controls divided into two groups according to their DRD2 genotype (A1/A2, n = 15; A2/A2, n = 55). Compared with individuals' homozygous for the A2 allele, the A1 carriers had significantly smaller areas of a specific part of the midbrain, encompassing the substantia nigra bilaterally. Our findings showed an association of the DRD2 TaqIA polymorphism with the changed volumes of a specific subcortical region strongly involved in the dopaminergic system.     

Compaction properties of an intrinsically disordered protein: Sic1 and its kinase-inhibitor domain

IDPs in their unbound state can transiently acquire secondary and tertiary structure. Describing such intrinsic structure is important to understand the transition between free and bound state, leading to supramolecular complexes with physiological interactors. IDP structure is highly dynamic and, therefore, difficult to study by conventional techniques. This work focuses on conformational analysis of the KID fragment of the Sic1 protein, an IDP with a key regulatory role in the cell-cycle of Saccharomyces cerevisiae. FT-IR spectroscopy, ESI-MS, and IM measurements are used to capture dynamic and short-lived conformational states, probing both secondary and tertiary protein structure. The results indicate that the isolated Sic1 KID retains dynamic helical structure and populates collapsed states of different compactness. A metastable, highly compact species is detected. Comparison between the fragment and the full-length protein suggests that chain length is crucial to the stabilization of compact states of this IDP. The two proteins are compared by a length-independent compaction index.     

Probing the location of the substrate binding site of ascorbate oxidase near type 1 copper: an investigation through spectroscopic, inhibition and docking studies

The present investigation addresses the problem of the binding mode of phenolic inhibitors and the substrate ascorbate to the active site of ascorbate oxidase. The results from both types of compounds indicate that the binding site is located in a pocket near the type 1 copper center. This information is of general interest for blue multicopper oxidases. Docking calculations performed on the ascorbate oxidase-ascorbate complex show that binding of the substrate occurs in a pocket near type 1 Cu, and is stabilized by at least five hydrogen bonding interactions with protein residues, one of which involves the His512 Cu ligand. Similar docking studies show that the isomeric fluorophenols, which act as competitive inhibitors toward ascorbate, bind to the enzyme in a manner similar to ascorbate. The docking calculations are supported by 19F NMR relaxation measurements performed on fluorophenols in the presence of the enzyme, which show that the bound inhibitors undergo enhanced relaxation by the paramagnetic effect of a nearby Cu center. Unambiguous support to the location of the inhibitor close to type 1 Cu was obtained by comparative relaxation measurements of the fluorophenols in the presence of the ascorbate oxidase derivative where a Zn atom selectively replaces the paramagnetic type 2 Cu. The latter experiments show that contribution to relaxation of the bound inhibitors by the type 2 Cu site is negligible.     

Increasing relevance of spring temperatures for Norway spruce trees in Davos,Switzerland, after the 1950s

Key message

Relevance of spring temperatures for tree-ring growth steadily increased since 1950s. Closely linked tree-ring growth and net CO ₂ exchange driven by spring temperatures.

Abstract

We investigated long-term (over 100 years) tree-ring width (TRW) variabilities as well as short-term (10 years) variations in net ecosystem productivity (NEP) in response to climate to assess the driving factors for stem growth of Norway spruce in a subalpine forest at Davos in Switzerland. A tree-ring width index (TRWi) chronology for the period from 1750 to 2006 was constructed and linked with climate data from 1876 to 2006, and with NEP available for the period from 1997 to 2006. Based on TRWi, we found that only two out of the 257 years exhibited extreme negative TRWi, compared to 29 years with extreme positive anomalies, observed mainly in recent decades. Annual temperature, annual precipitation, as well as autumn and winter temperature signals were well preserved in the TRWi chronology over the last 130 years. Spring temperatures became increasingly relevant for TRWi, explaining less than 1 % of the variation in TRWi for the period from 1876 to 2006, but 8 % for the period from 1950 to 2006 (p = 0.032), and even 47 % for 1997–2006 (p = 0.028). We also observed a strong positive relationship between annual TRWi and annual NEP (r = 0.661; p = 0.037), both strongly related to spring temperatures (r = 0.687 and r = 0.678 for TRWi and NEP, respectively; p = 0.028; p = 0.032). Moreover, we found strong links between monthly NEP of March and annual TRWi (r = 0.912; p = 0.0001), both related to March temperatures (r = 0.767, p = 0.010 and r = 0.724, p = 0.018, respectively). Thus, under future climate warming, we expect stem growth of these subalpine trees and also ecosystem carbon (C) sequestration to increase, as long as water does not become a limiting factor.     

Ecological Niche Modelling of Potential West Nile Virus Vector Mosquito Species and Their Geographical Association with Equine Epizootics in Italy

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.     

Development of single nucleotide polymorphisms in Phaseolus vulgaris and related Phaseolus spp

In this study, new single nucleotide polymorphism (SNP) markers were developed for common bean (Phaseolus vulgaris L.) and related Phaseolus species. The applied strategy presents new and interesting aspects, such as the choice of accessions used, which was aimed at capturing a large portion of the genetic diversity present in the common bean, with particular focus on wild and domesticated materials from Mesoamerica and the identification of loci for sequencing. Indeed, the primer pairs for 34 loci were designed with the main strategy being to search for single-copy orthologous genes among the legumes (for use in other legume species and comparative analyses). The 10 remaining loci were selected as being near to domestication quantitative trait loci or detected as putatively under selection during domestication in previous studies. To provide an efficient and inexpensive genotyping platform for geneticists and breeders, we used sequence data to develop 60 new SNP markers for KASPar assay genotyping. The same sample was also genotyped with SNP markers developed for common bean in other studies for the same assay. This allowed testing for systematic bias according to the criteria chosen to select the genotypes in which the genetic diversity is surveyed during SNP discovery. Finally, we show that most of the SNP markers worked well in a set of accessions of other species belonging to the Phaseolus genus. The genetic resources developed will be very useful not only for breeding, but also for biodiversity conservation management and evolutionary studies on legumes.     

A Bioartificial Renal Tubule Device Embedding Human Renal Stem/Progenitor Cells

We present a bio-inspired renal microdevice that resembles the in vivo structure of a kidney proximal tubule. For the first time, a population of tubular adult renal stem/progenitor cells (ARPCs) was embedded into a microsystem to create a bioengineered renal tubule. These cells have both multipotent differentiation abilities and an extraordinary capacity for injured renal cell regeneration. Therefore, ARPCs may be considered a promising tool for promoting regenerative processes in the kidney to treat acute and chronic renal injury. Here ARPCs were grown to confluence and exposed to a laminar fluid shear stress into the chip, in order to induce a functional cell polarization. Exposing ARPCs to fluid shear stress in the chip led the aquaporin-2 transporter to localize at their apical region and the Na⁺K⁺ATPase pump at their basolateral portion, in contrast to statically cultured ARPCs. A recovery of urea and creatinine of (20±5)% and (13±5)%, respectively, was obtained by the device. The microengineered biochip here-proposed might be an innovative “lab-on-a-chip” platform to investigate in vitro ARPCs behaviour or to test drugs for therapeutic and toxicological responses.     

Biodiversity assessment: a case study in predicting richness from the potential distributions of plant species in the forests of south-western Australia

Distributions were predicted for 1430 native plant species as part of a biodiversity assessment in the forests of south‐west Western Australia. From these predicted distributions, an index of plant species richness was generated for the forest area. The most common predictors for distribution were found to be climatic surfaces incorporating some aspect of seasonality in temperature and precipitation. Although coarse, the index confirmed existing knowledge of areas of high biodiversity within the study area and introduced a new area, the Blackwood Plateau, for consideration as a conservation reserve with high species richness. An additional survey was conducted to sample actual species richness in 11 test sites. When actual richness was regressed against predicted richness, a significant correlation was obtained if both annuals and geophytes were excluded from the analysis. With refinement of the model and further data collection targeted at areas of low effort, the species richness index is proposed as a useful tool for conservation planning.     

Poly(1,2,3-benzotriazolyl)borate complexes with copper(I) and tri-organophosphane: an unprecedented κ-coordination of [H2B(btz)2] (btz=1,2,3-benzotriazolyl) in the X-ray crystal structure of [Cu(PBn3)2{(btz)BH2(btz)}]

New copper(I) triorganophosphane derivatives [Cu(PR₃)_n{H₂B(btz)₂}] and [Cu(PR₃)_n{HB(btz)₃}] (n=1 or 2) have been synthesized from the reaction of CuCl with PR₃ (R=phenyl, cyclohexyl, benzyl, o-, m-, or p-tolyl) or PMePh₂ and potassium dihydrobis(1,2,3-benzotriazolyl)borate K[H₂B(btz)₂] or potassium hydrotris(1,2,3-benzotriazolyl)borate K[HB(btz)₃]. The complexes obtained have been characterized by elemental analyses and FT-IR in the solid state and by NMR (¹H and ³¹P{¹H}) spectroscopy and conductivity measurements in solution. Solution data are consistent with partial dissociation of complexes occurring throughout breaking of the CuP bond. Single crystal structural characterizations were undertaken for two of them. The structurally authenticated arrays are, (a) [Cu(PBn₃)₂{(btz)BH₂(btz)}] with a three coordinate P₂Cu(N) coordination sphere and the donor [H₂B(btz)₂] coordinated throughout only one N3 atom. (b) [Cu(P-m-tolyl₃)_n{(btz)₃BH}] with a four coordinate PCuN₃ coordination sphere with the tris(benzotriazolyl)borate acting as tripodal donor throughout all its N2 atoms.