Molecular cloning and functional expression of the alpha-scorpion toxin BotIII: pivotal role of the C-terminal region for its interaction with voltage-dependent sodium channels

Alpha scorpion toxins bind to receptor site 3 on voltage-dependent sodium channels and inhibit their inactivation. The alpha-scorpion toxin BotIII is the most toxic protein of Buthus occitanus tunetanus. Its sequence differs only by three amino acid residues from that of AahII, the most active alpha-toxin. Due to their high affinity and selectivity for mammalian sodium channels, BotIII and AahII represent powerful tools for studying the molecular determinants of specificity for voltage-dependent sodium channels. Sequence analysis of BotIII gene has revealed two exons separated by a 381-bp intron and a signal peptide of 19 amino acids. We succeeded in expressing BotIII in significantly higher amounts than AahII the only expressed strict alpha anti-mammalian scorpion toxin reported in the literature. We have also modified specific amino acid residues of BotIII. The recombinant and the natural toxins differ by the amidation of the C-terminal residue. Toxicity and binding experiments indicated: (a) the affinity of rBotIII-OH and rAahII-OH (rBotIII-OH with the 3 mutations R10V, V51L, N64H) for the voltage-dependent sodium channels is reduced compared to the natural toxins. This data revealed the important role of the C-terminal amidation for the biological activity of BotIII and AahII; (b) the single mutation N64H is responsible for the difference of toxicity and affinity between rBotIII-OH and rAahII-OH; (c) the addition of the sequence GR to rBotIII-OH leads to the loss of biological activity. This study is in agreement with the important role attributed to the C-terminal sequence of alpha-toxins in their interaction with sodium channels receptors.     

Perception of cuticular hydrocarbons by the olfactory organs in Periplaneta americana (L.) (Insecta: Dictyoptera)

Despite the importance of cuticular hydrocarbons (CHs) in insect chemical communication, direct proof that they are detected and recognized by insects by contact or by olfactory receptors are rare. In Periplaneta americana, CHs induce aggregation. The aim of our study was to investigate how CHs are detected by P. americana antennae. Using solid phase microextraction and gas chromatography, the three main CHs of the species profile were identified in the volatiles emitted by these insects. Gas chromatography coupled to electroantennography recordings demonstrated that the antennae responded to these three CHs. Furthermore, CHs had an attraction effect in Y-olfactometer bioassays when presented at high concentrations. As CHs can be perceived by P. americana, at least from a short distance, they could play a role in attracting conspecifics during aggregation processes, in addition to inducing aggregation when direct contact is possible.     

Effect of the Aegean Sea barrier between Europe and Asia on differentiation in Juniperus drupacea (Cupressaceae)

Juniperus drupacea is an eastern Mediterranean mountain tree with a disjunct geographical range. We hypothesized that this disjunct occurrence (the Peloponnese in Europe and the Taurus and Lebanon Mountains in Asia) should be reflected in the patterns of genetic and morphological diversity and differentiation. Nuclear microsatellite markers (nSSR) and biometric variables of the cones and seeds were examined on material sampled from four populations in Europe and eight in Asia. The Asian populations were characterized by a higher level of genetic diversity than the European populations. The genetic differentiation among populations was moderate but significant (F_ST = 0.101, P < 0.001). According to the clustering performed with BAPS, six genetically and geographically groups of populations were found: I and II from the Peloponnese; III from the Taurus Mountains; IV and V from the Anti‐Taurus Mountains; and VI from the Lebanon Mountains. The level of genetic differentiation among these six groups (4.30%, P = 0.012) probably reflects long‐lasting genetic isolation during the Pleistocene, as limited genetic admixture was found. In accordance with genetic analysis, the biometric investigations indicated a high level of morphological divergence between the European and Asian populations of the species, with further differentiation between the populations from the Taurus and Lebanon Mountains.     

Rare taxa and dark microbial matter: novel bioactive actinobacteria abound in Atacama Desert soils

An “in house” taxonomic approach to drug discovery led to the isolation of diverse actinobacteria from hyper-arid, extreme hyper-arid and very high altitude Atacama Desert soils. A high proportion of the isolates were assigned to novel taxa, with many showing activity in standard antimicrobial plug assays. The application of more advanced taxonomic and screening strategies showed that strains classified as novel species of Lentzea and Streptomyces synthesised new specialised metabolites thereby underpinning the premise that the extreme abiotic conditions in the Atacama Desert favour the development of a unique actinobacterial diversity which is the basis of novel chemistry. Complementary metagenomic analyses showed that the soils encompassed an astonishing degree of actinobacterial ‘dark matter’, while rank-abundance analyses showed them to be highly diverse habitats mainly composed of rare taxa that have not been recovered using culture-dependent methods. The implications of these pioneering studies on future bioprospecting campaigns are discussed.     

Effect of heated naringenin on immunomodulatory properties and cellular antioxidant activity

Naringenin is one of the most popular flavonoids derived from citrus. It has been reported to be an effective anti-inflammatory compound. Citrus fruit may be used raw, cooked, stewed, or boiled. The present study was conducted to investigate the effect of thermal processes on naringenin in its immunomodulatory and cellular antioxidant activities. The effects of flavonoids on B and T cell proliferation were assessed on splenocytes stimulated or not with mitogens. However, their effects on cytotoxic T lymphocyte (CTL) and natural killer (NK) activities were assessed in splenocytes co-incubated with target cells. The amount of nitric oxide production and the lysosomal enzyme activity were evaluated in vitro on mouse peritoneal macrophages. Cellular antioxidant activity in splenocytes and macrophages was determined by measuring the fluorescence of the dichlorofluorescin (DCF). Our findings revealed that naringenin induces B cell proliferation and enhances NK activity. The highest concentration of native naringenin exhibits a significant proliferation of T cells, induces CTL activity, and inhibits cellular oxidation in macrophages. Conversely, it was observed that when heat-processed, naringenin improves the cellular antioxidant activity in splenocytes, increases the cytotoxic activity of NK cells, and suppresses the cytotoxicity of T cells. However, heat treatment maintains the anti-inflammatory potency of naringenin.     

A 1,100-year-old founder effect mutation in IL12B gene is responsible for Mendelian susceptibility to mycobacterial disease in Tunisian patients

Mendelian susceptibility to mycobacterial disease (MSMD) is a rare disorder predisposing apparently healthy individuals to infections caused by weakly virulent mycobacteria such as bacille Calmette–Guerin (BCG), environmental mycobacteria, and poorly virulent Salmonella strains. IL-12p40 deficiency is the first reported human disease due to a cytokine gene defect and is one of the deficiencies that cause MSMD. Nine mutant alleles only have been identified in the IL12B gene, and three of them are recurrent mutations due to a founder effect in specific populations. IL-12p40 deficiency has been identified especially in countries where consanguinity is high and where BCG vaccination at birth is universal. We investigated, in such settings, the clinical, cellular, and molecular features of six IL-12p40-deficient Tunisian patients having the same mutation in IL12B gene (c.298_305del). We found that this mutation is inherited as a common founder mutation arousing ~1,100 years ago. This finding facilitates the development of a preventive approach by genetic counseling and prenatal diagnosis especially in affected families.     

Association of Lymphotoxin Alpha Polymorphism with Type 1 Diabetes in a Tunisian Population

We investigated the association of the lymphotoxin (LT)-α gene polymorphism +249A/G with type 1 diabetes. The distribution of genotypes of the LT-α +249A/G single nucleotide polymorphism (SNP) was assessed in 115 diabetic patients and 123 normoglycemic control subjects, using PCR-restriction fragment length polymorphism analysis. Among unselected patients, the SNP was significantly associated with increased risk of diabetes (χ² = 8.44, p = 0.014) and was found to be more pronounced among female (χ² = 8.37, p = 0.02) than male (χ² = 6.11, p = 0.047) patients. A significant association was detected between LT-α +249A/G and increased risk of diabetes, in particular for young-onset patients (χ² = 6.92, p = 0.031). Moreover, we reported significant differences in levels of HbA1c, triglycerides, alanine transaminase, and anti-glutamic acid decarboxylase-65 among alleles. Additional studies with extended patient age groups and different ethnicities are needed to confirm our findings.