Long term bed rest with and without vibration exercise countermeasures: effects on human muscle protein dysregulation

The present investigation, the first in the field, was aimed at analyzing differentially, on individual samples, the effects of 55 days of horizontal bed rest, a model for microgravity, on myosin heavy and myosin light chain isoforms distribution (by SDS) and on the proteome (by 2-D DIGE and MS) in the vastus lateralis (VL), a mixed type II/I (～50:50%) head of the quadriceps and in the calf soleus (SOL), a predominantly slow (～35:65%) twitch muscle. Two separate studies were performed on six subjects without (BR) and six with resistive vibration exercise (RVE) countermeasures, respectively. Both VL and SOL underwent in BR decrements of ～15% in cross-sectional area and of ～22% in maximal torque that were prevented by RVE. Myosin heavy chain distribution showed increased type I and decreased type IIA in BR both in VL and in SOL, the opposite with RVE. A substantial downregulation of proteins involved in aerobic metabolism characterized both in SOL and VL in BR. RVE reversed the pattern more in VL than in SOL, whereas proteins involved in anaerobic glycolysis were upregulated. Proteins from the Z-disk region and from costamers were differently dysregulated during bed rest (both BR and RVE), particularly in VL.     

Structure of an archaeal non-discriminating glutamyl-tRNA synthetase: a missing link in the evolution of Gln-tRNAGln formation

The molecular basis of the genetic code relies on the specific ligation of amino acids to their cognate tRNA molecules. However, two pathways exist for the formation of Gln-tRNA^Gln. The evolutionarily older indirect route utilizes a non-discriminating glutamyl-tRNA synthetase (ND-GluRS) that can form both Glu-tRNA^Glu and Glu-tRNA^Gln. The Glu-tRNA^Gln is then converted to Gln-tRNA^Gln by an amidotransferase. Since the well-characterized bacterial ND-GluRS enzymes recognize tRNA^Glu and tRNA^Gln with an unrelated α-helical cage domain in contrast to the β-barrel anticodon-binding domain in archaeal and eukaryotic GluRSs, the mode of tRNA^Glu/tRNA^Gln discrimination in archaea and eukaryotes was unknown. Here, we present the crystal structure of the Methanothermobacter thermautotrophicus ND-GluRS, which is the evolutionary predecessor of both the glutaminyl-tRNA synthetase (GlnRS) and the eukaryotic discriminating GluRS. Comparison with the previously solved structure of the Escherichia coli GlnRS-tRNA^Gln complex reveals the structural determinants responsible for specific tRNA^Gln recognition by GlnRS compared to promiscuous recognition of both tRNAs by the ND-GluRS. The structure also shows the amino acid recognition pocket of GluRS is more variable than that found in GlnRS. Phylogenetic analysis is used to reconstruct the key events in the evolution from indirect to direct genetic encoding of glutamine.     

Phytoalexins of woody plants

Summary Phytoalexins accumulated in selected woody plants in response to microbial attack or stress are reviewed and listed with respect to their chemical structure and probable biogenetic origin. The host-pathogen systems from which they have been isolated are described. The review also considers the antimicrobial activity of the phytoalexins to the causal pathogens and other microorganisms.     

Metabolite profiling of mycorrhizal roots of Medicago truncatula

Metabolite profiling of soluble primary and secondary metabolites, as well as cell wall-bound phenolic compounds from roots of barrel medic (Medicago truncatula) was carried out by GC-MS, HPLC and LC-MS. These analyses revealed a number of metabolic characteristics over 56 days of symbiotic interaction with the arbuscular mycorrhizal (AM) fungus Glomus intraradices, when compared to the controls, i.e. nonmycorrhizal roots supplied with low and high amounts of phosphate. During the most active stages of overall root mycorrhization, elevated levels of certain amino acids (Glu, Asp, Asn) were observed accompanied by increases in amounts of some fatty acids (palmitic and oleic acids), indicating a mycorrhiza-specific activation of plastidial metabolism. In addition, some accumulating fungus-specific fatty acids (palmitvaccenic and vaccenic acids) were assigned that may be used as markers of fungal root colonization. Stimulation of the biosynthesis of some constitutive isoflavonoids (daidzein, ononin and malonylononin) occurred, however, only at late stages of root mycorrhization. Increase of the levels of saponins correlated AM-independently with plant growth. Only in AM roots was the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives) observed. The structures of the unknown cyclohexenone derivatives were identified by spectroscopic methods as glucosides of blumenol C and 13-hydroxyblumenol C and their corresponding malonyl conjugates. During mycorrhization, the levels of typical cell wall-bound phenolics (e.g. 4-hydroxybenzaldehyde, vanillin, ferulic acid) did not change; however, high amounts of cell wall-bound tyrosol were exclusively detected in AM roots. Principal component analyses of nonpolar primary and secondary metabolites clearly separated AM roots from those of the controls, which was confirmed by an hierarchical cluster analysis. Circular networks of primary nonpolar metabolites showed stronger and more frequent correlations between metabolites in the mycorrhizal roots. The same trend, but to a lesser extent, was observed in nonmycorrhizal roots supplied with high amounts of phosphate. These results indicate a tighter control of primary metabolism in AM roots compared to control plants. Network correlation analyses revealed distinct clusters of amino acids and sugars/aliphatic acids with strong metabolic correlations among one another in all plants analyzed; however, mycorrhizal symbiosis reduced the cluster separation and enlarged the sugar cluster size. The amino acid clusters represent groups of metabolites with strong correlations among one another (cliques) that are differently composed in mycorrhizal and nonmycorrhizal roots. In conclusion, the present work shows for the first time that there are clear differences in development- and symbiosis-dependent primary and secondary metabolism of M. truncatula roots.     

Polychaete diversity in the Scotia Arc benthic realm: Are polychaetes tracers for faunal exchange?

The Scotia Arc is the only shallow-water and island bridge linking nowadays Patagonia and the Antarctic. The Antarctic Circumpolar Current as an oceanographic peculiarity makes this region an interesting biogeographic transition zone, because this frontal system traditionally is said to isolate the Antarctic fauna from that of the adjacent northern ecosystems. Based on benthos samples from three expeditions onboard R/V Polarstern, we studied distribution patterns of 200 polychaete species and 34 major benthic taxa in order to evaluate the role of polychaetes in the benthic realm of this part of the Southern Ocean. ANOSIM test distinguished three station groups: the central eastern Scotia Sea, the continental shelf off South America and stations at the tip of the Antarctic Peninsula. These station groups differed in organism densities and diversities with stations at the tip of the Antarctic Peninsula hosting the most diverse and dense community. The polychaete diversity patterns in the three assemblages evidenced closer connectivity between the tip of the Antarctic Peninsula and the central eastern Scotia Sea than between the continental shelf off South America with either the stations off the tip of the Peninsula or the central eastern Scotia Sea. This is probably supported by the Polar Front, which divides the island chain into two branches. Species distribution and community patterns of polychaetes appear to be associated with oceanographic and sediment conditions in this region. Most of the shared species showed the capability to tolerate differences in hydrostatic pressure. We suggest that the islands of the Scotia Sea may constitute a bridge for exchange of benthic species, particularly for polychaetes with eurybathic distribution and high dispersal capabilities.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

High transformation frequencies obtained from a commercial wheat (Triticum aestivum L. cv. ‘Combi’) by microbombardment of immature embryos followed by GFP screening combined with PPT selection

Improved gene transfer techniques are necessary to obtain adequatenumbers of stable transgenic wheat plants needed for practical purposes.Considering that wheat transformation is genotype-dependent, we used cv. Combiin all experiments, which had been selected from agronomically important Germanspring wheat cultivars because of its high transformation ability. In mostwheatgene transfer attempts, immature embryos or embryogenic scutellar calli weremicrobombarded. We compared both methods under optimised conditions, usingbar, uidA, andgfp as markers in co-transformation attempts. Integrationof the genes mentioned above was proven by Southern blotting, expression levelswere measured by assays on phosphinothricin acetyltransferase and-glucuronidase activities, and by monitoring for green fluorescentproteinin most developmental stages. Following bombardment of scutellar calli, anaverage transformation frequency of 0.13% was attained. Using immature embryos,mean transformation frequency (1.06%) was 8-fold higher. In addition, embryotechniques were over 2 weeks faster than scutellar callus procedures.Introducing gfp as a vital marker led to an improvement ofembryo-based techniques. In a first screening, transientgfp-expressing embryos were transferred tophosphinothricincontaining callus medium. Only gfp-expressing calli whichdeveloped on it were cultured further on phosphinothricin containingregeneration medium. Shoots obtained from gfp-expressingcalli were rooted on phosphinothricin-free medium, and cultured exvitro. Average transformation frequency (4.93%) was 38-fold higherthan with scutellar callus techniques. Differences between the transformationstrategies used were of high statistical significance. Combining greenfluorescent protein screening with phosphinothricin selection in embryo-basedtechniques offers a promising system to obtain high wheat transformationfrequencies.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.