Diversity of culturable phyllosphere bacteria on beech and oak: the effects of lepidopterous larvae

The community composition of epiphytic heterotrophic bacteria on leaves of beech and oak, which were either damaged by lepidopterous larvae or remained undamaged, was investigated. In addition, the ability of these bacteria to utilize inorganic nitrogen was studied. The bacteria were isolated on nutrient agar and systematically identified with biochemical and physiological tests. Rarefaction plots and the Shannon-Wiener function revealed that species diversity was significantly higher on leaves of damaged beech compared to undamaged leaves, but no differences were found on leaves of oak. The portion of bacterial isolates showing a strong response to ammonia and nitrate was significantly larger on leaves of oak than on those of beech. Furthermore, significantly more isolates with a high capability to assimilate both nitrogen compounds were found on leaves attacked by the folivorous larvae compared to those not attacked on oak. It is suggested that the changes in the microbial community in response to folivorous insects might affect the extent of nutrient cycling exceeding eventually the scale of a leaf.     

BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture

Human bone marrow-derived mesenchymal stem cells (MSCs) have been shown to differentiate into distinct mesenchymal tissues including bone and cartilage. The capacity of MSCs to replicate undifferentiated and to mature into cartilaginous tissues suggests these cells as an attractive cell source for cartilage tissue engineering. Here we show that the stimulation of human bone marrow-derived MSCs with recombinant bone morphogenetic protein-2 (BMP2) results in chondrogenic lineage development under serum-free conditions. Histological staining of proteoglycan with Alcian blue and immunohistochemical staining of cartilage-specific type II collagen revealed the deposition of typical cartilage extracellular matrix components. Semi-quantitative real-time gene expression analysis of characteristic chondrocytic matrix genes, such as cartilage link protein, cartilage oligomeric matrix protein, aggrecan, and types I, II, and IX collagen, confirmed the induction of the chondrocytic phenotype in high-density culture upon stimulation with BMP2 and transforming growth factor-beta3 (TGFbeta3). Histologic staining of mineralized extracellular matrix with von Kossa, immunostaining of type X collagen (typical for hypertrophic chondrocytes), and gene expression analysis of osteocalcin and adipocyte-specific fatty acid binding protein (aP2) further documented that BMP2 induced chondrogenic lineage development and not osteogenesis and/or adipogenesis in human MSCs. These results suggest BMP2 as a promising candidate for tissue engineering approaches regenerating articular cartilage on the basis of mesenchymal progenitors from bone marrow.     

Increased expression of Fe-chelatase leads to increased metabolic flux into heme and confers protection against photodynamically induced oxidative stress

Fe-chelatase (FeCh, EC 4.99.1.1) inserts Fe²⁺ into protoporphyrin IX (Proto IX) to form heme, which influences the flux through the tetrapyrrole biosynthetic pathway as well as fundamental cellular processes. In transgenic rice (Oryza sativa), the ectopic expression of Bradyrhizobium japonicum FeCh protein in cytosol results in a substantial increase of FeCh activity compared to wild-type (WT) rice and an increasing level of heme. Interestingly, the transgenic rice plants showed resistance to oxidative stress caused not only by the peroxidizing herbicide acifluorfen (AF) as indicated by a reduced formation of leaf necrosis, a lower conductivity, lower malondialdehyde and H₂O₂ contents as well as sustained F_v/F_m compared to WT plants, but also by norflurazon, paraquat, salt, and polyethylene glycol. Moreover, the transgenic plants responded to AF treatment with markedly increasing FeCh activity. The accompanying increases in heme content and heme oxygenase activity demonstrate that increased heme metabolism attenuates effects of oxidative stress caused by accumulating porphyrins. These findings suggest that increases in heme levels and porphyrin scavenging capacity support a detoxification mechanism serving against porphyrin-induced oxidative stress. This study also implicates heme as possibly being a positive signal in plant stress responses.     

eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining

eIF5B is a eukaryal translational GTPase that catalyzes ribosomal subunit joining to form elongation‐competent ribosomes. Despite its central role in protein synthesis, the mechanistic details that govern the function of eIF5B or its archaeal and bacterial (IF2) orthologs remained unclear. Here, we present six high‐resolution crystal structures of eIF5B in its apo, GDP‐ and GTP‐bound form that, together with an analysis of the thermodynamics of nucleotide binding, provide a detailed picture of the entire nucleotide cycle performed by eIF5B. Our data show that GTP binding induces significant conformational changes in the two conserved switch regions of the G domain, resulting in the reorganization of the GTPase center. These rearrangements are accompanied by the rotation of domain II relative to the G domain and release of domain III from its stable contacts with switch 2, causing an increased intrinsic flexibility in the free GTP‐bound eIF5B. Based on these data, we propose a novel domain release mechanism for eIF5B/IF2 activation that explains how eIF5B and IF2 fulfill their catalytic role during ribosomal subunit joining.     

Exploring the multiscale signaling behavior of phototropin1 from Chlamydomonas reinhardtii using a full‐residue space kinetic Monte Carlo molecular dynamics technique

Devising analysis tools for elucidating the regulatory mechanism of complex enzymes has been a challenging task for many decades. It generally requires the determination of the structural‐dynamical information of protein solvent systems far from equilibrium over multiple length and time scales, which is still difficult both theoretically and experimentally. To cope with the problem, we introduce a full‐residue space multiscale simulation method based on a combination of the kinetic Monte Carlo and molecular dynamics techniques, in which the rates of the rate‐determining processes are evaluated from a biomolecular forcefield on the fly during the simulation run by taking into account the full space of residues. To demonstrate its reliability and efficiency, we explore the light‐induced functional behavior of the full‐length phototropin1 from Chlamydomonas reinhardtii (Cr‐phot1) and its various subdomains. Our results demonstrate that in the dark state the light oxygen voltage‐2‐Jα (LOV2‐Jα) photoswitch inhibits the enzymatic activity of the kinase, whereas the LOV1‐Jα photoswitch controls the dimerization with the LOV2 domain. This leads to the repulsion of the LOV1‐LOV2 linker out of the interface region between both LOV domains, which results in a positively charged surface suitable for cell–membrane interaction. By contrast, in the light state, we observe that the distance between both LOV domains is increased and the LOV1‐LOV2 linker forms a helix–turn–helix (HTH) motif, which enables gene control through nucleotide binding. Finally, we find that the kinase is activated through the disruption of the Jα‐helix from the LOV2 domain, which is followed by a stretching of the activation loop (A‐loop) and broadening of the catalytic cleft of the kinase. Proteins 2014; 82:2018–2040. © 2014 Wiley Periodicals, Inc.     

HNCA+, HNCO+, and HNCACB+ experiments: improved performance by simultaneous detection of orthogonal coherence transfer pathways

Three experiments, BEST–TROSY HNCA+, HNCO+ and HNCACB+ are presented for sequential backbone resonance assignment of ¹³C, ¹⁵N labelled proteins. The novelty of these experiments with respect to conventional pulse sequences is the detection of additional orthogonal coherence transfer pathways that results in enhanced sensitivity for sequential correlations without significantly compromising the intensity of intra-residue correlation peaks. In addition, a 2-step phase cycle separates peaks originating from the orthogonal coherence transfer pathways in 2 sub-spectra, thus providing similar information as obtained from performing a pair of sequential and intra-residue correlation experiments.