Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana

? Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. ? A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. ? We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. ? Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.     

Niches of temperate tree species converge towards nutrient‐richer conditions over ontogeny

Niche changes during a species’ lifespan are known as ontogenetic niche shifts. These shifts reflect changes in resource availability, requirements, organisms’ foraging ability and/or size‐dependent biotic interactions. In the plant kingdom, however, this issue remains poorly covered. We investigated nutritional niche shifts over the ontogeny of 23 temperate tree species (among nine phylogenetic families) by a synchronic approach. We used 1963 temporary phytoecological surveys conducted throughout metropolitan French forests. The realised niches of three life‐history stages (seedling: <0.5 m; sapling: >0.5 m and < 8 m; tree: >8 m) in each tree species were modelled on the basis of presence/absence data and the main factors of species distribution (energy, water and nutritional resources). We computed the nutritional optima and amplitudes by bootstrapping partial response curves for the C:N ratio and base saturation rate. We assessed changes in these niche parameters over ontogeny and also evaluated the relative importance of ontogenetic shifts in the differentiation of nutritional niche among the selected tree species. The tree stage was found to occur mainly at higher nutrient availability than the seedling (+16.3% on the nutritional gradient) or sapling (+11.1%) stages. In addition, nutritional niches of tree species exhibited, successively, a niche enlargement in eutrophic conditions and a niche restriction in oligotrophic conditions during growth. These global nutritional niche shifts observed over the species’ lifespan contributed moderately but significantly to the niche separation in temperate tree communities (up to 4.5%). We interpreted niche shifts as a response to an increase in nutritional requirements over ontogeny, leading to an intra‐specific selection where individuals established in eutrophic soils have the maximal fitness. Biotic interactions and temporal changes in the environment may secondarily enhance or counteract the process. The importance of ontogenetic niche shifts requires consideration in the study of species autecology and plant community organisation.     

Design,synthesis and biological evaluation of estradiol-PEG-linked platinum(II) hybrid molecules: Comparative molecular modeling study of three distinct families of hybrids

The synthesis of a series of 17β-estradiol-platinum(II) hybrid molecules is reported. The hybrids are made of a PEG linking chain of various length and a 2-(2′-aminoethyl)pyridine ligand. They are prepared from estrone in only 5 chemical steps with an overall yield of 22%. The length of the PEG chain does not influence the solubility of the compounds as it remains relatively constant throughout the series. MTT assays showed that the derivative with the longest PEG chain showed the best activity against two human breast cancer cell lines (MCF-7 and MDA-MB-231). The novel PEG-hybrids are also compared in terms of activities with two other families of 17β-estradiol-platinum(II) hybrids that we reported in previous studies. Molecular modeling study performed on a representative member of each family of hybrids reveals distinct molecular interactions with the estrogen receptor α which further corroborates their notably contrasting cytocidal activities on breast cancer cell lines. This study also shows that lipophilicity and the orientation of the tether chain between the estrogenic portion and the platinum(II) core contribute markedly to the biological activity of the various families of hybrids. The most active hybrids are those possessing an alkyl tether chain at position 16β of the steroid nucleus. For example, derivative 3 (p = 6) is about 16 times more potent on MCF-7 breast cancer cells than the corresponding 16α-PEG-hybrids (2b) made in this study.     

Characterization of lipoteichoic acid structures from three probiotic Bacillus strains: involvement of d-alanine in their biological activity

Probiotics represent a potential strategy to influence the host’s immune system thereby modulating immune response. Lipoteichoic Acid (LTA) is a major immune-stimulating component of Gram-positive cell envelopes. This amphiphilic polymer, anchored in the cytoplasmic membrane by means of its glycolipid component, typically consists of a poly (glycerol-phosphate) chain with d-alanine and/or glycosyl substitutions. LTA is known to stimulate macrophages in vitro, leading to secretion of inflammatory mediators such as Nitric Oxide (NO). This study investigates the structure–activity relationship of purified LTA from three probiotic Bacillus strains (Bacillus cereus CH, Bacillus subtilis CU1 and Bacillus clausii O/C). LTAs were extracted from bacterial cultures and purified. Chemical modification by means of hydrolysis at pH 8.5 was performed to remove d-alanine. The molecular structure of native and modified LTAs was determined by ¹H NMR and GC–MS, and their inflammatory potential investigated by measuring NO production by RAW 264.7 macrophages. Structural analysis revealed several differences between the newly characterized LTAs, mainly relating to their d-alanylation rates and poly (glycerol-phosphate) chain length. We observed induction of NO production by LTAs from B. subtilis and B. clausii, whereas weaker NO production was observed with B. cereus. LTA dealanylation abrogated NO production independently of the glycolipid component, suggesting that immunomodulatory potential depends on d-alanine substitutions. d-alanine may control the spatial configuration of LTAs and their recognition by cell receptors. Knowledge of molecular mechanisms behind the immunomodulatory abilities of probiotics is essential to optimize their use.     

CORM-3, a water soluble CO-releasing molecule,uncouples mitochondrial respiration via interaction with the phosphate carrier

Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O₂ consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.     

Expression analysis of ATAD3 isoforms in rodent and human cell lines and tissues

ATAD3 (ATPase family AAA-Domain containing protein 3) is a mitochondrial inner membrane ATPase with unknown but vital functions. Initial researches have focused essentially on the major p66-ATAD3 isoform, but other proteins and mRNAs are described in the data banks. Using a set of anti-peptide antibodies and by the use of rodent and human cell lines and organs, we tried to detail ATAD3 gene expression profiles and to verify the existence of the various ATAD3 isoforms. In rodent, the single ATAD3 gene is expressed as a major isoform of 67 kDa, (ATAD3l; long), in all cells and organs studied. A second isoform, p57-ATAD3s (small), is expressed specifically throughout brain development and in adult, and overexpressed around the peri-natal period. p57-ATAD3s is also expressed in neuronal and glial rodent cell lines, and during in vitro differentiation of primary cultured rat oligodendrocytes. Other smaller isoforms were also detected in a tissue-specific manner. In human and primates, ATAD3 paralogues are encoded by three genes (ATAD3A, 3B and 3C), each of them presenting several putative variants. Analyzing the expression of ATAD3A and ATAD3B with four specific anti-peptide antibodies, and comparing their expressions with in vitro expressed ATAD3 cDNAs, we were able to observe and define five isoforms. In particular, the previously described p72-ATAD3B is confirmed to be in certain cases a phosphorylated form of ATAD3As. Moreover, we observed that the ATAD3As phosphorylation level is regulated by insulin and serum. Finally, exploring ATAD3 mRNA expression, we confirmed the existence of an alternative splicing in rodent and of several mRNA isoforms in human.