Oxidative Stress in Cerebrospinal Fluid of Patients with Aseptic and Bacterial Meningitis

This study aimed to determine whether patients with aseptic and bacterial meningitis presented alterations in oxidative stress parameters of cerebrospinal fluid (CSF). A total of 30 patients were used in the research. The CSF oxidative stress status has been evaluated through many parameters, such as lipid peroxidation through thiobarbituric acid reactive substances (TBARS) and antioxidant defense systems such as superoxide dismutase (SOD), glutathione S-transferase (GST), reduced glutathione (GSH) and ascorbic acid. TBARS levels, SOD and GST activity increase in aseptic meningitis and in bacterial meningitis. The ascorbic acid concentration increased significantly in patients with both meningitis types. The reduced glutathione levels were reduced in CSF of patients with aseptic and bacterial meningitis. In present study we may conclude that oxidative stress contributes at least in part to the severe neurological dysfunction found in meningitis.     

New Arabidopsis thaliana Cytochrome c Partners: A Look Into the Elusive Role of Cytochrome c in Programmed Cell Death in Plants

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.Programmed cell death (PCD)¹ is a fundamental event for the development of multicellular organisms and the homeostasis of their tissues. It is an evolutionarily conserved mechanism present in organisms ranging from yeast to mammals (1–3).In mammals, cytochrome c (Cc) and dATP bind to apoptosis protease-activating factor-1 (Apaf-1) in the cytoplasm, a process leading to the formation of the Apaf-1/caspase-9 complex known as apoptosome. This apoptosome subsequently activates caspases-3 and -7 (4, 5). In other organisms, such as Caenorhabditis elegans or Drosophila melanogaster, however, Cc is not essential for the assembly and activation of the apoptosome (6) despite the presence of proteins homologous to Apaf-1—cell death abnormality-4 (CED-4) in C. elegans and Drosophila Apaf-1-related killer (Dark) in D. melanogaster—which have been found to be essential for caspase cascade activation. Furthermore, other organisms such as Arabidopsis thaliana lack Apaf-1 (7). In fact, only highly distant caspase homologues (metacaspases) (8, 9), serine proteases (saspases) (10), phytaspases (11) and VEIDases (12–14) with caspase-like activity have been detected in plants; however, their targets remain veiled and whether they are activated by Cc remains unclear.Intriguingly, the release of Cc from mitochondria into the cytoplasm during the onset of PCD is an evolutionarily conserved event found in organisms ranging from yeast (15) and plants (16) to flies (17), and mammals (18). However, understanding of the roles of this phenomenon in different species can be said to be uneven at best. In fact, the release of Cc from mitochondria has thus far been considered a random event in all organisms, save mammals. Thus, the participation of Cc in the onset and progression of PCD needs to be further elucidated.Even in the case of mammals, the role(s) of Cc in the cytoplasm during PCD remain(s) controversial. Recently, new putative functions of Cc, going beyond the already-established apoptosome assembly process, have been proposed in the nucleus (19, 20) and the endoplasmic reticulum (21–23). Neither these newly proposed functions nor other arising functions, such as oxidative stress (24), are as yet fully understood. This current state of affairs demands deeper exploration of the additional roles played by Cc in nonmammalian species.In this study, putative novel Cc-partners involved in plant PCD were identified. For this identification, a proteomic approach was employed based on affinity chromatography and using Cc as bait. The Cc-interacting proteins were identified using nano-liquid chromatography tandem mass spectrometry (NanoLC-MS/MS). These Cc-partners were then further confirmed in vivo through bimolecular fluorescence complementation (BiFC) in A. thaliana protoplasts and human HEK293T cells, as a heterologous system. Finally, the Cc-GLY2, Cc-NRP1 and Cc-TCL interactions were corroborated in vitro using surface plasmon resonance (SPR).These results indicate that Cc is able to interact with targets in the plant cell cytoplasm during PCD. Moreover, they provide new ways of understanding why Cc release is an evolutionarily well-conserved event, and allow us to propose Cc as a signaling messenger, which somehow controls different essential events during PCD.     

Chestnut cultivar diversification process in the Iberian Peninsula, Canary Islands, and Azores

This is a large-scale molecular study based on simple sequence repeat (SSR) loci of the diversification process in chestnut cultivars from Portugal and Spain, from the northern Iberian Peninsula to the Canary Islands and the Azores. A total of 593 grafted chestnut trees (Castanea sativa Mill.) were analysed with 10 SSRs: 292 from Portugal and 301 from Spain. Some of the trees studied were more than 300 years old. Accessions were analysed using a model-based Bayesian procedure to assess the geographical structure and to assign individuals to reconstructed populations based on the SSR genotypes. We found 356 different genotypes with a mean value of clonality of 33% owing to grafting. Mutations accounted for 6%, with hybridization being the main diversification process that can explain the great diversity found. Ten main cultivar groups were detected: four in northern Spain, five in the centre of the Iberian Peninsula, and one in southern Spain related to the centre of the Iberian Peninsula. This work demonstrated that cultivar origin and the diversification process was a combination of clonal propagation of selected seedlings, hybridization, and mutations, which allowed high levels of diversity to be maintained with respect to selected clones for fruit production. Furthermore, seedlings and graft sticks facilitated the transport to new destinations in the colonization process, transporting sometimes more than 3000 km if we consider the Azores and the Canary Islands.     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

Isolation and characterisation of nuclear mutants with enhanced mitochondrial mutability in the fission yeast Schizosaccharomyces pombe

In this paper we report the isolation and preliminary characterisation of nuclear mutants with increased mitochondrial mutability in fission yeast. Screening of about 2000 clones after nitrosoguanidine mutagenesis led to the isolation of ten mutator mutants. For one of them (mut-1) we show that the mutation is chromosomally encoded. The activity of the mutator is restricted to the mitochondrial genome, since it increases the mutation rate to mitochondrially encoded drug resistance considerably, whereas the mutability of nuclear genes is not altered.     

Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.     

One year of exercise training promotes distinct adaptations in right and left ventricle of female Sprague-Dawley rats

Journal of Physiology and Biochemistry - Aerobic exercise training induces a unique cardioprotective phenotype, but it is becoming clear that it does not promote the same structural, functional,...     

Diurnal patterns in Scots pine stem oleoresin pressure in a boreal forest

Coniferous tree stems contain large amounts of oleoresin under positive pressure in the resin ducts. Studies in North‐American pines indicated that the stem oleoresin exudation pressure (OEP) correlates negatively with transpiration rate and soil water content. However, it is not known how the OEP changes affect the emissions of volatile vapours from the trees. We measured the OEP, xylem diameter changes indicating changes in xylem water potential and monoterpene emissions under field conditions in mature Scots pine (Pinus sylvestris L.) trees in southern Finland. Contrary to earlier reports, the diurnal OEP changes were positively correlated with temperature and transpiration rate. OEP was lowest at the top part of the stem, where water potentials were also more negative, and often closely linked to ambient temperature and stem monoterpene emissions. However, occasionally OEP was affected by sudden changes in vapour pressure deficit (VPD), indicating the importance of xylem water potential on OEP as well. We conclude that the oleoresin storage pools in tree stems are in a dynamic relationship with ambient temperature and xylem water potential, and that the canopy monoterpene emission rates may therefore be also regulated by whole tree processes and not only by the conditions prevailing in the upper canopy.     

Mutations in PNKP Cause Recessive Ataxia with Oculomotor Apraxia Type 4

Hereditary autosomal-recessive cerebellar ataxias are a genetically and clinically heterogeneous group of disorders. We used homozygosity mapping and exome sequencing to study a cohort of nine Portuguese families who were identified during a nationwide, population-based, systematic survey as displaying a consistent phenotype of recessive ataxia with oculomotor apraxia (AOA). The integration of data from these analyses led to the identification of the same homozygous PNKP (polynucleotide kinase 3′-phosphatase) mutation, c.1123G>T (p.Gly375Trp), in three of the studied families. When analyzing this particular gene in the exome sequencing data from the remaining cohort, we identified homozygous or compound-heterozygous mutations in five other families. PNKP is a dual-function enzyme with a key role in different pathways of DNA-damage repair. Mutations in this gene have previously been associated with an autosomal-recessive syndrome characterized by microcephaly; early-onset, intractable seizures; and developmental delay (MCSZ). The finding of PNKP mutations associated with recessive AOA extends the phenotype associated with this gene and identifies a fourth locus that causes AOA. These data confirm that MCSZ and some forms of ataxia share etiological features, most likely reflecting the role of PNKP in DNA-repair mechanisms.