Physiological responses of Plantago algarbiensis and P. almogravensis shoots and plantlets to low pH and aluminum stress

We investigated the impact of low pH and aluminum (Al) stress on the growth, nutrients concentration, chlorophyll a fluorescence, photosynthetic pigment contents, proline and carbohydrate accumulation in shoots and plantlets (leaves and roots) of Plantago almogravensis and P. algarbiensis. Both species accumulated considerable and similar amounts of Al in their tissues, mainly in the roots. The presence of Al caused a significant reduction on root elongation in P. algarbiensis. Low pH and Al induced significant changes on nutrient accumulation, but no significant alterations on the maximum efficiency of PSII (F _v/F _m), quantum yield of PSII photochemistry (?_PSII), quantum yield of regulated energy dissipation (?_NPQ) and quantum yield of non-regulated energy dissipation (?_NO) were detected in both species in response to these stresses. However, Al increased significantly the non-photochemical quenching and the chlorophyll b content and decreased the PSII excitation pressure (1 ? q _p) in P. almogravensis leaves. Both stress treatments induced carbohydrate accumulation in the shoots and roots of this species, but not in leaves. In P. algarbiensis, low pH and Al decreased the photosynthetic pigment contents in the shoots, whereas Al stimulated the carbohydrate accumulation in the leaves. Although our data showed that both species are tolerant to Al³⁺ and H⁺, P. almogravensis appeared to be more adapted to maintain cellular physiology and growth under those conditions.     

Development of a chloroplast DNA marker for monitoring of transgene introgression in Brassica napus L.

Chloroplast molecular markers can provide useful information for high-resolution analysis of inter- and intra-specific variation in Brassicaceae and for differentiation between its species. Combining data generated from nuclear and chloroplast markers enables the study of seed and pollen movement, and assists in the assessment of gene-flow from genetically modified (GM) plants through hybridization studies. To develop chloroplast DNA markers for monitoring of transgene introgression in Brassica napus L., we searched for sequence variations in the chloroplast (cp) genome, and developed a simple cpDNA marker that is reliable, time-saving, and easily discriminates among 4 species (B. napus, B. rapa, Raphanus sativus, and Sinapis alba) based on PCR-product length polymorphism. This marker will be useful to identify maternal lineages and to estimate transgene movement of GM canola.     

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.     

Functional characterization of an uncoupling protein in goldfish white skeletal muscle

Uncoupling proteins (UCP) are able to increase H⁺ leakage across the inner mitochondrial membrane, thus dissipating the membrane potential and increasing oxygen consumption. Despite the identification of several UCP orthologs in birds, reptiles, amphibians and fish, little is known about their functional properties in fish. The aim of this work was to identify and characterize a UCP in mitochondria found in goldfish white skeletal muscle. Western blot analysis, using a polyclonal antibody raised against mammalian UCP3, showed a single band at approximately 32 kDa. During non-phosphorylating respiration, we observed that palmitate promoted a dose-dependent increase in oxygen consumption that is abolished by addition of BSA (fatty acid chelator). Interestingly, this palmitate-induced increase in oxygen consumption was not inhibited by GDP, a well-known UCP inhibitor. In phosphorylating mitochondria, palmitate lowered both ADP/O ratio (number of atoms of phosphorus incorporated as ATP per molecule of O₂ consumed) and the respiratory control ratio. Moreover, we found that different fatty acids can modulate mitochondrial membrane potential. In conclusion, our results suggest that goldfish UCP is functionally similar to the UCP found in other species, including mammals.     

Bacterial Diversity and Geochemical Profiles in Sediments from Eutrophic Azorean Lakes

In the Azores, the advanced trophic state of the lakes requires a fast intervention to achieve the good ecological status prescribed by the Water Framework Directive. Despite the considerable effort made to describe the phytoplankton growing on the water column, the lack of information regarding the microbial processes in sediments is still high. Thus, for the successful implementation of internal management actions, the present work explored the relationships between geochemical profiles and dominant members of the bacterial community in sediments from eutrophic Azorean lakes. Lake Azul geochemical profiles were quite homogeneous for all parameters, while in lake Furnas the total iron profile presented a peak below the aerobic layer. For lake Verde, the concentrations of all studied parameters (20 ± 2% loss-on-ignition; 2.10 ± 0.08 mg g^?1 total phosphorus; 1.31 ± 0.50 mg g^?1 total nitrogen; 8.06 ± 0.13 mg g^?1 total iron) in the uppermost sediment layer were approximately two times higher than the ones in sediments from other lakes, decreasing with sediment depth. The higher amounts of phosphorus and organic matter in lake Verde suggested a higher internal contribution of phosphorus to eutrophication. The dominant members of the sediment bacterial community, investigated by denaturing gradient gel electrophoresis, were mostly affiliated to Proteobacteria phylum (Alpha-, Delta-, and Gamma-subclasses), group Bacteroidetes/Chlorobi and phylum Chloroflexi. The Cyanobacteria phylum was solely detected in sediments from lake Verde and lake Furnas that presented the highest amounts of nitrogen and phosphorus both in the water column and sediments, while the other phyla were detected in sediments from the three studied lakes. In conclusion, management measurers to achieve the good ecological status until 2015 should be distinct for the different lakes taking into account the relative magnitude of the nutrient sources and the bacterial diversity in sediments.     

Rotenone-Dependent Changes of Anterograde Motor Protein Expression and Mitochondrial Mobility in Brain Areas Related to Neurodegenerative Diseases

The presence of protein aggregates is common in neurodegenerative disorders; however, the real cause and effect of these aggregates during neurodegeneration is still a matter of investigation. We hypothesize that impairment of intracellular traffic may appear in the absence of protein inclusions and might trigger protein aggregation. In the present study, we aimed to evaluate mitochondria mobility as well as protein and messenger RNA expression of KIF1B and KIF5 that are molecular motors for neuronal anterograde traffic, in hippocampus, substantia nigra, and locus coeruleus of 10-month-old Lewis rats and cultured cells, from these same areas, following exposure to low doses of rotenone that do not lead to protein inclusions. The present study showed alteration in KIF1B and KIF5 expression, as well as in mitochondria mobility prior to protein aggregation involved in neurodegenerative disorders. These findings suggest that change in intracellular trafficking might be critical and one of the primary events for impairment of cell physiology during neurodegeneration associated with protein inclusions.     

The effects of the Brazilian antDinoponera quadriceps venom on chemically induced seizure models

Arthropod venoms are potential sources of neuroactive substances, providing new tools for the design of drugs. The aim of this study was to evaluate the effects of Dinoponera quadriceps venom (DqV) on seizure models in mice induced by pentylenetetrazole (PTZ), pilocarpine, and strychnine. In the PTZ model, intraperitoneal treatment with DqV (0.5 mg/kg) increased the time until the first seizure and the percentage of survival (155.4 ± 27.7 s/12.5%, p < 0.05) compared to the control group (79.75 ± 3.97 s/0%), whereas endovenous treatment (0.1 and 0.5 mg/kg) decreased the time until the first seizure (0.1 mg/kg: 77.83 ± 5.3 s versus 101.0 ± 3.3 s in the control group; 0.5 mg/kg: 74.43 ± 3.9 s versus 101.0 ± 3.3 s for the control group, p < 0.05). We did not observe significant changes in the pilocarpine- and strychnine-induced seizure models. In assays that measured oxidative parameters in the PTZ model, intraperitoneal treatment with DqV (0.5 and 2.0 mg/kg) only decreased the levels of MDA and nitrite in the cortex. However, endovenous treatment with DqV (0.1 and 0.5 mg/kg) increased the levels of MDA in the cortex and hippocampus and at a dose of 0.5 mg/kg in the striatum. Moreover, increased in nitrite content was observed in all three of the brain regions analyzed. Taken together, the D. quadriceps venom caused both neuroprotective and neurotoxic effects in a PTZ-induced seizure model, and this effect was dependent on the route of administration used.     

Hypoxia–Ischemia Alters Nucleotide and Nucleoside Catabolism and Na+,K+-ATPase Activity in the Cerebral Cortex of Newborn Rats

It is well known that the levels of adenosine in the brain increase dramatically during cerebral hypoxic-ischemic (HI) insults. Its levels are tightly regulated by physiological and pathophysiological changes that occur during the injury acute phase. The aim of the present study was to examine the effects of the neonatal HI event on cytosolic and ecto-enzymes of purinergic system––NTPDase, 5′-nucleotidase (5′-NT) and adenosine deaminase (ADA)––in cerebral cortex of rats immediately post insult. Furthermore, the Na⁺/K⁺-ATPase activity, adenosine kinase (ADK) expression and thiobarbituric acid reactive species (TBARS) levels were assessed. Immediately after the HI event the cytosolic NTPDase and 5′-NT activities were increased in the cerebral cortex. In synaptosomes there was an increase in the ecto-ADA activity while the Na⁺/K⁺ ATPase activity presented a decrease. The difference between ATP, ADP, AMP and adenosine degradation in synaptosomal and cytosolic fractions could indicate that NTPDase, 5′-NT and ADA were differently affected after insult. Interestingly, no alterations in the ADK expression were observed. Furthermore, the Na⁺/K⁺-ATPase activity was correlated negatively with the cytosolic NTPDase activity and TBARS content. The increased hydrolysis of nucleotides ATP, ADP and AMP in the cytosol could contribute to increased adenosine levels, which could be related to a possible innate neuroprotective mechanism aiming at potentiating the ambient levels of adenosine. Together, these results may help the understanding of the mechanism by which adenosine is produced following neonatal HI injury, therefore highlighting putative therapeutical targets to minimize ischemic injury and enhance recovery.     

Tigutcystatin, a cysteine protease inhibitor from Triatoma infestans midgut expressed in response to Trypanosoma cruzi

The insect Triatoma infestans is a vector of Trypanosoma cruzi, the etiological agent of Chagas disease. A cDNA library was constructed from T. infestans anterior midgut, and 244 clones were sequenced. Among the EST sequences, an open reading frame (ORF) with homology to a cystatin type 2 precursor was identified. Then, a 288-bp cDNA fragment encoding mature cystatin (lacking signal peptide) named Tigutcystatin was cloned fused to a N-terminal His tag in pET-14b vector, and the protein expressed in Escherichia coli strain Rosetta gami. Tigutcystatin purified and cleaved by thrombin to remove His tag presented molecular mass of 11 kDa and 10,137 Da by SDS–PAGE and MALDI-TOF mass spectrometry, respectively. Purified Tigutcystatin was shown to be a tight inhibitor towards cruzain, a T. cruzi cathepsin L-like enzyme (K_i = 3.29 nM) and human cathepsin L (K_i = 3.78 nM). Tissue specific expression analysis showed that Tigutcystatin was mostly expressed in anterior midgut, although amplification in small intestine was also detected by semi quantitative RT-PCR. qReal time PCR confirmed that Tigutcystatin mRNA is significantly up-regulated in anterior midgut when T. infestans is infected with T. cruzi. Together, these results indicate that Tigutcystatin may be involved in modulation of T. cruzi in intestinal tract by inhibiting parasite cysteine proteases, which represent the virulence factors of this protozoan.