Molecular Basis for the Resistance of Human Mitochondrial 2-Cys Peroxiredoxin 3 to Hyperoxidation

Peroxiredoxins (Prxs) detoxify peroxides and modulate H₂O₂-mediated cell signaling in normal and numerous pathophysiological contexts. The typical 2-Cys subclass of Prxs (human Prx1–4) utilizes a Cys sulfenic acid (Cys-SOH) intermediate and disulfide bond formation across two subunits during catalysis. During oxidative stress, however, the Cys-SOH moiety can react with H₂O₂ to form Cys sulfinic acid (Cys-SO₂H), resulting in inactivation. The propensity to hyperoxidize varies greatly among human Prxs. Mitochondrial Prx3 is the most resistant to inactivation, but the molecular basis for this property is unknown. A panel of chimeras and Cys variants of Prx2 and Prx3 were treated with H₂O₂ and analyzed by rapid chemical quench and time-resolved electrospray ionization-TOF mass spectrometry. The latter utilized an on-line rapid-mixing setup to collect data on the low seconds time scale. These approaches enabled the first direct observation of the Cys-SOH intermediate and a putative Cys sulfenamide (Cys-SN) for Prx2 and Prx3 during catalysis. The substitution of C-terminal residues in Prx3, residues adjacent to the resolving Cys residue, resulted in a Prx2-like protein with increased sensitivity to hyperoxidation and decreased ability to form the intermolecular disulfide bond between subunits. The corresponding Prx2 chimera became more resistant to hyperoxidation. Taken together, the results of this study support that the kinetics of the Cys-SOH intermediate is key to determine the probability of hyperoxidation or disulfide formation. Given the oxidizing environment of the mitochondrion, it makes sense that Prx3 would favor disulfide bond formation as a protection mechanism against hyperoxidation and inactivation.     

The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates

Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.     

Brain changes in BDNF and S100B induced by ketogenic diets in Wistar rats

AimsWe investigated the effects of ketogenic diet (KD) on levels of tumor necrosis factor alpha (TNF-α, a classical pro-inflammatory cytokine), BDNF (brain-derived neurotrophic factor, commonly associated with synaptic plasticity), and S100B, an astrocyte neurotrophic cytokine involved in metabolism regulation.Main methodsYoung Wistar rats were fed during 8 weeks with control diet or two KD, containing different proportions of omega 6 and omega 3 polyunsaturated fatty acids. Contents of TNF-α, BDNF and S100B were measured by ELISA in two brain regions (hippocampus and striatum) as well as blood serum and cerebrospinal fluid.Key findingsOur data suggest that KD was able to reduce the levels of BDNF in the striatum (but not in hippocampus) and S100B in the cerebrospinal fluid of rats. These alterations were not affected by the proportion of polyunsaturated fatty acids offered. No changes in S100B content were observed in serum or analyzed brain regions. Basal TNF-α content was not affected by KD.SignificanceThese findings reinforce the importance of this diet as an inductor of alterations in the brain, and such changes might contribute to the understanding of the effects (and side effects) of KD in brain disorders.     

Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1) antibodies (Abs) represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Western blotting (WB) for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF) and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF) indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I) and gag (p24) proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.     

Identification of Legionella spp. in Environmental Water Samples by ScanVIT-Legionella™ Method in Spain

Rapid and more sensitive methods for the detection and quantification of viable Legionella cells have been developed. In this paper, a comparative analysis of environmental water samples using the ScanVIT-Legionella? method and the traditional “gold standard” method of culturing is realised indicating the usefulness of the ScanVIT method. The ScanVIT-Legionella? method was performed on environmental water samples from different locations of Huesca region (Spain). Legionella micro-colonies should appear green colour and Legionella pneumophila micro-colonies appear red. Twenty-one environmental water samples analysed by standard culture plus five control samples (Two sterile water samples with Legionella as positive controls and three sterile water samples as negative controls). All of them were used to apply ScanVIT-Legionella? method. From of 21 environmental samples eleven were positive, six negative with both methods and four samples were negative for culture method and positive for ScanVIT-Legionella? method. The positive control samples were positive and the negative were negative for both methods. A comparative analysis of the results obtained with two methods showed a strong positive determination coefficient (R² = 0.99753). The results demonstrate the usefulness of the ScanVIT-Legionella? method as a rapid diagnostic tool in order to provide a diagnosis as quick as possible. ScanVIT-Legionella? method offers a series of advantages such as quickly diagnosis, higher sensitivity and the possibility to identify Legionella spp. and L. pneumophila simultaneously.     

Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.     

Serologic survey of West Nile virus in horses from Central-West,Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.     

Bacterially Induced Weathering of Ultramafic Rock and Its Implications for Phytoextraction

The bioavailability of metals in soil is often cited as a limiting factor of phytoextraction (or phytomining). Bacterial metabolites, such as organic acids, siderophores, or biosurfactants, have been shown to mobilize metals, and their use to improve metal extraction has been proposed. In this study, the weathering capacities of, and Ni mobilization by, bacterial strains were evaluated. Minimal medium containing ground ultramafic rock was inoculated with either of two Arthrobacter strains: LA44 (indole acetic acid [IAA] producer) or SBA82 (siderophore producer, PO₄ solubilizer, and IAA producer). Trace elements and organic compounds were determined in aliquots taken at different time intervals after inoculation. Trace metal fractionation was carried out on the remaining rock at the end of the experiment. The results suggest that the strains act upon different mineral phases. LA44 is a more efficient Ni mobilizer, apparently solubilizing Ni associated with Mn oxides, and this appeared to be related to oxalate production. SBA82 also leads to release of Ni and Mn, albeit to a much lower extent. In this case, the concurrent mobilization of Fe and Si indicates preferential weathering of Fe oxides and serpentine minerals, possibly related to the siderophore production capacity of the strain. The same bacterial strains were tested in a soil-plant system: the Ni hyperaccumulator Alyssum serpyllifolium subsp. malacitanum was grown in ultramafic soil in a rhizobox system and inoculated with each bacterial strain. At harvest, biomass production and shoot Ni concentrations were higher in plants from inoculated pots than from noninoculated pots. Ni yield was significantly enhanced in plants inoculated with LA44. These results suggest that Ni-mobilizing inoculants could be useful for improving Ni uptake by hyperaccumulator plants.