Chicoric Acid Is an Antioxidant Molecule That Stimulates AMP Kinase Pathway in L6 Myotubes and Extends Lifespan in Caenorhabditis elegans

Chicoric acid (CA) is a caffeoyl derivative previously described as having potential anti-diabetic properties. As similarities in cellular mechanism similarities between diabetes and aging have been shown, we explored on L6 myotubes the effect of CA on the modulation of intracellular pathways involved in diabetes and aging. We also determined its influence on lifespan of Caenorhabditis elegans worm (C. elegans). In L6 myotubes, CA was a potent reactive oxygen species (ROS) scavenger, reducing ROS accumulation under basal as well as oxidative stress conditions. CA also stimulated the AMP-activated kinase (AMPK) pathway and displayed various features associated with AMPK activation: CA (a) enhanced oxidative enzymatic defences through increase in glutathion peroxidase (GPx) and superoxide dismutase (SOD) activities, (b) favoured mitochondria protection against oxidative damage through up-regulation of MnSOD protein expression, (c) increased mitochondrial biogenesis as suggested by increases in complex II and citrate synthase activities, along with up-regulation of PGC-1α mRNA expression and (d) inhibited the insulin/Akt/mTOR pathway. As AMPK stimulators (e.g. the anti-diabetic agent meformin or polyphenols such as epigallocatechingallate or quercetin) were shown to extend lifespan in C. elegans, we also determined the effect of CA on the same model. A concentration-dependant lifespan extension was observed with CA (5–100 μM). These data indicate that CA is a potent antioxidant compound activating the AMPK pathway in L6 myotubes. Similarly to other AMPK stimulators, CA is able to extend C. elegans lifespan, an effect measurable even at the micromolar range. Future studies will explore CA molecular targets and give new insights about its possible effects on metabolic and aging-related diseases.     

Isolation of plant DNA: A fast,inexpensive, and reliable method

We describe here a simple method to isolate DNA of high molecular weight from a wide variety of plant materials, such as trees, herbaceous plants, cell suspension cultures, calli, seeds, dried embryos, ferns and lichens. The crucial step of the extraction is the use of an acidic extraction medium. When necessary, the sample was separated on a fast RPC-5 column providing us with highly purified DNA suitable not only for restriction endonuclease analyses but also for PCR experiments, RLFP analyses, or detection of adducts.     

13C]-Specific labeling of 8-2' linked (-)-cis-blechnic, (-)-trans-blechnic and (-)-brainic acids in the fern Blechnum spicant

In vivo administration experiments using stable (13C) and radio (14C) labeled precursors established that the optically active 8-2' linked lignans, (-)-cis-blechnic, (-)-trans-blechnic and (-)-trans-brainic acids, were directly derived from L-phenylalanine, cinnamate, and p-coumarate but not either from tyrosine or acetate. The radiochemical time course data suggest that the initial coupling product is (-)-cis-blechnic acid, which is then apparently converted into both (-)-trans-blechnic and (-)-trans-brainic acids in vivo. These findings provide additional evidence for vascular plant proteins engendering distinct but specific phenolic radical-radical coupling modes, i.e., for full control over phenylpropanoid coupling in vivo, whether stereoselective or regiospecific.     

Origin and evolution of transmembrane Chl-binding proteins: hydrophobic cluster analysis suggests a common one-helix ancestor for prokaryotic (Pcb) and eukaryotic (LHC) antenna protein superfamilies

All chlorophyll (Chl)-binding proteins constituting the photosynthetic apparatus of both prokaryotes and eukaryotes possess hydrophobic domains, corresponding to membrane-spanning alpha-helices (MSHs). Hydrophobic cluster analysis of representative members of the different Chl protein superfamilies revealed that all Chl proteins except the five-helix reaction center II proteins and the small subunits of photosystem I possess related domains. As a major conclusion, we found that the eukaryotic antennae likely share a common precursor with the prokaryotic Chl a/b antennae from Chl-b-containing oxyphotobacteria. From these data, we propose a global scheme for the evolution of these proteins from a one-MSH ancestor.     

The structure of the neutral polysaccharide gum secreted by Rhizobium strain cb744

A previous investigation of the structure of the extracellular polysaccharide gum from the nitrogen-fixing Rhizobium strain cb744 (a member of the slow-growing Cowpea group) indicated that there were two β-(1→4)-linked d-glucopyranosyl residues for each α-(1→4)-linked d-mannopyranosyl residue, and that each mannose was substituted at O-6 by a β-d-galactopyranosyl residue having 71% of the galactose present as 4-O-methylgalactose. The present study shows that, although the gum appeared to have a simple tetrasaccharide repeating unit, it is composed of two closely associated components. One is a (1→4)-linked α-d-mannan substituted at each O-6 by a β-d-galactopyranosyl residue (71% 4-O-methylated). The second component is a (1→4)-linked β-d-glucan. The existence of the two polysaccharides was established by separation of the β-d-galactosidase-treated gum on a column of concanavalin A-Sepharose 4B. The d configurations were determined and the anomeric attribution of the linkages confirmed by the use of enzymes. The interaction between the two gum components is discussed.     

Effects of supplemental oxygen administration on coronary blood flow in patients undergoing cardiac catheterization

Patients with heart disease are frequently treated with supplemental oxygen. Although oxygen can exhibit vasoactive properties in many vascular beds, its effects on the coronary circulation have not been fully characterized. To examine whether supplemental oxygen administration affects coronary blood flow (CBF) in a clinical setting, we measured in 18 patients with stable coronary heart disease the effects of breathing 100% oxygen by face mask for 15 min on CBF (via coronary Doppler flow wire), conduit coronary diameter, CBF response to intracoronary infusion of the endothelium-dependent dilator ACh and to the endothelium-independent dilator adenosine, as well as arterial and coronary venous concentrations of the nitric oxide (NO) metabolites nitrotyrosine, NO(2)(-), and NO(3)(-). Relative to breathing room air, breathing of 100% oxygen increased coronary resistance by approximately 40%, decreased CBF by approximately 30%, increased the appearance of nitrotyrosine in coronary venous plasma, and significantly blunted the CBF response to ACh. Oxygen breathing elicited these changes without affecting the diameter of large-conduit coronary arteries, coronary venous concentrations of NO(2)(-) and NO(3)(-), or the coronary vasodilator response to adenosine. Administering supplemental oxygen to patients undergoing cardiac catheterization substantially increases coronary vascular resistance by a mechanism that may involve oxidative quenching of NO within the coronary microcirculation.     

Paleogenomics or the search for remnant duplicated copies of the yeast DUP240 gene family in intergenic areas

Duplication, resulting in gene redundancy, is well known to be a driving force of evolutionary change. Gene families are therefore useful targets for approaching genome evolution. To address the gene death process, we examined the fate of the 10-member-large S288C DUP240 family in 15 Saccharomyces cerevisiae strains. Using an original three-step method of analysis reported here, both slightly and highly degenerate DUP240 copies, called pseudo-open reading frames (ORFs) and relics, respectively, were detected in strain S288C. It was concluded that two previously annotated ORFs correspond, in fact, to pseudo-ORFs and three additional relics were identified in intergenic areas. Comparative intraspecies analysis of these degenerate DUP240 loci revealed that the two pseudo-ORFs are present in a nondegenerate state in some other strains. This suggests that within a given gene family different loci are the target of the gene erasure process, which is therefore strain dependent. Besides, the variable positions observed indicate that the relic sequence may diverge faster than the flanking regions. All in all, this study shows that short conserved protein motifs provide a useful tool for detecting and accurately mapping degenerate gene remnants. The present results also highlight the strong contribution of comparative genomics for gene relic detection because the possibility of finding short conserved protein motifs in intergenic regions (IRs) largely depends on the choice of the most closely related paralog or ortholog. By mapping new genetic components in previously annotated IRs, our study constitutes a further refinement step in the crucial stage of genome annotation and provides a strategy for retracing ancient chromosomal reshaping events and, hence, for deciphering genome history.     

Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

Introduction  

Extracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.     

Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin

Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest that Notch ligands require endocytosis, ubiquitylation, and epsin endocytic adaptors to activate signaling, but the exact role of ligand endocytosis remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins, and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose that the mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.