Enhanced ammonia detoxification to urea in hepatocytes transduced with human aquaporin-8 gene.

Hepatic ammonia detoxification to urea is critical for the prevention of hyperammonemia and neurological damage. Hepatocyte mitochondrial aquaporin-8 (AQP8) channels have been involved in ammonia-derived ureagenesis. Herein, we studied whether the adenoviral gene transfer of human AQP8 (hAQP8) to hepatocyte mitochondria enhances ammonia conversion to urea. Using primary cultured rat hepatocytes, we first confirmed the mitochondrial expression of hAQP8 and then, using unlabeled or 15 N-labeled ammonia, we demonstrated that the urea synthesis was significantly enhanced in hAQP8-transduced hepatocytes. Studies using isolated hAQP8-expressing mitochondria also showed an increased ammonia metabolism. hAQP8 transduction was able to recover the impaired ammonia-derived ureagenesis in hepatotoxin-treated hepatocytes. Our data suggest that mitochondrially-expressed hAQP8 enhances and improves hepatocyte ammonia conversion to urea, a finding with potential therapeutic implications for liver disease with impaired ammonia detoxification.

Peach Fruit Development: A Comparative Proteomic Study Between Endocarp and Mesocarp at Very Early Stages Underpins the Main Differential Biochemical Processes Between These Tissues.

Peach (Prunus persica) is an important economically temperate fruit. The development follows double sigmoid curve with four phases (S1-S4). We centered our work in the early development. In addition to S1, we studied the very early stage (E) characterized by the lag zone of the exponential growing phase S1, and the second stage (S2) when the pit starts hardening. "Dixiland" peach fruit were collected at 9 (E), 29 (S1), and 53 (S2) days after flowering (DAF) and endocarp and mesocarp were separated. There was a pronounced decrease in total protein content along development in both tissues. Quantitative proteomic allowed the identification of changes in protein profiles across development and revealed the main biochemical pathways sustaining tissue differentiation. Protein metabolism was the category most represented among differentially proteins in all tissues and stages. The decrease in protein synthesis machinery observed during development would be responsible of the protein fall, rather than a proteolytic process; and reduced protein synthesis during early development would reroute cell resources to lignin biosynthesis. These changes were accompanied by net decrease in total amino acids in E1-S1 and increase in S1-S2 transitions. Amino acid profiling, showed Asn parallels this trend. Concerted changes in Asn and in enzymes involved in its metabolism reveal that increased synthesis and decreased catabolism of Asn may conduct to an Asn increase during very early development and that the ß-Cyano-Alanine synthase/ß-Cyano-Alanine hydratase could be the pathway for Asn synthesis in "Dixiland" peach fruit. Additionally, photosynthetic machinery decays during early development in mesocarp and endocarp. Proteins related to photosynthesis are found to a higher extent in mesocarp than in endocarp. We conclude mesocarpic photosynthesis is possible to occur early on the development, first providing both carbon and reductive power and latter only reductive power. Together with proteomic, histological tests and anatomical analysis help to provide information about changes and differences in cells and cell-walls in both tissues. Collectively, this work represents the first approach in building protein databases during peach fruit development focusing on endocarp and mesocarp tissues and provides novel insights into the biology of peach fruit development preceding pit hardening.

Improved water use efficiency and shorter life cycle of Nicotiana tabacum due to modification of guard and vascular companion cells.

Severe droughts are predicted for the twenty-first century, which contrast with the increased demand for plant materials. Thus, to sustain future generations, a great challenge is to improve crop yield and water use efficiency (WUE), which is the carbon gained per water lost. Here, expression of maize NADP-malic enzyme (NADP-ME) in the guard and vascular companion cells of Nicotiana tabacum results in enhanced WUE, earlier flowering and shorter life cycle. Transgenic lines exhibit reduced stomatal aperture than wild-type (WT). Nevertheless, an increased net CO2 fixation rate is observed, which results in less water consumption and more biomass production per water used. Transgenic lines export sugars to the phloem at higher rate than WT, which leads to higher sugars levels in phloem exudates and veins. Leaf quantitative proteomic profiling revealed drastic differences in proteins related to cell cycle, flowering, hormone signaling and carbon metabolism between transgenic lines and WT. We propose that the increased sugar export from leaves in the transgenic lines alleviates sugar negative feedback on photosynthesis and thus, stomatal closure takes place without a penalty in CO2 assimilation rate. This results in improved WUE and accelerated overall life cycle, key traits for plant productivity in the near future world.

Integration of Temperature and Blue-Light Sensing in Acinetobacter baumannii Through the BlsA Sensor.

BlsA is a BLUF photoreceptor present in Acinetobacter baumannii, responsible for modulation of motility, biofilm formation and virulence by light. In this work, we have combined physiological and biophysical evidences to begin to understand the basis of the differential photoregulation observed as a function of temperature. Indeed, we show that blsA expression is reduced at 37°C, which correlates with negligible photoreceptor levels in the cells, likely accounting for absence of photoregulation at this temperature. Another point of control occurs on the functionality of the BlsA photocycle itself at different temperatures, which occurs with an average quantum yield of photoactivation of the signaling state of 0.20 ± 0.03 at 15°C < T < 25°C, but is practically inoperative at T > 30°C, as a result of conformational changes produced in the nanocavity of FAD. This effect would be important when the photoreceptor is already present in the cell to avoid almost instantaneously further signaling process when it is no longer necessary, for example under circumstances of temperature changes possibly faced by the bacteria. This complex interplay between light and temperature would provide the bacteria clues of environmental location and dictate/modulate light photosensing in A. baumannii.

Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii.

Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle.IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur.

Unravelling early events in the Taphrina deformans-Prunus persica interaction: an insight into the differential responses in resistant and susceptible genotypes.

Leaf peach curl is a devastating disease affecting leaves, flowers and fruits, caused by the dimorphic fungus Taphrina deformans. To gain insight into the mechanisms of fungus pathogenesis and plant responses, leaves of a resistant and two susceptible Prunus persica genotypes were inoculated with blastospores (yeast), and the infection was monitored during 120 h post inoculation (h.p.i.). Fungal dimorphism to the filamentous form and induction of reactive oxygen species (ROS), callose synthesis, cell death and defence compound production were observed independently of the genotype. Fungal load significantly decreased after 120 h.p.i. in the resistant genotype, while the pathogen tended to grow in the susceptible genotypes. Metabolic profiling revealed a biphasic re-programming of plant tissue in susceptible genotypes, with an initial stage co-incident with the yeast form of the fungus and a second when the hypha is developed. Transcriptional analysis of PRs and plant hormone-related genes indicated that pathogenesis-related (PR) proteins are involved in P. persica defence responses against T. deformans and that salicylic acid is induced in the resistant genotype. Conducted experiments allowed the elucidation of common and differential responses in susceptible versus resistant genotypes and thus allow us to construct a picture of early events during T. deformans infection.

Maize cytosolic NADP-malic enzyme (ZmCytNADP-ME): a phylogenetically distant isoform specifically expressed in embryo and emerging roots.

Two maize plastidic NADP-malic enzyme isoforms have been characterized: the bundle sheath-located photosynthetic isoform (ZmC(4)-NADP-ME) and a constitutively expressed one (Zm-nonC(4)-NADP-ME). In this work, the characterization of the first maize cytosolic NADP-ME (ZmCytNADP-ME) is presented, which transcript is exclusively found in embryo and emerging roots. ZmCytNADP-ME expression in roots decreases with development, while Zm-nonC ( 4 ) -NADP-ME increases concomitantly. On the other hand, ZmCytNADP-ME accumulation is differentially modulated by several stress conditions and shows coordination with that of Zm-nonC ( 4 ) -NADP-ME in maize young roots. Recombinant ZmCytNADP-ME displays clearly distinct kinetic parameters and metabolic regulation than the plastidic isoforms. The particular properties and the specific-expression pattern of this novel isoform suggest that it may be involved in the control of cytosolic malate levels in emerging roots, e.g. during hypoxia. ZmCytNADP-ME is phylogenetically related to other cytosolic mono and dicot NADP-MEs, and data indicate that it belongs to an ancestral unique group among plant NADP-MEs.

NADP-malic enzyme and Hsp70: co-purification of both proteins and modification of NADP-malic enzyme properties by association with Hsp70.

Different preparations of antibodies against 62 kDa NADP-malic enzyme (NADP-ME) from purified maize leaves cross-react with a 72 kDa protein from diverse tissues in many species. A 72 kDa protein, suggested to be a non-photosynthetic NADP-ME, has been purified from several plant species. However, to date, a cDNA coding for this putative 72 kDa NADP-ME has not been isolated. The screening of maize and tobacco leaf expression libraries using antibodies against purified 62 kDa NADP-ME allowed the identification of a heat shock protein (Hsp70). In addition, tandem mass spectrometry (MS/MS) studies indicate that along with NADP-ME, a 72 kDa protein, identified as an Hsp70 and reacting with the antibodies, is also purified from maize roots. On the other hand, the screening of a maize root cDNA library revealed the existence of a cDNA that encodes a mature 66 kDa NADP-ME. These results suggest that the 72 kDa protein is not actually an NADP-ME but in fact an Hsp70, at least in maize and tobacco. Probably, NADP-ME-Hsp70 association, taking place at least when preparing crude extracts, can lead to a co-purification of the proteins and can thus explain the cross-reaction of the antibodies. In the present work, we analyse and discuss a probable interaction of NADP-ME with Hsp70.