Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch root nodule development

We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.     

Zn induced structural aggregation patterns of β-amyloid peptides by first-principle simulations and XAS measurements

We show in this paper that in the presence of Zn ions a peculiar structural aggregation pattern of β-amyloid peptides in which metal ions are sequentially coordinated to either three or four histidines of nearby peptides is favored. To stabilize this configuration a deprotonated imidazole ring from one of the histidines forms a bridge connecting two adjacent Zn ions. Though present in zeolite imidazolate frameworks, remarkably in biological compounds this peculiar Zn-imidazolate-Zn topology is only found in enzymes belonging to the Cu,Zn-superoxide dismutase family in the form of an imidazolate bridging Cu and Zn. The results we present are obtained by combining X-ray absorption spectroscopy experimental data with detailed first-principle molecular dynamics simulations.     

Taxanes from Taxus x media

The roots of Taxus x media gave two new taxoids, the structures of which were established as 10-deacetyl-10-dehydro-7-acetyl taxol A and 10-deacetylyunnanxane on the basis of spectroscopic data.     

Blood Lipids in Antarctic and in Temperate-Water Fish Species

Antarctic fish live in very cold water and have adapted to this exceptional environment. Hemoglobin is absent or very low; yet these fish still have erythrocytes, and from these we prepared ghost-like membranes. We studied for the first time the lipid composition of ghost membranes and of plasma in Antarctic fish (C. hamatus and T. bernacchii) and compared our results with those obtained for temperate-water fish (C. auratus and A. anguilla taken from Lake Trasimeno, Perugia, Italy). The membranes of Antarctic fish were richer in glycerophospholipid (especially phosphatidylethanolamine), whereas the membranes of temperate-water fish were richer in sphingomyelin. Unsaturated fatty acids were particularly abundant in Antarctic fish: C. hamatus had long-chain unsaturated fatty acid (especially C22:6 ω-3), whereas T. bernacchii had shorter unsaturated fatty acyl chains (c16:1, ω-7). On the other hand, C. auratus and A. anguilla were particularly rich in C16:0, which constituted more than one-half of the total fatty acid. Plasma lipids (both phospholipid and cholesterol) were much more abundant in temperate-water fish. The differences in phospholipid content were mainly due to choline glycerolipids. Measures of membrane fluidity inferred from the fluorescence anisotropy of DPH indicated that the membranes from Antarctic fish were more fluid at any measured temperature than those obtained from fish living in temperate waters. The ability to live in a very cold environment has therefore been achieved by the two Antarctic species tested in this paper by different strategies, but with the same results on fluidity.     

Procyanidins from grape seeds protect endothelial cells from peroxynitrite damage and enhance endothelium-dependent relaxation in human artery: new evidences for cardio-protection

The peroxynitrite scavenging ability of Procyanidins from Vitis vinifera L. seeds was studied in homogeneous solution and in human umbilical endothelial cells (EA.hy926 cell line) using 3-morpholinosydnonimine (SIN-1) as peroxynitrite generator. In homogeneous phase procyanidins dose-dependently inhibited 2',7'-dichloro-dihydrofluorescein (DCFH) oxidation induced by SIN-1 with an IC50 value of 0.28 microM. When endothelial cells (EC) were exposed to 5 mM SIN-1, marked morphological alterations indicating a necrotic cell death (cell viability reduced to 16 +/- 2.5%) were observed. Cell damage was suppressed by procyanidins, with a minimal effective concentration of 1 microM (cell morphology and integrity completely recovered at 20 microM). Cellular localization of procyanidins in EC was confirmed using a new staining procedure and site-specific peroxyl radical inducers: AAPH and cumene hydroperoxide (CuOOH). Endothelial cells (EC) pre-incubated with procyanidins (20 microM) and exposed to FeCl3/K3Fe(CN)6 showed a characteristic blue staining, index of a site-specific binding of procyanidins to EC. Procyanidins dose-dependently inhibit the AAPH induced lipid oxidation and reverse the consequent loss of cell viability, but were ineffective when oxidation was driven at intracellular level (CuOOH). This demonstrates that the protective effect is due to their specific binding to the outer surface of EC thus to quench exogenous harmful radicals. Procyanidins dose-dependently relaxed human internal mammary aortic (IMA) rings (with intact endothelium) pre-contracted with norepinephrine (NE), showing a maximal vasorelaxant effect (85 +/- 9%) at 50 microM (catechin: 18 +/- 2% relaxation at 50 microM). This effect was completely abolished when IMA-rings were de-endothelized and when IMA-rings with intact endothelium were pretreated with L-NMMA or with the soluble guanylate cyclase inhibitor, ODQ. Pre-incubation with indomethacin reduces (by almost 50%) the vasodilating effect of procyanidins, indicating the involvement also of a COX-dependent mechanism. This was confirmed in another set of experiments, where procyanidins dose-dependently stimulate the prostacyclin (PGI2) release, reaching a plateau between 25 and 50 microM. Finally, pre-incubation of IMA-rings with procyanidins (from 6.25 to 25 microM) resulted in a dose-dependent prevention of the endothelin-1 (ET-1) vasoconstriction. The ability of procyanidins to prevent peroxynitrite attack to vascular cells, by layering on the surface of coronary EC, and to enhance endothelial NO-synthase-mediated relaxation in IMA rings provide further insight into the molecular mechanisms through which they exert cardioprotective activity in ischemia/reperfusion injury in vivo.     

bis-Dehydroxy-Curcumin Triggers Mitochondrial-Associated Cell Death in Human Colon Cancer Cells through ER-Stress Induced Autophagy

Background

The activation of autophagy has been extensively described as a pro-survival strategy, which helps to keep cells alive following deprivation of nutrients/growth factors and other stressful cellular conditions. In addition to cytoprotective effects, autophagy can accompany cell death. Autophagic vacuoles can be observed before or during cell death, but the role of autophagy in the death process is still controversial. A complex interplay between autophagy and apoptosis has come to light, taking into account that numerous genes, such as p53 and Bcl-2 family members, are shared between these two pathways.

Methodology/Principal Findings

In this study we showed a potent and irreversible cytotoxic activity of the stable Curcumin derivative bis-DeHydroxyCurcumin (bDHC) on human colon cancer cells, but not on human normal cells. Autophagy is elicited by bDHC before cell death as demonstrated by increased autophagosome formation -measured by electron microscopy, fluorescent LC3 puncta and LC3 lipidation- and autophagic flux -measured by interfering LC3-II turnover. The accumulation of poly-ubiquitinated proteins and ER-stress occurred upstream of autophagy induction and resulted in cell death. Cell cycle and Western blot analyses highlighted the activation of a mitochondrial-dependent apoptosis, which involves caspase 7, 8, 9 and Cytochrome C release. Using pharmacological inhibitions and RNAi experiments, we showed that ER-stress induced autophagy has a major role in triggering bDHC-cell death.

Conclusion/Significance

Our findings describe the mechanism through which bDHC promotes tumor selective inhibition of proliferation, providing unequivocal evidence of the role of autophagy in contrasting the proliferation of colon cancer cells.     

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners,STIM1 and STIM2

Our recent work identified store-operated Ca²⁺ entry (SOCE) as the critical Ca²⁺ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370–5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca²⁺ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca²⁺ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca²⁺-content during repeated KCl-induced Ca²⁺ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.     

Oxo-rhenium(V) compounds containing bis(3,5-dimethylpyrazol-1-yl)acetate scorpionate ligand

Ligand-exchange reactions of bidentate donor agents such as malonic acid (H₂mal) or 1,3-propanediol (H₂diol) with labile Re(O)(bdmpza)Cl₂ (bdmpza = bis(3,5-dimethylpyrazol-1-yl)acetate) precursors in the presence of triethylamine yield the mixed-ligand complexes Re(O)(bdmpza)(mal) (2) and Re(O)(bdmpza)(diol) (3), respectively. Compounds 2 and 3 can also be obtained starting directly from [Re(O)Cl₄][NBu₄], Libdmpza and the appropriate bidentate ligand in the presence of triethylamine. X-ray analyses of the two compounds show in both cases a distorted octahedral geometry around the rhenium atom comprising two pyrazolyl nitrogens and the bidentate ligand in the equatorial plane, and the oxo and the carboxylate oxygens on the apices. Differently from 2 and 3, X-ray diffraction analysis of the asymmetric precursor (OC-6-42)Re(O)(bdmpza)Cl₂ (1) reveals a slightly distorted octahedral geometry with the apical positions taken by the carboxylate oxygen and a chloride atom, and with the equatorial plane occupied by the nitrogens of the bis-pyrazolyl ligand, a chloride and the oxygen atom.     

Tendril Coiling in Grapevine: Jasmonates and a New Role for GABA?

Grapevine (Vitis vinifera L., Vitaceae) belongs to the genus Vitis, and is characterized as a vine due to the presence of tendrils, which are located opposite to leaves. Tendrils are thigmo-responsive organs, able to carry out delicate mechanosensory responses upon touch and related stimuli. These organs are an adaptation of the plant to climb with the help of support to higher places and finally remain at a position with favorable light quality. In previous studies on Bryonia dioica (Cucurbitaceae), phytohormones of the jasmonate class were identified as the endogenous hormone signals to initiate coiling of the tendrils. Strikingly, this is still the only example for jasmonate-induced tendril coiling. In grapevine, three compounds (12-oxo-phytodienoic acid, jasmonic acid (JA), and JA isoleucine conjugate) of the jasmonate class were found at higher concentrations in non-coiled tendrils when compared with coiled ones. Upon treatment with phytohormones, we could confirm the activity of jasmonates on tendril coiling in grapevine. However, not jasmonates but a non-proteinogenic amino acid, γ-aminobutyric acid (GABA), was detected to accumulate in grapevine tendrils at significantly higher levels than in all other tissues, independent of their coiling status. For GABA we detected a significant, transient positive effect on tendril coiling. Use of a GABA synthesis blocker, 3-mercaptopropionic acid, caused reduced GABA- but not JA-induced coiling scores. No additive effect of JA plus GABA was detectable on the tendrils’ coiling score. Thus, for grapevine, our data demonstrate a strong activity of jasmonates in tendril coiling induction even without mechanical stimuli and, furthermore, the data also suggest that GABA can independently promote tendril coiling.

     

High order DNA structure as inferred by optical fluorimetry and scanning calorimetry

New quantitative insights on the native high order chromatin-DNA structure existing within interphase nuclei are obtained by monitoring the effects of two common well-characterized fixatives, glutaraldehyde and ethanol/acetic acid mixture, at the level of the intranuclear DNA distribution and structures. Reproducible distinct levels of DNA fluorescence intensity and their intranuclear distribution are apparent in unfixed and fixed thymocytes by using DAPI and quantitative optical microscopy based on a charge coupled device. The fluorescent histograms correlated with the calorimetric thermograms on the very same thymocytes fixed and unfixed, establish an unequivocal baseline for the different levels of structural organization of the chromatin within the intact nucleus; namely their number, DNA packing ratio and fiber diameter. A systematic comparison among all the numerous models, being so far proposed for the quinternary and quaternary levels of DNA folding, to identifies the rope or ribbon-like and the chromonema as the ones that best fit with the in situ distribution. This revised version was published online in June 2006 with corrections to the Cover Date.