Lack of evidence for inhibitory processes in over-selectivity

Stimulus over-selectivity can be defined as control over behavior being exerted by one aspect of the environment at the expense of other equally salient aspects of the environment, and is a common problem for discrimination learning under conditions of cognitive strain, and in intellectual disorders. Non-clinical participants exposed to a concurrent task load were trained and tested on a two-component trial-and-error discrimination task to investigate whether inhibition plays a role in producing under-selectivity by using both summation and retardation tests. Experiment 1 found evidence for the over-selectivity effect, and replicated the finding that revaluation of a previously over-selected stimulus allows emergence of control by a previous under-selected stimulus, despite the latter stimulus receiving no direct conditioning. The under-selected cue was not found to gain any conditioned inhibitory status, as reflected by summation (Experiment 2), and retardation (Experiment 3), tests. The results extend the literature explaining over-selectivity as a post-acquisition failure.     

Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics

α-Synuclein (AS) is associated with both sporadic and familial forms of Parkinson disease (PD). In sporadic disease, wild-type AS fibrillates and accumulates as Lewy bodies within dopaminergic neurons of the substantia nigra. The accumulation of misfolded AS is associated with the death of these neurons, which underlies many of the clinical features of PD. In addition, a rare missense mutation in AS, A30P, is associated with highly penetrant, autosomal dominant PD, although the pathogenic mechanism is unclear. A30P AS fibrillates more slowly than the wild-type (WT) protein in vitro and has been reported to preferentially adopt a soluble, protofibrillar conformation. This has led to speculation that A30P forms aggregates that are distinct in structure compared with wild-type AS. Here, we perform a detailed comparison of the chemical shifts and secondary structures of these fibrillar species, based upon our recent characterization of full-length WT fibrils. We have assigned A30P AS fibril chemical shifts de novo and used them to determine its secondary structure empirically. Our results illustrate that although A30P forms fibrils more slowly than WT in vitro, the chemical shifts and secondary structure of the resultant fibrils are in high agreement, demonstrating a conserved β-sheet core.     

Fenretinide prevents lipid-induced insulin resistance by blocking ceramide biosynthesis

Fenretinide is a synthetic retinoid that is being tested in clinical trials for the treatment of breast cancer and insulin resistance, but its mechanism of action has been elusive. Recent in vitro data indicate that fenretinide inhibits dihydroceramide desaturase, an enzyme involved in the biosynthesis of lipotoxic ceramides that antagonize insulin action. Because of this finding, we assessed whether fenretinide could improve insulin sensitivity and glucose homeostasis in vitro and in vivo by controlling ceramide production. The effect of fenretinide on insulin action and the cellular lipidome was assessed in a number of lipid-challenged models including cultured myotubes and isolated muscles strips incubated with exogenous fatty acids and mice fed a high-fat diet. Insulin action was evaluated in the various models by measuring glucose uptake or disposal and the activation of Akt/PKB, a serine/threonine kinase that is obligate for insulin-stimulated anabolism. The effects of fenretinide on cellular lipid levels were assessed by LC-MS/MS. Fenretinide negated lipid-induced insulin resistance in each of the model systems assayed. Simultaneously, the drug depleted cells of ceramide, while promoting the accumulation of the precursor dihydroceramide, a substrate for the reaction catalyzed by Des1. These data suggest that fenretinide improves insulin sensitivity, at least in part, by inhibiting Des1 and suggest that therapeutics targeting this enzyme may be a viable therapeutic means for normalizing glucose homeostasis in the overweight and diabetic.     

Determination of sphingosine-1-phosphate lyase activity by gas chromatography coupled to electron impact mass spectrometry

Sphingosine-1-phosphate lyase (SGPL1) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldolic cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. In this article we report on an easy and sensitive procedure to determine SPL activity. The assays uses C17-sphinganine-1-phosphate as substrate and the aldehyde product, pentadecanal, is quantified as its pentafluorobenzyloxime derivative by GC/MS. Derivatization of pentadecanal is performed as a one-step reaction, and the oxime product is directly injected for GC/MS analysis without any further purification. Acquisition in selected ion monitoring mode allows very high sensitivity, with a limit of detection of 281fmol. The assay is linear with both protein concentration and incubation time up to 20μg and 40min, respectively. The K(m) value obtained (6μM) is similar to that for the natural substrate sphingosine-1-phosphate. Using this method, FTY720 and deoxypyridoxine phosphate inhibited SPL with similar potencies to those reported.     

A polyphasic approach for the characterization of endophytic Alternaria strains isolated from grapevines

A polyphasic approach was set up and applied to characterize 20 fungal endophytes belonging to the genus Alternaria, recovered from grapevine in different Italian regions.Morphological, microscopical, molecular and chemical investigations were performed and the obtained results were combined in a pooled cluster analysis. Following morphological analyses, all strains were grouped according to their three-dimensional sporulation pattern on PCA and to the colony characteristics on different substrates. After DNA extraction, all strains were analyzed by RAPD-PCR and the resulting profiles were subjected to cluster analysis. The metabolites extracted from the 20 Alternaria endophytes were analyzed by a HPLC and the resulting metabolite profiles were subjected to multivariate statistic analyses. In comparison with reference ‘small-spored’ Alternaria species, the 20 strains were segregated into two morphological groups: one belonging to the A. arborescens species-group and a second to the A. tenuissima species-group. RAPD analysis also showed that grapevine endophytes belonged to either the A. arborescens or the A. tenuissima species-group and that they were molecularly distinct from strains belonging to A. alternata. Chemotaxonomy gave the same grouping: the grapevine endophytic strains belong to A. arborescens or A. tenuissima species-groups producing known metabolites typical of these species-groups. Interestingly, the 20 grapevine endophytes were able to produce also a number of unknown metabolites, whose characterization could be useful for a more precise segregation of the two species-groups.The results show how complementary morphological, molecular and chemical data can clarify relationships among endophyte species-groups of low morphological divergence.     

Constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle MHC class I

Six major histocompatibility complex (MHC) classical class I genes have been identified in cattle, and up to three of these are expressed in variable combinations on different haplotypes. The origin and functional significance of this genetic complexity is unknown. However, an improved assembly of the cattle genome, an expanded database of full-length cDNA sequences and high-resolution frequency data concerning expressed class I genes in an economically important cattle breed combine to provide a new opportunity to study the significance of cattle MHC class I diversity. Analysis of these new data supports assignment of alleles to six discrete genes and further shows that all these classical genes share a common ancestor with a single non-classical gene, NC1. While haplotype structure is variable, with thirteen gene configurations identified, there are nevertheless clear constraints relating to both the number and combination of genes. Haplotypes expressing two classical genes are most frequently observed, and the classical class I gene 2 is almost invariably present. The frequency data support the dominance of gene 2, showing that close to 100?% of individuals carry at least one copy. This indicates a hierarchy in the functional importance of particular genes and haplotype structures. Haplotype frequency in cattle populations is therefore likely to impact on differential disease susceptibilities. This knowledge will be important for development of informed breeding strategies aimed at increasing the ability of cattle to survive in the face of future unpredictable pathogen exposure.     

A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting in primed plant defenses

For an efficient defense response against pathogens, plants must coordinate rapid genetic reprogramming to produce an incompatible interaction. Nitrate Trasnporter2 (NRT2) gene family members are sentinels of nitrate availability. In this study, we present an additional role for NRT2.1 linked to plant resistance against pathogens. This gene antagonizes the priming of plant defenses against the bacterial pathogen Pseudomonas syringae pv tomato DC3000 (Pst). The nrt2 mutant (which is deficient in two genes, NRT2.1 and NRT2.2) displays reduced susceptibility to this bacterium. We demonstrate that modifying environmental conditions that stimulate the derepression of the NRT2.1 gene influences resistance to Pst independently of the total level of endogenous nitrogen. Additionally, hormonal homeostasis seemed to be affected in nrt2, which displays priming of salicylic acid signaling and concomitant irregular functioning of the jasmonic acid and abscisic acid pathways upon infection. Effector-triggered susceptibility and hormonal perturbation by the bacterium seem to be altered in nrt2, probably due to reduced sensitivity to the bacterial phytotoxin coronatine. The main genetic and metabolic targets of coronatine in Arabidopsis (Arabidopsis thaliana) remain largely unstimulated in nrt2 mutants. In addition, a P. syringae strain defective in coronatine synthesis showed the same virulence toward nrt2 as the coronatine-producing strain. Taken together, the reduced susceptibility of nrt2 mutants seems to be a combination of priming of salicylic acid-dependent defenses and reduced sensitivity to the bacterial effector coronatine. These results suggest additional functions for NRT2.1 that may influence plant disease resistance by down-regulating biotic stress defense mechanisms and favoring abiotic stress responses.     

Synthesis and biological evaluation of colchicine C-ring analogues tethered with aliphatic linkers suitable for prodrug derivatisation

Colchicine was modified at the 10-OCH₃ position of the C-ring by reaction with heterocyclic amines or commercially available amines to afford a library of target colchicinoids in high yields (62–99%). Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in resistant A2780AD or HeLa cells with mutant Class III β-tubulin. Importantly, unlike colchicine, the analogues in this study are amenable for prodrug derivatisation and with potential for tumor-selective delivery.     

Fatty acid-binding protein 4 impairs the insulin-dependent nitric oxide pathway in vascular endothelial cells

ABSTRACT: BACKGROUND: Recent studies have shown that fatty acid-binding protein 4 (FABP4) plasma levels are associated with impaired endothelial function in type 2 diabetes (T2D). In this work, we analysed the effect of FABP4 on the insulin-mediated nitric oxide (NO) production by endothelial cells in vitro. METHODS: In human umbilical vascular endothelial cells (HUVECs), we measured the effects of FABP4 on the insulin-mediated endothelial nitric oxide synthase (eNOS) expression and activation and on NO production. We also explored the impact of exogenous FABP4 on the insulin-signalling pathway (insulin receptor substrate 1 (IRS1) and Akt). RESULTS: We found that eNOS expression and activation and NO production are significantly inhibited by exogenous FABP4 in HUVECs. FABP4 induced an alteration of the insulin-mediated eNOS pathway by inhibiting IRS1 and Akt activation. These results suggest that FABP4 induces endothelial dysfunction by inhibiting the activation of the insulin-signalling pathway resulting in decreased eNOS activation and NO production. CONCLUSION: These findings provide a mechanistic linkage between FABP4 and impaired endothelial function in diabetes, which leads to an increased cardiovascular risk.     

Impact of natalizumab on cognitive performances and fatigue in relapsing multiple sclerosis: a prospective, open-label, two years observational study

Background and Objectives

Natalizumab reduces the relapse rate and magnetic resonance imaging activity in patients with Relapsing-Remitting Multiple Sclerosis (RRMS). So far the influence of natalizumab on cognitive functions and fatigue in MS remains uncertain. The aim of this prospective, open-label, observational study was to evaluate the possible effects of natalizumab on cognition and fatigue measures in RRMS patients treated for up to two years.

Methods

Cognitive performances were examined by the Rao''s Brief Repeatable Battery (BRB), the Stroop test (ST) and the Cognitive Impairment Index (CII), every 12 months. Patients who failed in at least 3 tests of the BRB and the ST were classified as cognitively impaired (CI). Fatigue Severity Scale (FSS) was administered every 12 months to assess patient''s self-reported fatigue. One hundred and 53 patients completed 1 and 2 year-natalizumab treatment, respectively.

Results

After 1 year of treatment the percentage of CI patients decreased from 29% (29/100) at baseline to 19% (19/100) (p = 0.031) and the mean baseline values of CII (13.52±6.85) and FSS (4.01±1.63) scores were significantly reduced (10.48±7.12, p<0.0001 and 3.61±1.56, p = 0.008). These significant effects were confirmed in the subgroup of patients treated up to two years.

Conclusions

These results demonstrate that a short-term NTZ treatment may significantly improve cognitive performances and fatigue in RRMS patients.