Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.     

Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli

DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product.     

Crucial role of intracellular effectors on glycogenolysis in the isolated rat heart: Potential consequences on the myocardial tolerance to ischemia

The role played by glycogenolysis in the ischemic heart has been recently put into question because it is suspected that a slowing down of this process could be beneficial for the tolerance of the myocardium to ischemia. The role of the intracellular effectors that control the rate of glycogenolysis has therefore regained interest. We aimed to understand the role played by those intracellular effectors which are directly related to the energy balance of the heart. To this end, we review some of the previously published data on this subject and we present new data obtained from P-31 and C-13 NMR spectroscopic measurement on isolated rat heart. Two conditions of ischemia were studied: 15 min global no-flow and 25 min low-flow ischemia. The hearts were isolated either from control animals or from rats pre-treated with isoproterenol (5 mg.kg^–1 b.w. i.p.) 1 h before the perfusion in order to C-13 label glycogen stores. Our main results are as follows: (1) the biochemically determined glycogenolysis rate during the early phase of ischemia (up to 10–15 min) was larger in no-flow ischemia than in low-flow conditions for both groups, (2) direct measurement of the glycogenolysis rate, as determined by C-13 NMR, after labelling of the glycogen pool in the hearts from isoproterenol-treated rats, confirms the estimations from the biochemical data, (3) glycogenolysis was slower in the hearts from pre-treated animals than in control hearts for both conditions of ischemia, (4) the total activity of glycogen phosphorylase (a + b) increased, by 50%, after 5 min no-flow ischemia, whereas it decreased by 42% after the same time of low-flow ischemia. However, the ratio phosphorylase a/a + b was not altered, whatever the conditions, (5) the concentration of inorganic phosphate (Pi) increased sharply during the first minutes of ischemia, to values above 8–10 mM, under all conditions studied. The rate of increase was larger during no-flow ischemia than during low-flow ischemia. The concentration of Pi was thereafter higher in controls than in the hearts from isoproterenol-treated animals.The calculated cytosolic concentration of free 5 AMP increased sharply at the onset of ischemia, reaching in a few minutes values above 30 M in controls and significantly lower values, around 15 M, in the hearts from isoproterenol-treated rats. (6) The hearts from isoproterenol-treated rats displayed a reduced intracellular acidosis, when compared to controls, under both conditions of ischemia.We conclude that the intracellular effectors, mainly free AMP, play an essential role in the control of glycogenolysis via allosteric control of phosphorylase b activity. The alteration in the concentration of free Pi, the substrate of both forms of phosphorylase, can also be considered as determinant in the control of the rate of glycogenolysis.The attenuation of ischemia-induced intracellular acidosis in the hearts from isoproterenol-treated rats could be a consequence of a reduced glycogenolytic rate and is likely to be related to a better resumption of the mechanical function on reperfusion.     

How Many Nucleotides Are Required to Resolve a Phylogenetic Problem? The Use of a New Statistical Method Applicable to Available Sequences

The evolution of bootstrap proportions (BP) with sequence length was studied using a 28S ribosomal RNA data set. For different sequence lengths, informative sites were jackknifed several times. Bootstrapping was subsequently performed on each of these subsamples. For each node, BPs so obtained were plotted against sequence length, showing the evolution of the robustness with increasing number of informative sites. For robust nodes (BP of 100%), the pattern of BPs is unvarying and is described by a simple function BP = 100(1− e^{−b(x − x′)}), where x is the number of informative sites and b and x′ are two parameters estimated using a nonlinear regression procedure. When a node has a BP <100% and the pattern of BPs fits this function, it is possible to estimate the number of informative sites required to obtain a given average BP. The method also identifies nonrobust nodes (nonascending clusters of BP dots), for which it seems to be more cost effective and fruitful to turn to other species and/or genes rather than to continue sequencing longer gene lengths from the same species to reach a BP of 95%.     

Early response of herbaceous vegetation to Rhododendron ponticum subsp. baeticum invasion in European Atlantic forests

Questions

Rhododendron ponticum subsp. baeticum is an invasive shrub of growing concern in continental Europe, but little is known about its impact on native plant communities. Here we ask: do environmental conditions differ between forest stands invaded by it and uninvaded stands? Do these differences correlate with R. ponticum's cover? Are these differences associated with differences in taxonomic and functional diversity of vascular plant species of the herb layer? Can these vegetation changes be explained by the sorting of certain life-history traits by R. ponticum-induced environmental changes?

Location

Several forests invaded by R. ponticum in the French Atlantic domain.

Methods

We recorded vegetation composition and a number of environmental variables in 400-m² plots that were established in 64 paired forest stands (32 invaded vs 32 uninvaded). We compiled traits from existing databases. We computed several metrics of taxonomic and functional diversity. We compared environmental variables and diversity metrics between invaded and uninvaded stands. We used correlation and regression analyses to relate them with R. ponticum's cover. We ran RLQ and fourth-corner analyses to explore the relationships between R. ponticum invasion, environmental variables, species traits, and vegetation composition.

Results

Independent of its abundance, R. ponticum invasion was associated with lower light arrival at the forest floor and increased litter thickness. Concomitantly, species richness and diversity and trait diversity were reduced. The major driver of species assemblages was soil pH, which strongly interacted with the invasion gradient. R. ponticum did not sort species according to traits associated with shade tolerance and thick-litter tolerance. However, tree and shrub saplings were more abundant in invaded than uninvaded stands, at the expense of graminoid and fern species.

Conclusions

As R. ponticum becomes the dominant shrub, it exerts new selection forces on life-history traits of extant species, mostly via reduced light availability, increased litter thickness, and physical competition, thereby reducing taxonomic and functional diversity of the herb layer, without impeding tree and shrub self-regeneration, at least in the short term.     

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging

Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1β and TGF-β1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii^−/−) mice. The effects of arg-ii^−/− on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii^−/− cells, activates fibroblasts to produce various cytokines including TGF-β1 and collagen, which is abolished by IL-1β receptor antagonist or TGF-β type I receptor blocker. Conversely, TGF-β1 or IL-1β also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1β and TGF-β1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii^−/− mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1β and TGF-β1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.     

Genetic variation in the Y chromosome and sex-biased DNA methylation in somatic cells in the mouse

Mammalian Genome - Several lines of evidence suggest that the presence of the Y chromosome influences DNA methylation of autosomal loci. To better understand the impact of the Y chromosome on...     

The Ontogeny of Vocal Sequences: Insights from a Newborn Wild Chimpanzee (Pan troglodytes schweinfurthii)

International Journal of Primatology - Observations of early vocal behaviours in non-human primates (hereafter primates) are important for direct comparisons between human and primate vocal...     

Antibiofilm Activity of Invasive Plants against Candida albicans: Focus on Baccharis halimifolia Essential Oil and Its Compounds

The extracts of five invasive plants were investigated for antifungal and antibiofilm activities against Candida albicans, C. glabrata, C. krusei, and C. parapsilosis. The antifungal activity was evaluated using the microdilution assay and the antibiofilm effect by measurement of the metabolic activity. Ethanol and ethanol-water extracts of Reynoutria japonica leaves inhibited 50 % of planktonic cells at 250 μg mL⁻¹ and 15.6 μg mL⁻¹, respectively. Ethanol and ethanol-water extracts of Baccharis halimifolia inhibited >75 % of the mature biofilm of C. albicans at 500 μg mL⁻¹. The essential oil (EO) of B. halimifolia leaves was the most active (50 % inhibition (IC₅₀) at 4 and 74 μg mL⁻¹against the maturation phase and 24 h old-biofilms of C. albicans, respectively). Oxygenated sesquiterpenes were the primary contents in this EO (62.02 %), with β-caryophyllene oxide as the major component (37 %). Aromadendrene oxide-(2), β-caryophyllene oxide, and (±)-β-pinene displayed significant activities against the maturation phase (IC₅₀=9–310 μ mol l⁻¹) and preformed 24 h-biofilm (IC₅₀=38–630 μ mol l⁻¹) of C. albicans with very low cytotoxicity for the first two compounds. C. albicans remained the most susceptible species to this EO and its components. This study highlighted for the first time the antibiofilm potential of B. halimifolia, its EO and some of its components.