Binding of aflatoxins to the 20S proteasome: effects on enzyme functionality and implications for oxidative stress and apoptosis

Aflatoxins (AF) are contaminants of improperly stored foods; they are potent genotoxic and carcinogenic compounds, exerting their effects through damage to DNA. They can also induce mutations that increase oxidative damage. The goal of this study was to evaluate the possibility that a third mechanism could be involved in the carcinogenic action of aflatoxins, namely, direct binding to key enzymes involved in the regulatory pathways of the cell cycle, thereby modulating enzyme functionality. The 20S constitutive and immunoproteasome peptidase and proteolytic activities were assayed in the presence of aflatoxins B1, G1 and M1. All three toxins activated multiple peptidase activities of the proteasome. Aflatoxin (AF) M1 was the most potent activator of proteasome activity, while the constitutive 20S proteasome was specifically stimulated by AFG1. Furthermore, the effects of AFB1 on cultured hepatoma cells were investigated and the various proteasomal activities determined with cell lysates were differently affected. Taking into account the key role of the proteasome in cellular defense against oxidative stress, the carbonyl group content and the activities of antioxidant enzymes in cell lysates were analyzed. The proapoptotic effect of AFB1 was also investigated by measuring caspase-3 activity and cellular levels of p27 and IkappaBalpha.     

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (d_XY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.     

Polygyny in the tree swallow Tachycineta bicolor: a result of the cost of searching for an unmated male

The costs imposed by parental care duties on an individual's future survival and reproduction generate conflicts because parents should attempt to minimize their investment in the present brood, and exploit the parental care of the other parent. This conflict is likely to contribute to cases of both polygamy and desertion. Here, we study the costs of polygyny in the tree swallow Tachycineta bicolor , using observations on 52 nests that were attended by polygynous males over 14 y. Both females mated to polygynous males paid reproductive costs at several stages of the nesting cycle. Clutches laid by social mates of polygynous versus monogamous males did not differ in size. However, initial brood sizes for polygynously mated females were lower because a higher proportion of their eggs failed to hatch. Likewise, fledging success was lower and nest predation rates were higher, perhaps reflecting the direct or indirect effects of reduced male attention. These results demonstrate that females pay a productivity cost when breeding with reduced male parental care. In contrast, polygynous males fledge on average more young than monogamous ones and clearly benefit from the association. We suggest that a mate-search cost is leading to the few cases of polygamous males: although females are likely evaluating males for their prospective dedication to the breeding attempt, in a short-lived bird with a short breeding season, the cost to females of searching for a more dedicated male is the risk of not breeding at all.     

Micronuclei, genetic polymorphisms and cardiovascular disease mortality in a nested case-control study in Italy

AIM: To validate the predictive value of micronuclei (MN) in peripheral blood lymphocytes (PBL) and glutathione-S-transferases (GSTs) polymorphisms (GSTM1 and GSTT1) for mortality risk (MR) of cardiovascular diseases (CVD). METHODS: Blood samples from 1650 healthy subjects selected from the general population were collected between June 1991 and November 1993, and slides were immediately prepared for MN assessment. The vital status, or the cause of death, was monitored for all subjects until January 2005. At the end of the follow-up, 111 deaths were recorded and 39 CVD cases were observed (age range=42-88 years). Two thousand binucleated (BN) cells/subject were scored for the MN assay and GSTs genotypes were assessed on the DNA extracted from the blood or serum samples. RESULTS: A significantly higher MN frequency was recorded for the case group in comparison with the control group (n=67, Kruskall-Wallis test, p=0.006) and GSTT1 null genotype was significantly less frequent in CVD patients (chi(2)-test, p=0.036). The influence of other factors were evaluated using a unconditional logistic regression that confirmed a significant association of GSTT1 positive genotype with an increased OR for CVD (OR=6.29, 95% CI 1.32-29.95) beside a significant effect of age (OR=1.13, 95% CI 1.03-1.26 year(-1)). Finally, subjects with an higher MN frequency showed a higher MR for CVD (Log-rank test, p=0.001). CONCLUSIONS: MN confirmed to be a suitable cytogenetic biomarker for early prediction of CVD death. The GSTT1 positive genotype is associated with an increased MR for CVD.     

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome,a disease with low mutational burden

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34⁺) and normal (CD3⁺) cells isolated from the patients’ blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4⁺ response and 11 (34%) induced a CD8⁺ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.     

Racemic crystallography of synthetic protein enantiomers used to determine the X‐ray structure of plectasin by direct methods

We describe the use of racemic crystallography to determine the X‐ray structure of the natural product plectasin, a potent antimicrobial protein recently isolated from fungus. The protein enantiomers L ‐plectasin and D ‐plectasin were prepared by total chemical synthesis; interestingly, L ‐plectasin showed the expected antimicrobial activity, while D ‐plectasin was devoid of such activity. The mirror image proteins were then used for racemic crystallization. Synchrotron X‐ray diffraction data were collected to atomic resolution from a racemic plectasin crystal; the racemate crystallized in the achiral centrosymmetric space group P1 with one L ‐plectasin molecule and one D ‐plectasin molecule forming the unit cell. Dimer‐like intermolecular interactions between the protein enantiomers were observed, which may account for the observed extremely low solvent content (13%–15%) and more highly ordered nature of the racemic crystals. The structure of the plectasin molecule was well defined for all 40 amino acids and was generally similar to the previously determined NMR structure, suggesting minimal impact of the crystal packing on the plectasin conformation.     

T cell activation induces CuZn superoxide dismutase (SOD)-1 intracellular re-localization,production and secretion

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H₂O₂), more stable and able to freely diffuse through cell membranes. Copper–zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H₂O₂ and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H₂O₂ generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non-T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors.     

Excitability of the Motor Cortex in De Novo Patients with Celiac Disease

Introduction

Celiac disease (CD) may initially present as a neurological disorder or may be complicated by neurological changes. To date, neurophysiological studies aiming to an objective evaluation of the potential central nervous system involvement in CD are lacking.

Objective

To assess the profile of cortical excitability to Transcranial Magnetic Stimulation (TMS) in a group of de novo CD patients.

Materials and methods

Twenty CD patients underwent a screening for cognitive and neuropsychiatric symptoms by means of the Mini Mental State Examination and the Structured Clinical Interview for DSM-IV Axis I Disorders, respectively. Instrumental exams, including electroencephalography and brain computed tomography, were also performed. Cortico-spinal excitability was assessed by means of single and paired-pulse TMS using the first dorsal interosseus muscle of the dominant hand. TMS measures consisted of resting motor threshold, motor evoked potentials, cortical silent period (CSP), intracortical inhibition (ICI) and facilitation (ICF). None of the CD was on gluten-free diet. A group of 20 age-matched healthy controls was used for comparisons.

Results

CD showed a significantly shorter CSP (78.0 vs 125.0 ms, p<0.025), a reduced ICI (0.3 vs 0.2, p<0.045) and an enhanced ICF (1.1 vs 0.7, p<0.042) compared to controls. A dysthymic disorder was identified in five patients. The effect size between dysthymic and non-dysthymic CD patients indicated a low probability of interference with the CSP (Cohen''s d -0.414), ICI (-0.278) and ICF (-0.292) measurements.

Conclusion

A pattern of cortical excitability characterized by “disinhibition” and “hyperfacilitation” was found in CD patients. Immune system dysregulation might play a central role in triggering changes of the motor cortex excitability.     

Epoxidation of fatty acids with membrane-supported peroxygenase

Peroxygenase is an enzyme that can convert a double bond to an epoxide. Peroxygenase activity from oat (Avena sativa) seeds was immobilized on synthetic membranes. The immobilized preparation was tested on oleic acid in aqueous and heptane media. The order of oxidant activity was tert-butyl hydroperoxide>cumene hydroperoxide>H₂O₂–Urea-H₂O₂. The immobilized preparation could be reused. Oleic acid was a preferred substrate compared to its trans analogue, elaidic acid.     

Grazing history influences biodiversity: a case study on ground-dwelling arachnids (Arachnida: Araneae, Opiliones) in the Natural Park of Alpi Marittime (NW Italy)

Alpine pastures are typical examples of “high nature value farmlands”, representing important habitats harbouring unique biological communities. Disturbance induced by overgrazing influences significantly ecosystem processes, in which invertebrates play a major role. To develop new models of sustainable management of pastures, more knowledge is needed of the animal communities, essential to the ecological functioning of pasture ecosystems. The apparent poor ecological state of several pastures in the Natural Regional Park of Alpi Marittime (NW-Italy) lead us to evaluate the influence of grazing history on biodiversity, using spider and harvestmen assemblages as key groups. Four different pastoral types characterized by four different grazing histories were identified using the Daget-Poissonet method. Spiders and harvestmen were collected by means of pitfall traps. Arachnid assemblages were characterized in terms of composition, abundance, species richness, richness of endemic species and taxonomic relatedness. Generalized linear mixed models (GLMM) were used to test differences among assemblages occurring in each pastoral type. Furthermore, we tested differences in terms of plant communities (species richness and percentage of zoogenic species). Specificity and fidelity of every spider and harvestmen species within pastoral types were explored by the IndVal (Indicator Value) procedure. Fifty-eight species of spiders and seven species of harvestmen were collected (2,304 individuals). Pastoral types related to intensive grazing were characterized by the dominance of diurnal wanderer spiders (namely Lycosidae) while, conversely, nocturnal wanderers (mainly Gnaphosidae) were more abundant in extensive pastoral types. Results show that both species richness and spider abundance were higher in abandoned areas of extensive grazing, while endemic assemblages were richer in extensive grazed areas, which also hosted the most diverse plant community. Furthermore, most of the indicator species of spiders of this type were endemic, characterized by more demanding ecological requirements.