Differential toxicity of TAR DNA‐binding protein 43 isoforms depends on their submitochondrial localization in neuronal cells

TAR DNA ‐binding protein 43 (TDP ‐43) is an RNA ‐binding protein and a major component of protein aggregates found in amyotrophic lateral sclerosis and several other neurodegenerative diseases. TDP ‐43 exists as a full‐length protein and as two shorter forms of 25 and 35 kD a. Full‐length mutant TDP ‐43s found in amyotrophic lateral sclerosis patients re‐localize from the nucleus to the cytoplasm and in part to mitochondria, where they exert a toxic role associated with neurodegeneration. However, induction of mitochondrial damage by TDP ‐43 fragments is yet to be clarified. In this work, we show that the mitochondrial 35 kD a truncated form of TDP ‐43 is restricted to the intermembrane space, while the full‐length forms also localize in the mitochondrial matrix in cultured neuronal NSC ‐34 cells. Interestingly, the full‐length forms clearly affect mitochondrial metabolism and morphology, possibly via their ability to inhibit the expression of Complex I subunits encoded by the mitochondrial‐transcribed mRNA s, while the 35 kD a form does not. In the light of the known differential contribution of the full‐length and short isoforms to generate toxic aggregates, we propose that the presence of full‐length TDP ‐43s in the matrix is a primary cause of mitochondrial damage. This in turn may cause oxidative stress inducing toxic oligomers formation, in which short TDP ‐43 forms play a major role.

     

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction

Citrus canker is a plant disease caused by Gram‐negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm‐enriched fraction was performed for XAC cells grown in pathogenicity‐inducing (XAM‐M) and pathogenicity‐non‐inducing (nutrient broth) media using two‐dimensional electrophoresis combined with liquid chromatography‐tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up‐regulated proteins related to cellular envelope metabolism included glucose‐1‐phosphate thymidylyltransferase, dTDP‐4‐dehydrorhamnose‐3,5‐epimerase and peptidyl‐prolyl cis–trans‐isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real‐time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up‐regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60‐kDa chaperonin and glyceraldehyde‐3‐phosphate dehydrogenase were identified, suggesting the presence of post‐translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence‐related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies.     

Effect of cement industry flue gas simulation on the physiology and photosynthetic performance of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Cement plants account for significant emissions of CO₂ and other pollutants into the atmosphere. As a means for its mitigation, we tested the effect of a cement industry-based flue gas simulation (FGS — 18% CO₂, 9% O₂, 300 ppm NO₂, 140 ppm SO₂) on the green alga, Chlorella sorokiniana. Culture pH, cell density, cell viability and productivity, specific growth rates, photosynthetic performance, and biochemical composition were monitored. The treatments consisted of different FGS volumes (0.1, 0.3, 0.8, 1.5, 6, and 48 L day^?1) that were applied in a series of laboratory-scale semi-continuous batch cultures under controlled conditions. Controls were exposed to 18% CO₂ enriched air. Cell density showed that C. sorokiniana was able to grow in all treatments, but compared to the controls, low pH (~ 5.0) caused by 48 L FGS day^?1 led to 27% decrease in specific growth rate. Increasing FGS exposure decreased maximum and operational quantum yields obtained by pulse amplitude modulated fluorometry, while photochemical quenching remained constant (~ 0.93). The α and rETR _max parameters calculated from rapid light curves decreased with increasing FGS exposure. Total proteins and carbohydrates (per cell basis) increased after 6 and 48 L FGS day^?1, which can be advantageous for biotechnological applications, but cell productivity (cells L^?1 day^?1) decreased. Despite the effects in physiology, C. sorokiniana could withstand a pH range of 6.0–5.0 imposed by 48 L FGS day^?1. Overall, C. sorokiniana can be considered a robust species in flue gas bioremediation.     

Virulence factors in food,clinical and reference Enterococci: A common trait in the genus?

The occurrence of several virulence traits (cytolysin, adhesins and hydrolytic enzymes) was investigated in a collection of 164 enterococci, including food and clinical isolates (from human and veterinary origin), as well as type and reference strains from 20 enterococcal species. Up to fifteen different cyl genotypes were found, as well as silent cyl genes. The occurrence of the cyl operon and haemolytic potential seems to be widespread in the genus. A significant association of this virulent trait with clinical isolates was found (p < 0.05). High levels of incidence were also observed for genes encoding surface adhesins (esp, efaA(fs), efaA(fm)), agg and gelE, irrespectively of species allocation and origin of strains. Although gelE behaves as silent in the majority of the strains, gelatinase activity predominates in clinical isolates, whereas lipase and DNase were mainly detected in food isolates pointing to their minor role as virulence determinants. No hyaluronidase activity was detected for all strains. Numerical hierarchic data analysis grouped the strains in three main clusters, two of them including a total of 50 strains with low number of virulence determinants (from 2 to 7) and the other with 114 strains with a high virulence potential (up to 12 determinants). No statistical association was found between virulence clusters and species allocation (p > 0.10), strongly suggesting that virulence determinants are a common trait in the genus Enterococcus. Clinical strains seem to be significantly associated with high virulence potential, whereas food, commensal and environmental strains harbour fewer virulence determinants (p < 0.01). A high level of relative diversity in virulence patterns was observed (Shannon's index varies from 0.95 to 1.0 among clusters), reinforcing the strain-specific nature of the association of virulence factors. Although a low risk seems to be associated with the use of enterococci in long-established artisanal cheeses, screening of virulence traits and their cross-synergies must be performed, particularly for commercial starters, probiotic strains and products to be used by high risk population groups.     

Tobacco Smoke Exposure During Pregnancy Increases Maternal Blood Lead Levels Affecting Neonate Birth Weight

To assess the effect of lead exposure from cigarette smoke on fetal growth, blood lead concentrations were measured using inductively coupled plasma mass spectrometry in 150 healthy pregnant women. Mean lead concentrations in plasma and whole blood were significantly higher in the smoking group compared with the nonsmoking group in each trimester of pregnancy (p?<?0.001). Logistic regression analysis showed the highest impact of the number of cigarettes smoked per day for serum lead concentration (β?=?0.238; p?<?0.05), while in whole blood, it was duration of smoking before conception (β?=?0.297; p?<?0.001). Birth weight of the smoking mothers' infants was significantly lower (mean?±?SEM, 3,192?±?50.8 and 3,569?±?49.6 g, respectively; p?<?0.001) and negatively correlated with lead levels in plasma (r?=??0.38; p?<?0.001) and in whole blood (r?=??0.27; p?<?0.001). Therefore, it is suggested that smoking during pregnancy increases lead concentrations in maternal blood. Fetal exposure to low doses of lead in utero may be a serious risk factor causing lower birth weight.     

Mutations at residues 282, 286, and 293 of phage lambda integrase exert pathway-specific effects on synapsis and catalysis in recombination

Bacteriophage lambda integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. The att sites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: the attL site probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA with attR.     

A SAS-6-Like Protein Suggests that the Toxoplasma Conoid Complex Evolved from Flagellar Components

SAS-6 is required for centriole biogenesis in diverse eukaryotes. Here, we describe a novel family of SAS-6-like (SAS6L) proteins that share an N-terminal domain with SAS-6 but lack coiled-coil tails. SAS6L proteins are found in a subset of eukaryotes that contain SAS-6, including diverse protozoa and green algae. In the apicomplexan parasite Toxoplasma gondii, SAS-6 localizes to the centriole but SAS6L is found above the conoid, an enigmatic tubulin-containing structure found at the apex of a subset of alveolate organisms. Loss of SAS6L causes reduced fitness in Toxoplasma. The Trypanosoma brucei homolog of SAS6L localizes to the basal-plate region, the site in the axoneme where the central-pair microtubules are nucleated. When endogenous SAS6L is overexpressed in Toxoplasma tachyzoites or Trypanosoma trypomastigotes, it forms prominent filaments that extend through the cell cytoplasm, indicating that it retains a capacity to form higher-order structures despite lacking a coiled-coil domain. We conclude that although SAS6L proteins share a conserved domain with SAS-6, they are a functionally distinct family that predates the last common ancestor of eukaryotes. Moreover, the distinct localization of the SAS6L protein in Trypanosoma and Toxoplasma adds weight to the hypothesis that the conoid complex evolved from flagellar components.     

Differentiation of five tuna species by a multiplex primer-extension assay

A novel methodology based on analysis of mtDNA-cytb diagnostic sites was performed to discriminate four closely related species of Thunnus (Thunnus alalunga, Thunnus albacares, Thunnus obesus and Thunnus thynnus) and one species of Euthynnus (Katsuwonus pelamis) genus in raw and canned tuna. The primers used in the preliminary PCR designed in well conserved region upstream and downstream of the diagnosis sites successfully amplified a 132bp region from the cytb gene of all the species taken into consideration. The sites of diagnosis have been interrogate simultaneously using a multiplex primer-extension assay (PER) and the results were confirmed by fragment sequencing. The applicability of the multiplex PER assay to commercial canned tuna samples was also demonstrated. The proposed test could be useful for detection of fraud and for seafood traceability.     

Nitric Oxide Mediates the Glutamate-dependent Pathway for Neurotransmission in Sepia officinalis Chromatophore Organs

Chromatophore organs are complex and unique structures responsible for the variety of body coloration patterns used by cephalopods to communicate and camouflage. They are formed by a pigment-containing cytoelastic sacculus, surrounded by muscle fibers directly innervated from the brain. Muscle contraction and relaxation are responsible for expansion and retraction of the pigment-containing cell. Their functioning depends on glutamate and Phe-Met-Arg-Phe-NH₂-related peptides, which induce fast and slow cell expansion, respectively, and 5-hydroxytryptamine, which induces retraction. Apart from these three substances and acetylcholine, which acts presynaptically, no other neuroactive compounds have so far been found to be involved in the neuroregulation of chromatophore physiology, and the detailed signaling mechanisms are still little understood. Herein, we disclose the role of nitric oxide (NO) as mediator in one of the signaling pathways by which glutamate activates body patterning. NO and nitric-oxide synthase have been detected in pigment and muscle fibers of embryo, juvenile, and adult chromatophore organs from Sepia officinalis. NO-mediated Sepia chromatophore expansion operates at slower rate than glutamate and involves cGMP, cyclic ADP-ribose, and ryanodine receptor activation. These results demonstrate for the first time that NO is an important messenger in the long term maintenance of the body coloration patterns in Sepia.