Protein Phosphatases 2C Regulate the Activation of the Snf1-Related Kinase OST1 by Abscisic Acid in Arabidopsis

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.     

Development of an engineered bioluminescent reporter phage for detection of bacterial blight of crucifers

Bacterial blight, caused by the phytopathogen Pseudomonas cannabina pv. alisalensis, is an emerging disease afflicting important members of the Brassicaceae family. The disease is often misdiagnosed as pepper spot, a much less severe disease caused by the related pathogen Pseudomonas syringae pv. maculicola. We have developed a phage-based diagnostic that can both identify and detect the causative agent of bacterial blight and differentiate the two pathogens. A recombinant "light"-tagged reporter phage was generated by integrating bacterial luxAB genes encoding luciferase into the genome of P. cannabina pv. alisalensis phage PBSPCA1. The PBSPCA1::luxAB reporter phage is viable and stable and retains properties similar to those of the wild-type phage. PBSPCA1::luxAB rapidly and sensitively detects P. cannabina pv. alisalensis by conferring a bioluminescent signal response to cultured cells. Detection is dependent on cell viability. Other bacterial pathogens of Brassica species such as P. syringae pv. maculicola, Pseudomonas marginalis, Pectobacterium carotovorum, Xanthomonas campestris pv. campestris, and X. campestris pv. raphani either do not produce a response or produce significantly attenuated signals with the reporter phage. Importantly, the reporter phage detects P. cannabina pv. alisalensis on diseased plant specimens, indicating its potential for disease diagnosis.     

Gender Differences in Survival among Adult Patients Starting Antiretroviral Therapy in South Africa: A Multicentre Cohort Study

Background

Increased mortality among men on antiretroviral therapy (ART) has been documented but remains poorly understood. We examined the magnitude of and risk factors for gender differences in mortality on ART.

Methods and Findings

Analyses included 46,201 ART-naïve adults starting ART between January 2002 and December 2009 in eight ART programmes across South Africa (SA). Patients were followed from initiation of ART to outcome or analysis closure. The primary outcome was mortality; secondary outcomes were loss to follow-up (LTF), virologic suppression, and CD4+ cell count responses. Survival analyses were used to examine the hazard of death on ART by gender. Sensitivity analyses were limited to patients who were virologically suppressed and patients whose CD4+ cell count reached >200 cells/µl. We compared gender differences in mortality among HIV+ patients on ART with mortality in an age-standardised HIV-negative population.Among 46,201 adults (65% female, median age 35 years), during 77,578 person-years of follow-up, men had lower median CD4+ cell counts than women (85 versus 110 cells/µl, p<0.001), were more likely to be classified WHO stage III/IV (86 versus 77%, p<0.001), and had higher mortality in crude (8.5 versus 5.7 deaths/100 person-years, p<0.001) and adjusted analyses (adjusted hazard ratio [AHR] 1.31, 95% CI 1.22–1.41). After 36 months on ART, men were more likely than women to be truly LTF (AHR 1.20, 95% CI 1.12–1.28) but not to die after LTF (AHR 1.04, 95% CI 0.86–1.25). Findings were consistent across all eight programmes. Virologic suppression was similar by gender; women had slightly better immunologic responses than men. Notably, the observed gender differences in mortality on ART were smaller than gender differences in age-standardised death rates in the HIV-negative South African population. Over time, non-HIV mortality appeared to account for an increasing proportion of observed mortality. The analysis was limited by missing data on baseline HIV disease characteristics, and we did not observe directly mortality in HIV-negative populations where the participating cohorts were located.

Conclusions

HIV-infected men have higher mortality on ART than women in South African programmes, but these differences are only partly explained by more advanced HIV disease at the time of ART initiation, differential LTF and subsequent mortality, and differences in responses to treatment. The observed differences in mortality on ART may be best explained by background differences in mortality between men and women in the South African population unrelated to the HIV/AIDS epidemic. Please see later in the article for the Editors'' Summary.     

Immunohistochemical evidence of the presence of aromatase P450 in the rat hypophysis

In order to analyze whether aromatase is present in the hypophysis of adult rats, we have performed an immunohistochemical study in young adult male and female rats. Our study has revealed that the hypophysis of adult rats contains aromatase, although marked differences are found between the sexes. The hypophyses of male rats have cells immunoreactive for the enzyme, 34.40% of these hypophyseal cells showing reaction. By contrast, cells from female rats show very little reaction, only 0.84% of them being reactive. No significant differences in the percentage of immunoreactive cells between one phase and another are observed during the estrous cycle. Our results point to the immunohistochemical expression of aromatase in the hypophysis of adult rats and at the same time suggest that its expression is sex-dependent. The enzyme may therefore be involved in the regulation of adenohypophyseal cytology by androgens.     

Soil CO2 efflux in a mixed pine-oak forest in Valsaín (central Spain)

Soil-surface CO2 efflux and its spatial and temporal variation were investigated in a southern Mediterranean, mixed pine-oak forest ecosystem on the northern slopes of the Sierra de Guadarrama in Spain from February 2006 to July 2006. Measurements of soil CO2 efflux, soil temperatures, and moisture were conducted in nine 1963-m2 sampling plots distributed in a gradient around the ecotone between Pinus sylvestris L. and Quercus pyrenaica Lam. forest stands. Total soil organic matter, Walkey-Black C, particulate organic matter, organic matter fraction below 53 microm, total soil nitrogen content, total soil organic carbon content, and pH were also measured under three representative mature oak, pine, and mixed pine-oak forest stands. Soil respiration showed a typical seasonal pattern with minimums in winter and summer, and maximums in spring, more pronounced in oak and oak-pine stands. Soil respiration values were highest in pine stands during winter and in oak stands during spring and summer. Soil respiration was highly correlated with soil temperatures in oak and pine-oak stands when soil moisture was above a drought threshold of 15%. Below this threshold value, soil moisture was a good predictor of soil respiration in pine stands. Greater soil organic matter, particulate organic matter, Walkey-Black C, total organic C, and total N content in pine compared to oak sites potentially contributed to the greater total soil CO2 efflux in these stands during the winter. Furthermore, opposing trends in the organic matter fraction below 53 microm and soil respiration between plots suggest that in oak stands, the C forms are less affected by possible changes in use. The effects of soil properties on soil respiration were masked by differences in soil temperature and moisture during the rest of the year. Understanding the spatial and temporal variation even within small geographic areas is essential to assess C budgets at ecosystem level accurately. Thus, this study bears important implications for the study of large-scale ecosystem dynamics, particularly in response to climatic change.     

Field patterns of leaf plasticity in adults of the long-lived evergreen Quercus coccifera

BACKGROUND AND AIMS: Quercus coccifera, as a long-lived sprouter, responds plastically to environmental variation. In this study, the role of foliar plasticity as a mechanism of habitat selection and modification within the canopy and across contrasted habitats was characterized. An examination was made of the differential contribution of inner and outer canopy layers to the crown plasticity expressed in the field by adult individuals and its dependence on environmental and genetic factors. METHODS: Within-crown variation in eight foliar traits was examined in nine populations dominated by Q. coccifera. The difference between mean trait values at the inner and outer canopy layers was used as a proxy for crown plasticity to light. Correlations between geographic distances, environmental differences (climatic and edaphic) and phenotypic divergence (means and plasticities) were assessed by partial Mantel tests. A subset of field measurements was compared with data from a previous common garden experiment. KEY RESULTS: Phenotypic adjustment of sun leaves contributed significantly to the field variation in crown plasticity. Plasticity in leaf angle, lobation, xanthophyll cycle pigments and beta-carotene content was expressed in sun and shade leaves concurrently and in opposite directions. Phenotypic plasticity was more strongly correlated with environmental variation than mean trait values. Populations of taller plants with larger, thinner (higher specific leaf area) and less spiny leaves exhibited greater plasticity. In these populations, the midday light environment was more uniform at the inner than at the outer canopy layers. Field and common garden data ranked populations in the same order of plasticity. CONCLUSIONS: The expression of leaf plasticity resulted in a phenotypic differentiation that suggests a mechanism of habitat selection through division of labour across canopy layers. Signs of plasticity-mediated habitat modification were found only in the most plastic populations. Intracanopy plasticity was sensitive to environmental variation but also exhibited a strong genetic component.     

Using multiple landscape genetic approaches to test the validity of genetic clusters in a species characterized by an isolation‐by‐distance pattern

Bayesian clustering methods are typically used to identify barriers to gene flow, but they are prone to deduce artificial subdivisions in a study population characterized by an isolation‐by‐distance pattern (IbD). Here we analysed the landscape genetic structure of a population of wild boars (Sus scrofa) from south‐western Germany. Two clustering methods inferred the presence of the same genetic discontinuity. However, the population in question was characterized by a strong IbD pattern. While landscape‐resistance modelling failed to identify landscape features that influenced wild boar movement, partial Mantel tests and multiple regression of distance matrices (MRDMs) suggested that the empirically inferred clusters were separated by a genuine barrier. When simulating random lines bisecting the study area, 60% of the unique barriers represented, according to partial Mantel tests and MRDMs, significant obstacles to gene flow. By contrast, the random‐lines simulation showed that the boundaries of the inferred empirical clusters corresponded to the most important genetic discontinuity in the study area. Given the degree of habitat fragmentation separating the two empirical partitions, it is likely that the clustering programs correctly identified a barrier to gene flow. The differing results between the work published here and other studies suggest that it will be very difficult to draw general conclusions about habitat permeability in wild boar from individual studies.     

Inactivation of mitochondrial carbamoyl phosphate synthetase induced by ascorbate, oxygen, and Fe3+ in the presence of acetylglutamate: protection by ATP and HCO3- and lack of inactivation of ornithine transcarbamylase

Of the two mitochondrial enzymes of the urea cycle, carbamoyl phosphate synthetase (CPS) was and ornithine transcarbamylase (OTC) was not inactivated by the Fe3+-oxygen-ascorbate model system for mixed-function oxidation [R. L. Levine, (1983) J. Biol. Chem. 258, 11828-11833]. The susceptibility of OTC was not increased by its substrates, products, or inhibitors, whereas that of CPS was markedly increased by acetylglutamate (its allosteric activator) when ATP was absent. Thus, acetylglutamate binds in the absence of ATP and exposes to oxidation essential groups of the enzyme. We estimate for this binding a KD value of 1.6 mM, which greatly exceeds the KD values (less than 10 microM) determined in the presence of ATP and bicarbonate. ATP, and even more, mixtures of ATP and bicarbonate protected CPS from inactivation. Acetylglutamate exposes the site for the ATP molecule that yields Pi, and it appears that ATP protects by binding at this site. Experiments of limited proteolysis with elastase suggest that oxidation prevents this binding of ATP and show that it accelerates cleavage of CPS by the protease, thus supporting the idea that oxidation may precede proteolysis. Trypsin, chymotrypsin, and papain also hydrolyze the oxidized enzyme considerably faster than the native enzyme. Our results also support the idea that oxidative inactivation is site specific and requires sites on the enzyme for Me2+ and, possibly, for a nucleotide.