Chondrocyte hypertrophy in skeletal development,growth, and disease

Most of our bones form through the process of endochondral ossification, which is tightly regulated by the activity of the cartilage growth plate. Chondrocyte maturation through the various stages of growth plate physiology ultimately results in hypertrophy. Chondrocyte hypertrophy is an essential contributor to longitudinal bone growth, but recent data suggest that these cells also play fundamental roles in signaling to other skeletal cells, thus coordinating endochondral ossification. On the other hand, ectopic hypertrophy of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis. Thus, a better understanding of the processes that control chondrocyte hypertrophy in the growth plate as well as in articular cartilage is required for improved management of both skeletal growth disorders and osteoarthritis. This review summarizes recent findings on the regulation of hypertrophic chondrocyte differentiation, the cellular mechanisms involved in hypertrophy, and the role of chondrocyte hypertrophy in skeletal physiology and pathophysiology. Birth Defects Research (Part C) 102:74–82, 2014. © 2014 Wiley Periodicals, Inc.     

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1‐ethyl‐3‐methylimidazolium ethylsulfate

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water‐soluble IL 1‐ethyl‐3‐methylimidazolium ethylsulfate ([emim][EtSO₄]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl–agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis‐Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO₄]. When concentration of the IL was augmented, the values of V_max for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase–glyoxyl–agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO₄]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first‐order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO₄]. Finally, thermal stability was scarcely affected by the presence of the IL. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:790–796, 2014     

Human recombinant domain antibodies against multiple sclerosis antigenic peptide CSF114(Glc)

Multiple sclerosis (MS) is a chronic auto‐immune disease characterized by a damage to the myelin component of the central nervous system. Self‐antigens created by aberrant glycosylation have been described to be a key component in the formation of auto‐antibodies. CSF114(Glc) is a synthetic glucopeptide detecting in vitro MS‐specific auto‐antibodies, and it is actively used in diagnostics and research to monitor and quantify MS‐associated Ig levels. We reasoned that antibodies raised against this probe could have been relevant for MS. We therefore screened a human Domain Antibody library against CSF114(Glc) using magnetic separation as a panning method. We obtained and described several clones, and the one with the highest signals was produced as a 6×His‐tagged protein to properly study the binding properties as a soluble antibody. By surface plasmon resonance measurements, we evidenced that our clone recognized CSF114(Glc) with high affinity and specific for the glucosylated peptide. Kinetic parameters of peptide–clone interaction were calculated obtaining a value of K_D in the nanomolar range. Harboring a human framework, this antibody should be very well tolerated by human immune system and may represent a valuable tool for MS diagnosis and therapy, paving the way to new research strategies. Copyright © 2014 John Wiley & Sons, Ltd.     

Architecture and function of metallopeptidase catalytic domains

The cleavage of peptide bonds by metallopeptidases (MPs) is essential for life. These ubiquitous enzymes participate in all major physiological processes, and so their deregulation leads to diseases ranging from cancer and metastasis, inflammation, and microbial infection to neurological insults and cardiovascular disorders. MPs cleave their substrates without a covalent intermediate in a single‐step reaction involving a solvent molecule, a general base/acid, and a mono‐ or dinuclear catalytic metal site. Most monometallic MPs comprise a short metal‐binding motif (HEXXH), which includes two metal‐binding histidines and a general base/acid glutamate, and they are grouped into the zincin tribe of MPs. The latter divides mainly into the gluzincin and metzincin clans. Metzincins consist of globular ～130–270‐residue catalytic domains, which are usually preceded by N‐terminal pro‐segments, typically required for folding and latency maintenance. The catalytic domains are often followed by C‐terminal domains for substrate recognition and other protein–protein interactions, anchoring to membranes, oligomerization, and compartmentalization. Metzincin catalytic domains consist of a structurally conserved N‐terminal subdomain spanning a five‐stranded β‐sheet, a backing helix, and an active‐site helix. The latter contains most of the metal‐binding motif, which is here characteristically extended to HEXXHXXGXX(H,D). Downstream C‐terminal subdomains are generally shorter, differ more among metzincins, and mainly share a conserved loop—the Met‐turn—and a C‐terminal helix. The accumulated structural data from more than 300 deposited structures of the 12 currently characterized metzincin families reviewed here provide detailed knowledge of the molecular features of their catalytic domains, help in our understanding of their working mechanisms, and form the basis for the design of novel drugs.     

Computational identification of post‐translational modification‐based nuclear import regulations by characterizing nuclear localization signal‐import receptor interaction

The binding affinity between a nuclear localization signal (NLS) and its import receptor is closely related to corresponding nuclear import activity. PTM‐based modulation of the NLS binding affinity to the import receptor is one of the most understood mechanisms to regulate nuclear import of proteins. However, identification of such regulation mechanisms is challenging due to the difficulty of assessing the impact of PTM on corresponding nuclear import activities. In this study we proposed NIpredict, an effective algorithm to predict nuclear import activity given its NLS, in which molecular interaction energy components (MIECs) were used to characterize the NLS‐import receptor interaction, and the support vector regression machine (SVR) was used to learn the relationship between the characterized NLS‐import receptor interaction and the corresponding nuclear import activity. Our experiments showed that nuclear import activity change due to NLS change could be accurately predicted by the NIpredict algorithm. Based on NIpredict, we developed a systematic framework to identify potential PTM‐based nuclear import regulations for human and yeast nuclear proteins. Application of this approach has identified the potential nuclear import regulation mechanisms by phosphorylation of two nuclear proteins including SF1 and ORC6. Proteins 2014; 82:2783–2796. © 2014 Wiley Periodicals, Inc.     

Patterns and drivers of Araucaria araucana forest growth along a biophysical gradient in the northern Patagonian Andes: Linking tree rings with satellite observations of soil moisture

Araucaria araucana (Araucaria) is a long‐lived conifer growing along a sharp west–east biophysical gradient in the Patagonian Andes. The patterns and climate drivers of Araucaria growth have typically been documented on the driest part of the gradient relying on correlations with meteorological records, but the lack of in situ soil moisture observations has precluded an assessment of the growth responses to soil moisture variability. Here, we use a network of 21 tree‐ring width chronologies to investigate the spatiotemporal patterns of tree growth through the entire gradient and evaluate their linkages with regional climate and satellite‐observed surface soil moisture variability. We found that temporal variations in tree growth are remarkably similar throughout the gradient and largely driven by soil moisture variability. The regional spatiotemporal pattern of tree growth was positively correlated with precipitation (r = 0.35 for January 1920–1974; P < 0.01) and predominantly negatively correlated with temperature (r = ?0.38 for January–March 1920–1974; P < 0.01) during the previous growing season. These correlations suggest a temporally lagged growth response to summer moisture that could be associated with known physiological carry‐over processes in conifers and to a response to moisture variability at deeper layers of the rooting zone. Notably, satellite observations revealed a previously unobserved response of Araucaria growth to summer surface soil moisture during the current rather than the previous growing season (r = 0.65 for 1979–2000; P < 0.05). This new response has a large spatial footprint across the mid‐latitudes of the South American continent (35°–45°S) and highlights the potential of Araucaria tree rings for palaeoclimatic applications. The strong moisture constraint on tree growth revealed by satellite observations suggests that projected summer drying during the coming decades may result in regional growth declines in Araucaria forests and other water‐limited ecosystems in the Patagonian Andes.     

Humerus development in moles (Talpidae,Mammalia)

The humerus of fossorial moles has a highly derived anatomy, reflecting the ecological specialization of these animals for digging. It is short and broad, with enlarged muscle attachment sites and pronounced articulations compared to non‐fossorial sister taxa and other mammals. Both condyles are rotated in opposite directions, resulting in a torsion which is unique among eutherian mammals. The development of this exceptional bone was studied in embryonic stages of the fossorial Iberian mole (Talpa occidentalis) from mesenchymal condensation to incipient ossification based on histological serial sections using 3D reconstruction methods. For comparison, embryonic stages of the semi‐fossorial Japanese shrew mole (Urotrichus talpoides) as well as a sister taxon of moles, the terrestrial North American least shrew (Cryptotis parva), were studied. Results show that the humerus of Talpa already shows its derived anatomy with broadened muscle attachment sites and distinct articulations at early cartilaginous stages, when ossification has just started in the mid‐diaphyseal region. The torsion takes place simultaneously with the medial rotation of the forelimbs. The supracondylar foramen is closed in all studied Talpa embryos, but patent in Cryptotis and Urotrichus. This is an example of developmental penetrance, suggesting that variation of adult elements can be found at early stages as well.     

Reticulon 3 interacts with NS4B of the hepatitis C virus and negatively regulates viral replication by disrupting NS4B self‐interaction

The non‐structural protein 4B (NS4B) of the hepatitis C virus (HCV) is an endoplasmic reticulum (ER) membrane protein comprising two consecutive amphipathic α‐helical domains (AH1 and AH2). Its self‐oligomerization via the AH2 domain is required for the formation of the membranous web that is necessary for viral replication. Previously, we reported that the host‐encoded ER‐associated reticulon 3 (RTN3) protein is involved in the formation of the replication‐associated membranes of (+)RNA enteroviruses during viral replication. In this study, we demonstrated that the second transmembrane region of RTN3 competed for, and bound to, the AH2 domain of NS4B, thus abolishing NS4B self‐interaction and leading to the downregulation of viral replication. This interaction was mediated by two crucial residues, lysine 52 and tyrosine 63, of AH2, and was regulated by the AH1 domain. The silencing of RTN3 in Huh7 and AVA5 cells harbouring an HCV replicon enhanced the replication of HCV, which was counteracted by the overexpression of recombinant RTN3. The synthesis of viral RNA was also increased in siRNA‐transfected human primary hepatocytes infected with HCV derived from cell culture. Our results demonstrated that RTN3 acted as a restriction factor to limit the replication of HCV.