The oxidized form of vitamin C,dehydroascorbic acid,regulates neuronal energy metabolism

Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose‐6‐phosphate dehydrogenase, the rate‐limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time‐ and dose‐dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.

     

Physiological characteristics underpinning successful cryopreservation of endemic and endangered species of Bromeliaceae from the Brazilian Atlantic Forest

The germination requirements and the basis of the optimal water content before and after cryopreservation were studied for ten endangered Brazilian species of Bromeliaceae. Constant and alternating temperature regimes were used to determine the best conditions for seed germination. The relationship between seed water content and relative humidity was evaluated using water sorption isotherms at 15 °C. Seeds were cryostored at four water contents (3, 5, 7 and 9%) and three storage periods (0, 180 and 365 days), and loss in viability and vigour were estimated. Fresh seeds of all species showed maximum germination in < 30 days at temperatures between 20 and 30 °C, indicating the absence of a physical/morphological dormancy. A sigmoidal relationship between seed water content and relative humidity was observed with no apparent differences in sorption characteristics among the species. The optimum water content for cryopreservation of most of these species was c. 7%. Ultra‐drying (3% seed water content) had a detrimental effect on seed viability and vigour. Our experiments suggested orthodox storage behaviour for all species of Bromeliaceae examined as they are able to survive desiccation and freezing. This study has shown the feasibility of ex situ conservation in seed cryobanks of endangered bromeliads from the Brazilian Atlantic Forest to support future reintroduction of these species in nature. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 567–576.     

Temporal variation in microbial and plant biomass during summer in a Mediterranean high-mountain dry grassland

Aims

We assessed the temporal changes on microbial biomass in relation to changes in soil moisture, dissolved organic carbon and plant biomass during the summer season in a Mediterranean high-mountain grassland.

Methods

Temporal variations were tested by two-way ANOVA. The relationships among microbial biomass, plant biomass, soil water content, soil organic carbon, dissolved organic carbon and total soil nitrogen during the summer season were assessed by means of structural equation modeling.

Results

Microbial biomass did not show variation, while dissolved organic carbon and root biomass decreased throughout the summer. Aboveground plant biomass peaked in the middle of the summer, when soil water content was at its minimum. Soil water content directly and negatively affected soil microbial biomass, and positively affected dissolved organic carbon. Moreover soil microbial biomass and dissolved organic carbon were negatively related. Plant biomass effects on soil microbial biomass were driven by root biomass, which indirectly affected soil microbial biomass through effects on soil organic carbon and soil nitrogen.

Conclusions

The temporal dynamic of microbial biomass during the summer season appeared to differ from previous observations in temperate alpine communities, and indicated the drought resistance of the microbial community during the summer in Mediterranean high-mountain grasslands. During the dry period, microbial biomass may play an alternative role in soil carbon conservation.     

Geranyllinalool Synthases in Solanaceae and Other Angiosperms Constitute an Ancient Branch of Diterpene Synthases Involved in the Synthesis of Defensive Compounds

Many angiosperm plants, including basal dicots, eudicots, and monocots, emit (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, which is derived from geranyllinalool, in response to biotic challenge. An Arabidopsis (Arabidopsis thaliana) geranyllinalool synthase (GLS) belonging to the e/f clade of the terpene synthase (TPS) family and two Fabaceae GLSs that belong to the TPS-g clade have been reported, making it unclear which is the main route to geranyllinalool in plants. We characterized a tomato (Solanum lycopersicum) TPS-e/f gene, TPS46, encoding GLS (SlGLS) and its homolog (NaGLS) from Nicotiana attenuata. The K_m value of SlGLS for geranylgeranyl diphosphate was 18.7 µm, with a turnover rate value of 6.85 s^–1. In leaves and flowers of N. attenuata, which constitutively synthesize 17-hydroxygeranyllinalool glycosides, NaGLS is expressed constitutively, but the gene can be induced in leaves with methyl jasmonate. In tomato, SlGLS is not expressed in any tissue under normal growth but is induced in leaves by alamethicin and methyl jasmonate treatments. SlGLS, NaGLS, AtGLSs, and several other GLSs characterized only in vitro come from four different eudicot families and constitute a separate branch of the TPS-e/f clade that diverged from kaurene synthases, also in the TPS-e/f clade, before the gymnosperm-angiosperm split. The early divergence of this branch and the GLS activity of genes in this branch in diverse eudicot families suggest that GLS activity encoded by these genes predates the angiosperm-gymnosperm split. However, although a TPS sequence belonging to this GLS lineage was recently reported from a basal dicot, no representative sequences have yet been found in monocot or nonangiospermous plants.Geranyllinalool is an acyclic diterpene alcohol with a wide distribution in the plant kingdom; it has been identified as component of essential oils of distantly related plant species such as Jasmin grandiflorum, Michelia champaca, and Homamelis virginiana (Sandeep, 2009). Geranyllinalool is the precursor of 4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), a volatile C₁₆-homoterpene emitted from the foliage of many angiosperm species including Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), maize (Zea mays), fava bean (Vicia faba), lima bean (Phaseolus lunatus), alfalfa (Medicago sativa), and Eucalyptus spp. (Van Poecke et al., 2001; Ament et al., 2004; Williams et al., 2005; Hopke et al., 1994; Leitner et al., 2010; Webster et al., 2010). In addition, various hydroxygeranyllinalool glycosides have been isolated from many Solanaceous species such as Capsicum annuum, Lycium chinense, and at least 26 Nicotiana species (Yahara et al., 1993; Iorizzi et al., 2001; Snook et al., 1997).The biosynthetic pathway leading to geranyllinalool, as for all other terpenoids, begins with the condensation of isopentenyl diphosphate and its allylic isomer, dimethylallyl diphosphate. Sequential condensation of one isopentenyl diphosphate molecule with three dimethylallyl diphosphate molecules produces geranylgeranyl diphosphate (GGPP), the C-20 intermediate of the diterpenoid pathway. Next, a terpene synthase (TPS) catalyzes a two-step reaction in which carbocation formation of the C20 precursor is followed by an allylic rearrangement that results in the production of the tertiary alcohol geranyllinalool (Herde et al., 2008).Although geranyllinalool and its derivatives, TMTT and geranyllinalool glycosides, have been reported in a wide variety of plant species, a geranyllinalool synthase (GLS) involved in TMTT biosynthesis was only recently identified in Arabidopsis (Herde et al., 2008). AtTPS04 belongs to the TPS-e/f subfamily along with the previously identified Clarkia spp. linalool synthases (Chen et al., 2011). More recently, two TPSs from Vitis vinifera and one from the daisy Grindelia hirsutula, also members of the TPS-e/f subfamily, were found to exhibit GLS activity in vitro (Martin et al., 2010; Zerbe et al., 2013). However, no in planta information has been presented for these, nor any evidence showing their involvement in TMTT biosynthesis.The common characteristic of the TPS-e/f GLSs so far identified is that they lack a predicted plastidial transit peptide, and direct evidence for nonplastidial localization was obtained in Arabidopsis by observing the AtTPS04-GUS fusion protein in the cytosol and endoplasmic reticulum (Herde et al., 2008). On the other hand, two TPS-g subfamily proteins from the closely related Fabaceae species Medicago truncatula and Phaseolus lunata (MtTPS03 and PlTPS2, respectively) were shown to be plastidic and to catalyze the formation of geranyllinalool in vitro when GGPP was provided as a substrate and also when expressed in a heterologous plant species (Arimura et al., 2008; Brillada et al., 2013). However, the same enzymes also produced linalool and nerolidol when supplied with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), respectively (Arimura et al., 2008; Brillada et al., 2013). Given the present paucity of in vivo and in vitro studies of geranyllinalool biosynthesis in plants, it is not clear whether geranyllinalool in plants is typically produced via TPS-g or TPS-e/f type TPSs, or both.The role of geranyllinalool itself in plant tissues is not well established. Often geranyllinalool coexists in floral or vegetative tissues with its homoterpene derivative TMTT. The contribution of TMTT to the floral scent of insect-pollinated species suggests a putative role in attraction of pollinators (Tholl et al., 2011). On the other hand, in many angiosperm species, including tomato, TMTT is a component of volatile blends released from vegetative tissues upon herbivore attack, sometimes in parallel with its constitutive emission from floral tissues (Hopke et al., 1994; Ament et al., 2004; de Boer et al., 2004; Kant et al., 2004; Williams et al., 2005, Herde et al., 2008). The latter case suggests that TMTT might play a defensive role in both vegetative and floral tissues. TMTT production from insect-infested plants is considered as an indirect defense mechanism because TMTT attracts insect predators of the insect herbivores (Brillada et al., 2013). Interestingly, production of TMTT, and the homoterpene (E)-4,8-dimethyl-1,3,7-nonatriene, from herbivore-attacked lima bean plants has been found to correlate with enhanced expression of defense genes in neighboring nonaffected control plants (Arimura et al., 2000). In these cases, homoterpenes are believed to act as stress-responsive signals that enable intraspecies plant-to-plant communication.A plant defense role has also been suggested for 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs) present in leaves and flowers of Nicotiana species, with higher concentrations measured in buds (Heiling et al., 2010; Jassbi et al., 2010). Several studies have found negative correlation between total leaf HGL-DTG content and the mass of the larvae that feed on them (Jassbi et al., 2008; Dinh et al., 2013). Eleven HGL-DTGs that differ in sugar moieties and number of malonylesters have been isolated from Nicotiana attenuata. The sugar groups of these compounds are Glc and rhamnose and are conjugated to the hydroxygeranyllinalool skeleton via bonds at C3 and C17 hydroxylated carbons. Additional sugars may be added to these sugars on their hydroxyl groups at C2, C4, and C6, and manolyl esters are typically formed at the C6 hydroxyl group of the glucoses. The concentrations of these HGL-DTGs are higher in young and reproductive tissues. While their total levels appear to be constant, the concentration of individual compounds change upon herbivore attack, with a proportionally greater increase in malonylated compounds. Unlike many other defense-related specialized metabolites, the N. attenuata HGL-DTGs are not found on the leaf surface or the trichomes, but, instead, they accumulate inside the leaves (Heiling et al., 2010).Here, we show that in the Solanaceae species cultivated tomato and N. attenuata, geranyllinalool is synthesized by TPSs that belong to the TPS-e/f subfamily and that the corresponding genes are related to Arabidopsis TPS04. The tomato and N. attenuata enzymes were biochemically characterized, and the kinetic parameters were determined. We also describe a detailed quantitative expression of these genes in different parts of the plant. In addition, we establish that the expression of the geranyllinalool synthase genes correlates well with the induced emission of TMTT in tomato leaves after alamethicin and methyl jasmonate (MeJA) treatments and with the total concentrations of HGL-DTGs in N. attenuata leaves and floral organs.     

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.     

Successful laparoscopic insemination with a very low number of flow cytometrically sorted boar sperm in field conditions

The aim of this study was to develop a useful procedure for laparoscopic insemination (LI) with sex-sorted boar spermatozoa that yields adequate fertility results in farm conditions. In experiment 1, we evaluated the effects of single (oviducts) and double (oviducts and tips of the uterine horns) LI with X-sorted sperm on the reproductive performance of sows. Sows (N = 109) were inseminated once as follows: (1) single LI with 0.5 × 10⁶ unsorted sperm per oviduct; (2) single LI with 0.5 × 10⁶ sex-sorted sperm per oviduct; or (3) double LI with 0.5 × 10⁶ sex-sorted sperm per oviduct and 0.5 × 10⁶ sex-sorted sperm per uterine horn. The farrowing rates were lower (P < 0.05) in sows inseminated with sex-sorted sperm (43.2% and 61.9% for the single and double insemination groups, respectively) than in sows from the unsorted group (91.3%). Within the sex-sorted groups, the farrowing rate tended (P = 0.09) to be greater in sows inseminated using double LI. There were no differences in the litter size among groups. In experiment 2, we evaluated the effect of the number of sex-sorted sperm on the reproductive performance of sows when using double LI. Sows (N = 109) were inseminated with sex-sorted sperm once using double LI with: (1) 0.5 × 10⁶ sperm per oviduct and 1 × 10⁶ sperm per uterine horn; or (2) 1 × 10⁶ sperm per oviduct and 2 × 10⁶ sperm per uterine horn. Similarly high pregnancy (90%) and farrowing (80%) rates were achieved in both groups. The sows inseminated with the highest number of sperm tended (P = 0.09) to have more piglets (10.8 ± 0.7 vs. 9.2 ± 0.6). A high female proportion (number of female births divided by the total of all births ≥0.92) was obtained in both experiments using X-sorted sperm. Our results indicate that the double LI procedure, using between 3 and 6 × 10⁶ sex-sorted sperm per sow produces adequate fertility at the farm level, making sperm-sexing technology potentially applicable in elite breeding units.     

Transferable Denitrification Capability of Thermus thermophilus

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8. In contrast, many isolates of this species that have not been intensively grown under laboratory conditions keep the capability to grow anaerobically with one or more electron acceptors. The use of nitrogen oxides, especially nitrate, as electron acceptors is one of the most widespread capabilities among these facultative strains. In this process, nitrate is reduced to nitrite by a reductase (Nar) that also functions as electron transporter toward nitrite and nitric oxide reductases when nitrate is scarce, effectively replacing respiratory complex III. In many T. thermophilus denitrificant strains, most electrons for Nar are provided by a new class of NADH dehydrogenase (Nrc). The ability to reduce nitrite to NO and subsequently to N₂O by the corresponding Nir and Nor reductases is also strain specific. The genes encoding the capabilities for nitrate (nar) and nitrite (nir and nor) respiration are easily transferred between T. thermophilus strains by natural competence or by a conjugation-like process and may be easily lost upon continuous growth under aerobic conditions. The reason for this instability is apparently related to the fact that these metabolic capabilities are encoded in gene cluster islands, which are delimited by insertion sequences and integrated within highly variable regions of easily transferable extrachromosomal elements. Together with the chromosomal genes, these plasmid-associated genetic islands constitute the extended pangenome of T. thermophilus that provides this species with an enhanced capability to adapt to changing environments.     

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to “ribosomal stress” with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis.     

Coadministration of Branched-Chain Amino Acids and Lipopolysaccharide Causes Matrix Metalloproteinase Activation and Blood–Brain Barrier Breakdown

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a severe deficiency in the activity of the branched-chain α-keto acid dehydrogenase complex, leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine. Infections have a significant role in precipitating acute metabolic decompensation in patients with MSUD; however, the mechanisms underlying the neurotoxicity in this disorder are poorly understood. In this study, we subjected rats to the coadministration of lipopolysaccharide (LPS), which is a major component of gram-negative bacteria cell walls, and high concentrations of BCAA (H-BCAA) to determine their effects on the permeability of the blood–brain barrier (BBB) and on the levels of matrix metalloproteinases (MMP-2 and MMP-9). Our results demonstrated that the coadministration of H-BCAA and LPS causes breakdown of the BBB and increases the levels of MMP-2 and MMP-9 in the hippocampus of these rats. On the other hand, examination of the cerebral cortex of the 10- and 30-day-old rats revealed a significant difference in Evan’s Blue content after coadministration of H-BCAA and LPS, as MMP-9 levels only increased in the cerebral cortex of the 10-day-old rats. In conclusion, these results suggest that the inflammatory process associated with high levels of BCAA causes BBB breakdown. Thus, we suggest that BBB breakdown is relevant to the perpetuation of brain inflammation and may be related to the brain dysfunction observed in MSUD patients.