Immunolocalization of Non-Symbiotic Hemoglobins During Somatic Embryogenesis in Chicory

Hemoglobins are ancient O₂-binding proteins, ubiquitously found in eukaryotes. They have been categorized as symbiotic, nonsymbiotic and truncated hemoglobins. We have investigated the cellular localization of nonsymbiotic hemoglobin proteins during somatic embryogenesis in Cichorium hybrid leaves (Cichorium intybus L. var. sativum × C. endivia var. latifolia) using immunolocalization technique. These proteins were detected during the two steps of culture: induction and expression. In leaves, hemoglobins colocalised with plastids, which were dispersed in the parietal cytoplasm as well as in the two guard cells of a stomata, but not in epidermis cells. Upon induction of embryogenesis, in the dark, this pattern disappeared. During the induction phase, where competent cells reinitiate the cell cycle and prepare for mitosis, hemoglobins appeared initially near chloroplasts, and then in the vicinity of vascular vessels especially in the phloem and in cells surrounding the xylem vessels. When leaf fragments were transferred to another medium for the expression phase, hemoglobins were observed in the majority of the leaf blade cells and in small young embryos but not in the older ones. Hemoglobins were also detected in other leaves cells or tissues all along the process. The role of these nonsymbiotic hemoglobins during somatic embryogenesis is discussed.Key Words: chicory, immunolocalization, nonsymbiotic hemoglobin, somatic embryogenesis     

Directed evolution predicts cytochrome b G37V target site modification as probable adaptive mechanism towards the QiI fungicide fenpicoxamid in Zymoseptoria tritici

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Q_o site). We identified the G37V change within the cytochrome b Q_i site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Q_o site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs.     

Closed state of both binding domains of homodimeric mGlu receptors is required for full activity

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.     

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice

FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (-/-) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.     

The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase

The activation of monocytes involves a stimulation of glycolysis, release of potent inflammatory mediators, and alterations in gene expression. All of these processes are known to be further increased under hypoxic conditions. The activated monocytes express inducible 6-phosphofructo-2-kinase (iPFK-2), which synthesizes fructose 2,6-bisphosphate, a stimulator of glycolysis. During ischemia, AMP-activated protein kinase (AMPK) activates the homologous heart 6-phosphofructo-2-kinase isoform by phosphorylating its Ser-466. Here, we studied the involvement of AMPK and iPFK-2 in the stimulation of glycolysis in activated monocytes under hypoxia. iPFK-2 was phosphorylated on the homologous serine (Ser-461) and activated by AMPK in vitro. The activation of human monocytes by lipopolysaccharide induced iPFK-2 expression and increased fructose 2,6-bisphosphate content and glycolysis. The incubation of activated monocytes with oligomycin, an inhibitor of oxidative phosphorylation, or under hypoxic conditions activated AMPK and further increased iPFK-2 activity, fructose 2,6-bisphosphate content, and glycolysis. In cultured human embryonic kidney 293 cells, the expression of a dominant-negative AMPK prevented both the activation and phosphorylation of co-transfected iPFK-2 by oligomycin. It is concluded that the stimulation of glycolysis by hypoxia in activated monocytes requires the phosphorylation and activation of iPFK-2 by AMPK.     

The effects of a change in gravity on the dynamics of prehension

The objective of this study was to measure the forces applied on an object manipulated in different gravitational fields attained during parabolic flights. Eight subjects participated flights (ES) and four were inexperienced (NES). They had to move continuously an instrumented object up and down in three different gravitational conditions (1 g, 1.8 g, 0 g). In 1 g, the grip force precisely anticipated the fluctuations of load force which was maximum and minimum at the bottom and at the top of the arm trajectory respectively. When the gravity changed (0 g and 1.8 g), the grip-load force coupling persisted for all the subjects from the first parabola. While the ES immediately exerted a grip force appropriate to the gravity, the NES dramatically increased their grip when faced with hyper and microgravity for the first time. Then, they progressively released their grip until a continuous grip-load force relationship with regard to 1 g was established after the fifth parabola. We suggest that each new gravitational field is rapidly incorporated into an internal model within the CNS which can then be reused as required by the occasion.     

A simplified method for the detection of Y chromosome microdeletions in infertile men using a multiplex sequence-tagged site-based amplification

Sixteen sequence-tagged sites (STSs) were combined in five amplification reactions, to screen for deletions of DNA fragments located within the AZFa, AZFb, and AZFc regions of the Y chromosome. This multiplex strategy is fast and reliable, and most of the azoospermia-associated deletions reported so far are detected with this simplified method. Internal control STSs are included that allow discrimination between deletion and failure of amplification.     

An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.     

Two clusters of charged residues located in the electropositive face of the von Willebrand factor A1 domain are essential for heparin binding

The VWF A1 domain seems to possess two heparin binding regions (residues 565-587 and 633-648) displaying positively charged amino acids, but the overall polyanion-A1 domain interaction scheme remains essentially elusive. To probe this molecular reaction as well as the role of electrostatic forces in VWF-heparin interaction, we performed mutagenesis and molecular modeling experiments. Fifteen mutated rVWFs were expressed [R571A, K572A, R573A, K585A, R571A/K572A/R573A, R578A/R579A, R578A/R579A/K585A, R571A/K572A/R573A/R578A/R579A/K585A (6A), K642G, K643G, K644G, K645G, K642G/K645G, K643G/K644G, and K642G/K643G/K644G/K645G (4G)]. Experimental results indicate that the multimeric structure of the mutants was similar to that of wild-type (WT) rVWF and that all rVWFs displayed normal binding to four conformation-dependent mAbs directed against the A1 domain. Three variants displayed significant reductions in the level of heparin binding. The 6A variant showed 39.2 +/- 1.3% of the WT rVWF level (p < 0.005), while mutants K643G/K644G and 4G showed 63.6 +/- 3.2 and 53.3 +/- 5% of the WT rVWF level, respectively (p < 0.005). Computational investigations showed that one face of the A1 domain is strongly electropositive, indicating that electrostatic forces should be essential in steering heparin onto its binding site. In agreement with our experimental data, the most striking alterations of the electrostatic potential contours were seen for mutants 4G, K643G/K644G, and 6A. Our data suggest that two clusters, one at positions 571-573, 578, 579, and 585 and the other at positions 642-645, act in concert for the recognition of heparin, forming a single extended binding surface across the electropositive face of the VWF A1 domain. A structural model of the VWF A1 domain-heparin complex is proposed, taking into account both experimental and computer modeling data.     

Short inverse complementary amino acid sequences generate protein complexity

Inversions of short genomic sequences play a central role in the generation of protein complexity. More than half of the 1300 motifs registered in ProSite have protein inverse complementary sequences (princoms) among proteins registered in SwissProt. The observed number of princoms occurrences exceeds by far the expected number (p < 10(-10)). Princoms often endow their host proteins with a whole new range of biochemical and physiological capabilities, including the possibility of intramolecular and intermolecular disulfide bond formation. These results support the idea that, like the duplications, the inversions of small genomic fragments have been a fundamental mechanism for shaping genomes.