Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle

Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.     

Reduced levels of Cacna1c attenuate mesolimbic dopamine system function

Genetic variation in CACNA1C, which codes for the L‐type calcium channel (LTCC) Ca_v1.2, is associated with clinical diagnoses of bipolar disorder, depression and schizophrenia. Dysregulation of the mesolimbic‐dopamine (ML‐DA) system is linked to these syndromes and LTCCs are required for normal DAergic neurotransmission between the ventral tegmental area (VTA) and nucleus accumbens (NAc). It is unclear, however, how variations in CACNA1C genotype, and potential subsequent changes in expression levels in these regions, modify risk. Using constitutive and conditional knockout mice, and treatment with the LTCC antagonist nimodipine, we examined the role of Cacna1c in DA‐mediated behaviors elicited by psychomotor stimulants. Using fast‐scan cyclic voltammetry, DA release and reuptake in the NAc were measured. We find that subsecond DA release in Cacna1c haploinsufficient mice lacks normal sensitivity to inhibition of the DA transporter (DAT). Constitutive haploinsufficiency of Cacna1c led to attenuation of hyperlocomotion following acute administration of stimulants specific to DAT, and locomotor sensitization of these mice to the DAT antagonist GBR12909 did not reach the same level as wild‐type mice. The maintenance of sensitization to GBR12909 was attenuated by administration of nimodipine. Sensitization to GBR12909 was attenuated in mice with reduced Cacna1c selectively in the VTA but not in the NAc. Our findings show that Cacna1c is crucial for normal behavioral responses to DA stimulants and that its activity in the VTA is required for behavioral sensitization. Cacna1c likely exerts these effects through modifications to presynaptic ML‐DA system function.     

Bias in freshwater biodiversity sampling: the case of Iberian water beetles

Extensive distributional data bases are key tools in ecological research, and good-quality data are required to provide reliable conservation strategies and an understanding of biodiversity patterns and processes. Although the evaluation of data bases requires the incorporation of estimates of sampling effort and bias, no studies have focused on these aspects for freshwater biodiversity data. We used here a comprehensive data base of water beetles from the Iberian Peninsula and the Balearic Islands, and examine whether these data provide an unbiased, reliable picture of their diversity and distribution in the study area. Based on theoretical estimates using the Clench function on the accumulated number of records as a surrogate of sampling effort, about a quarter of the Iberian and Balearic 50 × 50 km Universal Transverse Mercator grid cells can be considered well prospected, with more than 70% of the theoretical species richness actually recorded. These well-surveyed cells are not evenly distributed across biogeographical and physicoclimatic subregions, reflecting some geographical bias in the distribution of sampling effort. Our results suggest that recording was skewed by relatively simple variables affecting collector activity, such as the perceived 'attractiveness' of mountainous landscapes and protected areas with recently described species, and accessibility of sampling sites (distance from main research centres). We emphasize the importance of these evaluation exercises, which are useful to locate areas needed of further sampling as well as to identify potential biases in the distribution of current biodiversity patterns.     

Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): Effects of metallic and organic contaminants

Juvenile Solea senegalensis (Senegalese sole) were exposed to freshly collected sediments from three sites of the Sado Estuary (West-Portuguese coast) in 28-day laboratory assays in order to assess the ecological risk from sediment contaminants, by measuring two genotoxicity biomarkers in peripheral blood: the percentage of Erythrocyte Nuclear Abnormalities (ENA) by use of an adaptation of the micronucleus test, and the percentage of DNA strand-breakage (DNA-SB) with the Comet assay. Sediments were surveyed for metallic (Cr, Ni, Cu, Zn, As, Cd and Pb) and organic (PAHs (polycyclic aromatic hydrocarbons), PCBs (polychlorinated biphenyls) and DDTs (dichloro-diphenyl-trichloroethane)) contaminants. Sediments from site A (farthest from hotspots of contamination) were found to be the least contaminated and weaker inducers of genotoxic damage, whereas sediments from sites B (urban influence) and C (affected by industrial effluents and agricultural runoffs) were responsible for a very significant increase in both ENA and DNA-SB, site B being most contaminated with metals and site C mainly with organic pollutants, especially PAHs and PCBs . Analysis of genotoxic effects showed a strong correlation between the concentrations of PAHs and PCBs and both biomarkers at sampling times T₁₄ and T₂₈, while the amounts of Cu, As, Cd and Pb were less strongly correlated, and at T₂₈ only, with ENA and DNA-SB. These results show that organic contaminants in sediment are stronger and faster acting genotoxic stressors. The results also suggest that metals may have an inhibitory effect on genotoxicity when interacting with organic contaminants, at least during early exposure. ENA and DNA-SB do not show a linear relationship, but a strong correlation exists between the overall increase in genotoxicity caused by exposure to sediment, confirming that they are different, and possibly non-linked effects that respond similarly to exposure. Although the Comet assay showed enhanced sensitivity, the two analyses are complementary and suitable for the biomonitoring of sediment contaminants in a benthic species like S. senegalensis.     

Phenotypic and genotypic identification of bacteria from the Burkholderia cepacia complex

AIM: Evaluation of the diagnostic value of pheno- and genotypic characteristics of B. cepacia strains collection. MATERIALS AND METHODS: Phenotypic and genetic methods of identification and differentiation of 25 strains of the B. cepacia complex. RESULTS: Polyphasic taxonomic approach utilizing multiple diagnostic tests was used for accurate identification of Burkholderia species. Algorithm for identification of microorganisms from the B. cepacia complex was developed. CONCLUSION: Combined use of phenotypic and molecular genetic tests, such as recA gene PCR, is recommended for differentiation of the B. cepacia complex genomovars.     

The interaction of Bacteroides fragilis with components of the human fibrinolytic system

Bacteroides fragilis is a minor component of the intestinal microbiota and the most frequently isolated from intra-abdominal infections and bacteremia. Previously, our group has shown that molecules involved in laminin-1 (LMN-1) recognition were present in outer membrane protein extracts of B. fragilis MC2 strain. One of these proteins was identified and showed 98% similarity to a putative B. fragilis plasminogen-binding protein precursor, deposited in the public database. Thus, the objective of this work was to overexpress and further characterize this novel adhesin. The ability of B. fragilis MC2 strain and purified protein to convert plasminogen into plasmin was tested. Our results showed that B. fragilis strain MC2 strain adhered to both LMN-1 and plasminogen and this adhesion was inhibited by either LMN-1 or plasminogen. Regarding the plasminogen activation activity, both the whole bacterial cell and the purified protein converted plasminogen into plasmin similar to streptokinase used as a positive control. Bacterial receptors that recognize plasminogen bind to it and enhance its activation, transforming a nonproteolytic bacterium into a proteolytic one. We present in vitro evidence for a pathogenic function of the plasminogen receptor in promoting adherence to laminin and also the formation of plasmin by B. fragilis .     

Glutamine homeostasis and mitochondrial dynamics

Glutamine is a multifaceted amino acid that plays key roles in many metabolic pathways and also fulfils essential signaling functions. Although classified as non-essential, recent evidence suggests that glutamine is a conditionally essential amino acid in several physiological situations. Glutamine homeostasis must therefore be exquisitely regulated and mitochondria represent a major site of glutamine metabolism in numerous cell types. Glutaminolysis is mostly a mitochondrial process with repercussions in organelle structure and dynamics suggesting a tight and mutual control between mitochondrial form and cell bioenergetics. In this review we describe an updated account focused on the critical involvement of glutamine in oxidative stress, mitochondrial dysfunction and tumour cell proliferation, with special emphasis in the initial steps of mitochondrial glutamine pathways: transport into the organelle and hydrolytic deamidation through glutaminase enzymes. Some controversial issues about glutamine catabolism within mitochondria are also reviewed.