Identification of three novel Toxoplasma gondii rhoptry proteins

The rhoptries are key secretory organelles from apicomplexan parasites that contain proteins involved in invasion and modulation of the host cell. Some rhoptry proteins are restricted to the posterior bulb (ROPs) and others to the anterior neck (RONs). As many rhoptry proteins have been shown to be key players in Toxoplasma invasion and virulence, it is important to identify, understand and characterise the biological function of the components of the rhoptries. In this report, we identified putative novel rhoptry genes by identifying Toxoplasma genes with similar cyclical expression profiles as known rhoptry protein encoding genes. Using this approach we identified two new rhoptry bulb (ROP47 and ROP48) and one new rhoptry neck protein (RON12). ROP47 is secreted and traffics to the host cell nucleus, RON12 was not detected at the moving junction during invasion. Deletion of ROP47 or ROP48 in a type II strain did not show major influence in in vitro growth or virulence in mice.     

Hypoxic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): role of branchial O2 chemoreceptors

In one series of experiments, heart frequency (f _H), blood pressure (P _a), gill ventilation frequency (f _R ), ventilation amplitude (V _AMP) and total gill ventilation (V _TOT) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO₂ ~140, normoxia to 17 mmHg, severe hypoxia), they increased f _R , V _AMP, V _TOT and P _a and decreased f _H. In a second series of experiments, air-breathing frequency (f _RA), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 μg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 μg NaCN in 1.0 mL water) on f _R , V _AMP, V _TOT, P _a and f _H we conclude that the O₂ receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O₂ levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.     

Camptosemin, a tetrameric lectin of Camptosema ellipticum: structural and functional analysis

Lectins have been classified into a structurally diverse group of proteins that bind carbohydrates and glycoconjugates with high specificity. They are extremely useful molecules in the characterization of saccharides, as drug delivery mediators, and even as cellular surface makers. In this study, we present camptosemin, a new lectin from Camptosema ellipticum. It was characterized as an N-acetyl-d-galactosamine-binding homo-tetrameric lectin, with a molecular weight around 26 kDa/monomers. The monomers were stable over a wide range of pH values and exhibited pH-dependent oligomerization. Camptosemin promoted adhesion of breast cancer cells and hemagglutination, and both activities were inhibited by its binding of sugar. The stability and unfolding/folding behavior of this lectin was characterized using fluorescence and far-UV circular dichroism spectroscopies. The results indicate that chemical unfolding of camptosemin proceeds as a two-state monomer-tetramer process. In addition, small-angle X-ray scattering shows that camptosemin behaves as a soluble and stable homo-tetramer molecule in solution.     

Omega-3 fatty acids deprivation affects ontogeny of glutamatergic synapses in rats: Relevance for behavior alterations

Essential omega-3 polyunsaturated fatty acids (ω3) are crucial to brain development and function, being relevant for behavioral performance. In the present study we examined the influence of dietary ω3 in the development of the glutamatergic system and on behavior parameters in rats. Female rats received isocaloric diets, either with ω3 (ω3 group) or a ω3 deficient diet (D group). In ontogeny experiments of their litters, hippocampal immunocontent of ionotropic NMDA and AMPA glutamatergic receptors subunits (NR2 A\B and GluR1, respectively) and the alpha isoform of the calcium-calmodulin protein kinase type II (αCaMKII) were evaluated. Additionally, hippocampal [³H]glutamate binding and uptake were assessed. Behavioral performance was evaluated when the litters were adult (60 days old), through the open-field, plus-maze, inhibitory avoidance and flinch-jump tasks. The D group showed decreased immunocontent of all proteins analyzed at 02 days of life (P2) in comparison with the ω3 group, although the difference disappeared at 21 days of life (except for αCaMKII, which content normalized at 60 days old). The same pattern was found for [³H]glutamate binding, whereas [³H]glutamate uptake was not affected. The D group also showed memory deficits in the inhibitory avoidance, increased in the exploratory pattern in open-field, and anxiety-like behavior in plus-maze. Taken together, our results suggest that dietary ω3 content is relevant for glutamatergic system development and for behavioral performance in adulthood. The putative correlation among the neurochemical and behavioral alterations caused by dietary ω3 deficiency is discussed.     

Effect of dolomite on the repair of bone defects in rats: histological study

The aim of the present study was to evaluate histologically and radiographically the tissue response to dolomite [CaMg(CO3)2] and its osteogenic potential in the repair of bone cavities in the calvaria of rats. A bone defect 10 mm in diameter and 1 mm deep was made in the calvaria of male Wistar rats. The defects were filled with dolomite, inorganic bovine bone (positive control), or coagulum (negative control). The animals were euthanized 7, 15, 30, and 60 days after surgery, and specimens were collected for radiographic and microscopic analyses. The bone defects were processed for paraffin embedding and H&E staining. The histological study revealed that dolomite stimulated a moderate inflammatory response, with programmed cell death in the first 15 days, compared to bovine bone which showed a moderate to intense acute response. In the chronic phase, the inflammatory response was characterized by the occurrence of macrophages organized as epithelioid cells in the dolomite group, and giant cells in the bovine-bone group. Fibrosis developed in all three groups; however, encapsulation of the fragments, reabsorption, and osteoconductive activity occurred only in the defects filled with bovine bone. The radiographic analysis showed that the bovine bone was most efficient in the repair of the defects, followed by the dolomite and the coagulum. This study demonstrated that the dolomite stimulated a moderate acute inflammatory response with programmed cell death, and a chronic inflammatory response by means of the phagocytic mononuclear system. Although osteo-conductive activity was not shown, the dolomite favored the repair process, compared to the coagulum group.     

HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.     

Chronic Social Isolation Compromises the Activity of Both Glutathione Peroxidase and Catalase in Hippocampus of Male Wistar Rats

Chronic neuroendocrine stress usually leads to the elevation of the stress hormones and increased metabolic rate, which is frequently accompanied by oxidative damage to the CNS. In the present study we hypothesized that chronic psychosocial isolation (CPSI) of male Wistar rats, characterized by decreased serum corticosterone (CORT), unaltered catecholamines (CTs), and low blood glucose (GLU), may also promote oxidative imbalance in the CNS, by targeting antioxidant defense system. To test it, we have examined the relation between these input signals and protein expression/activity of antioxidant enzymes (AOEs): superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GLR) in the hippocampus (HIPPO) of CPSI animals. We found that CPSI did not affect SODs or CAT, but decreased activity of GPx and compromised GLR, an enzyme highly dependent on blood GLU for its substrate precursor. Further, we have tested whether the CPSI experience altered AOEs response to a novelty stress, and found that it attenuated peroxide-metabolizing enzymes, CAT and GPx, and decreased GLR activity, even though blood GLU was restored. The altered ratios of hippocampal AOEs in CPSI animals, which were worsened under the combined stress conditions, may lead to the accumulation of peroxide products and oxidative imbalance. The mechanism by which CPSI generate oxidative imbalance in the HIPPO is most likely based on poor systemic energy conditions set by this stress. Such conditions may cause functional decline of CNS structures, such as HIPPO, and are likely to promote state linked to onset of many mood disorders.     

Effects of anti-TNF therapy on glucose metabolism in patients with ankylosing spondylitis,psoriatic arthritis or juvenile idiopathic arthritis

The objective of this study was to evaluate the influence of anti-tumor necrosis factor (anti-TNF) in juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS) or psoriatic arthritis (PsA). Sixty-two patients were investigated: 7 JIA; 37 AS; and 18 PsA. Caucasian race accounted for 79% and 29% were female. Mean age was 40.4 ± 12.6years. None of the patients had a history of diabetes, and none had used oral hypoglycemic agents or insulin. Treatment was with adalimumab, infliximab and etanercept. Glucose, inflammatory markers and prednisone dose were assessed at baseline, as well as after three and six months of treatment. The mean erythrocyte sedimentation rate was significantly lower at three months and six months than at baseline (13.7 ± 18.0 and 18 ± 22.5 vs. 27.9 ± 23.4 mm; p = 0.001). At baseline, three months and six months, we found the following: mean C-reactive protein levels were comparable (22.1 ± 22.7, 14.5 ± 30.7 and 16.0 ± 23.8 mg/L, respectively; p = 0.26); mean glucose levels remained unchanged (90.8 ± 22.2 mg/dl, 89.5 ± 14.6 mg/dl and 89.8 ± 13.6 mg/dl, respectively; p = 0.91); and mean prednisone doses were low and stable (3.9 ± 4.9 mg/day, 3.7 ± 4.8 mg/day and 2.6 ± 4.0 mg/day, respectively; p = 0.23). During the first six months of treatment, anti-TNF therapy does not seem to influence glucose metabolism in JIA, AS or PsA.     

A practical approach to FRET-based PNA fluorescence in situ hybridization

Given the demand for improved methods for detecting and characterizing RNA variants in situ, we developed a quantitative method for detecting RNA alternative splicing variants that combines in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes with confocal microscopy Förster resonance energy transfer (FRET). The use of PNA probes complementary to sequences flanking a given splice junction allows to specifically quantify, within the cell, the RNA isoform generating such splice junction as FRET efficiency measure. The FRET-based PNA fluorescence in situ hybridization (FP-FISH) method offers a conceptually new approach for characterizing at the subcellular level not only splice variant isoform structure, location, and dynamics but also potentially a wide variety of close range RNA–RNA interactions. In this paper, we explain the FP-FISH technique workflow for reliable and reproducible results.