Major trends in stem anatomy and growth forms in the perianth-bearing Piperales,with special focus on Aristolochia

Background and Aims

The order Piperales has the highest diversity of growth forms among the earliest angiosperm lineages, including trees, shrubs, climbers and herbs. However, within the perianth-bearing Piperales (Asarum, Saruma, Lactoris, Hydnora, Prosopanche, Thottea and Aristolochia), climbing species only occur in the most species-rich genus Aristolochia. This study traces anatomical and morphological traits among these lineages, to detect trends in growth form evolution and developmental processes.

Methods

Transverse stem sections of different developmental stages of representatives of Asarum, Saruma, Lactoris, Hydnora, Thottea and Aristolochia were compared and anatomical traits were linked to growth form evolution. Biomechanical properties of representative climbers were determined in three-point bending tests and are discussed based on the anatomical observations. Growth form evolution of the perianth-bearing Piperales was reconstructed by ancestral character state reconstruction using Mesquite.

Key Results

While species of Asarum and Saruma are exclusively herbaceous, species of the remaining genera show a higher diversity of growth habit and anatomy. This growth form diversity is accompanied by a more complex stem anatomy and appropriate biomechanical properties. The ancestral growth form of the perianth-bearing Piperales is reconstructed with either a shrub-like or herbaceous character state, while the following three backbone nodes in the reconstruction show a shrub-like character state. Accordingly, the climbing habit most probably evolved in the ancestor of Aristolochia.

Conclusions

Since the ancestor of the perianth-bearing Piperales has been reconstructed with a herb- or shrub-like habit, it is proposed that the climbing habit is a derived growth form, which evolved with the diversification of Aristolochia, and might have been a key feature for its diversification. Observed anatomical synapomorphies, such as the perivascular fibres in Lactoris, Thottea and Aristolochia, support the phylogenetic relationship of several lineages within the perianth-bearing Piperales. In addition, the hypothesis that the vegetative organs of the holoparasitic Hydnoraceae are most probably rhizomes is confirmed.     

Endocranial morphology of Palaeocene Plesiadapis tricuspidens and evolution of the early primate brain

Expansion of the brain is a key feature of primate evolution. The fossil record, although incomplete, allows a partial reconstruction of changes in primate brain size and morphology through time. Palaeogene plesiadapoids, closest relatives of Euprimates (or crown-group primates), are crucial for understanding early evolution of the primate brain. However, brain morphology of this group remains poorly documented, and major questions remain regarding the initial phase of euprimate brain evolution. Micro-CT investigation of the endocranial morphology of Plesiadapis tricuspidens from the Late Palaeocene of Europe—the most complete plesiadapoid cranium known—shows that plesiadapoids retained a very small and simple brain. Plesiadapis has midbrain exposure, and minimal encephalization and neocorticalization, making it comparable with that of stem rodents and lagomorphs. However, Plesiadapis shares a domed neocortex and downwardly shifted olfactory-bulb axis with Euprimates. If accepted phylogenetic relationships are correct, then this implies that the euprimate brain underwent drastic reorganization during the Palaeocene, and some changes in brain structure preceded brain size increase and neocortex expansion during evolution of the primate brain.     

Label-free measurement of histone lysine methyltransferases activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Histone lysine methyltransferases (HKMTs) are enzymes that play an essential role in epigenetic regulation. Thus, identification of inhibitors specifically targeting these enzymes represents a challenge for the development of new antitumor therapeutics. Several methods for measuring HKMT activity are already available. Most of them use indirect measurement of the enzymatic reaction through radioactive labeling or antibody-recognized products or coupled enzymatic assays. Mass spectrometry (MS) represents an interesting alternative approach because it allows direct detection and quantification of enzymatic reactions and can be used to determine kinetics and to screen small molecules as potential inhibitors. Application of mass spectrometry to the study of HKMTs has not been fully explored yet. We describe here the development of a simple reliable label-free MALDI-TOF MS-based assay for the detection and quantification of peptide methylation, using SET7/9 as a model enzyme. Importantly, the use of expensive internal standard often required in mass spectrometry quantitative analysis is not necessary in this assay. This MS assay allowed us to determine enzyme kinetic parameters as well as IC₅₀ for a known inhibitor of this enzyme. Furthermore, a comparative study with an antibody-based immunosorbent assay showed that the MS assay is more reliable and suitable for the screening of inhibitors.     

Hommes et environnements au Paléolithique supérieur en Ukraine continentale et en Crimée : introduction

Research, conducted under the ANR project “Mammouths”, on “the end of the mammoth steppe: Man/Environment relationship during late Pleniglacial in Eastern Europe”, is the subject of several contributions, a part of them is published in this volume, under the heading “Humans and environments during Upper Paleolithic in mainland Ukraine and Crimea”, in the French journal L’anthropologie.     

Accumulation of Free Oligosaccharides and Tissue Damage in Cytosolic α-Mannosidase (Man2c1)-deficient Mice

Free Man_7–9GlcNAc₂ is released during the biosynthesis pathway of N-linked glycans or from misfolded glycoproteins during the endoplasmic reticulum-associated degradation process and are reduced to Man₅GlcNAc in the cytosol. In this form, free oligosaccharides can be transferred into the lysosomes to be degraded completely. α-Mannosidase (MAN2C1) is the enzyme responsible for the partial demannosylation occurring in the cytosol. It has been demonstrated that the inhibition of MAN2C1 expression induces accumulation of Man_8–9GlcNAc oligosaccharides and apoptosis in vitro. We investigated the consequences caused by the lack of cytosolic α-mannosidase activity in vivo by the generation of Man2c1-deficient mice. Increased amounts of Man_8–9GlcNAc oligosaccharides were recognized in all analyzed KO tissues. Histological analysis of the CNS revealed neuronal and glial degeneration with formation of multiple vacuoles in deep neocortical layers and major telencephalic white matter tracts. Enterocytes of the small intestine accumulate mannose-containing saccharides and glycogen particles in their apical cytoplasm as well as large clear vacuoles in retronuclear position. Liver tissue is characterized by groups of hepatocytes with increased content of mannosyl compounds and glycogen, some of them undergoing degeneration by hydropic swelling. In addition, lectin screening showed the presence of mannose-containing saccharides in the epithelium of proximal kidney tubules, whereas scattered glomeruli appeared collapsed or featured signs of fibrosis along Bowman''s capsule. Except for a moderate enrichment of mannosyl compounds and glycogen, heterozygous mice were normal, arguing against possible toxic effects of truncated Man2c1. These findings confirm the key role played by Man2c1 in the catabolism of free oligosaccharides.     

New Selective Peptidyl Di(chlorophenyl) Phosphonate Esters for Visualizing and Blocking Neutrophil Proteinase 3 in Human Diseases

The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl^P(O-C₆H₄-4-Cl)₂. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.     

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α₄β₁ integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.     

Compositional patterns in reptilian genomes

Sauropsids form a complex group of vertebrates including squamates (lizards and snakes), turtles, crocodiles, sphenodon and birds (which are often considered as a separate class). Although avian genomes have been relatively well studied, the genomes of the other groups have remained only sparsely characterized. Moreover, the nuclear sequences available in databanks are still very limited. In the present study, we have analysed the compositional patterns, i.e. the GC (molar fraction of guanine and cytosine in DNA) distributions, of 31 reptilian (particularly snake) genomes by analytical ultracentrifugation of DNAs in CsCl gradients. The profiles were characterized by their modal buoyant density rho(o), mean buoyant density < rho>, asymmetry < rho>- rho(o), and heterogeneity H. The modal buoyant density distribution of reptilian DNAs clearly distinguishes two groups. The snakes fall in the same range of modal densities as most mammals, whereas crocodiles, turtles and lizards show higher values (>1.700 g/cm(3)). As far as the more important compositional properties of asymmetry and heterogeneity are concerned, previous studies showed that amphibians and fishes share relatively low values, whereas birds and mammals are characterized by highly heterogeneous and asymmetric patterns (with the exception of Muridae, which have a lower heterogeneity). The present results show that the snake genomes cover a broad range of asymmetry and heterogeneity values, whereas the genomes of crocodiles and turtles cover a narrow range that is intermediate between those of fishes/amphibians and those of mammals/birds.