Resolvin E2 formation and impact in inflammation resolution

Acute inflammation and its resolution are essential processes for tissue protection and homeostasis. In this context, specialized proresolving mediators derived from polyunsaturated fatty acids are of interest. In this study, we report that resolvin E2 (RvE2) from eicosapentaenoic acid is endogenously produced during self-limited murine peritonitis in both the initiation and resolution phases. RvE2 (1-10 nM) carries potent leukocyte-directed actions that include: 1) regulating chemotaxis of human neutrophils; and 2) enhancing phagocytosis and anti-inflammatory cytokine production. These actions appear to be mediated by leukocyte G-protein-coupled receptors as preparation of labeled RvE2 gave direct evidence for specific binding of radiolabeled RvE2 to neutrophils (K(d) 24.7 ± 10.1 nM) and resolvin E1 activation of recombinant G-protein-coupled receptors was assessed. In addition to the murine inflammatory milieu, RvE2 was also identified in plasma from healthy human subjects. RvE2 rapidly downregulated surface expression of human leukocyte integrins in whole blood and dampened responses to platelet-activating factor. Together, these results indicate that RvE2 can stimulate host-protective actions throughout initiation and resolution in the innate inflammatory responses.     

Transducin-like enhancer of Split-1 (TLE1) combines with Forkhead box protein G1 (FoxG1) to promote neuronal survival

Transducin-like enhancer of split-1 (TLE1) plays a critical role in the regulation of neurogenesis by inhibiting the differentiation of neural progenitor cells into neurons. Although TLE1 is also expressed highly in the postnatal brain and through adulthood, its role in postmitotic neurons is not clear. Using cultures of cerebellar granule neurons, we show that expression of TLE1 is reduced in neurons primed to die. Reestablishment of elevated TLE1 levels by ectopic expression protects neurons from death, whereas suppression of TLE1 expression in otherwise healthy neurons induces cell death. These results show that TLE1 is necessary for the maintenance of neuronal survival. Experiments using pharmacological inhibitors as well as expression of point mutants indicate that phosphorylation of TLE1 by casein kinase-2 (CK2) at Ser-239 and Ser-253 is necessary for its survival-promoting activity. TLE1-mediated survival is also inhibited by pharmacological inhibition of PI3K-Akt signaling but not by inhibitors of Raf-MEK-ERK signaling or other molecules, including histone deacetylases, calcium calmodulin kinase, or CK1. The survival-promoting activity of TLE1 depends critically on interaction with FoxG1, another protein involved in the regulation of neurogenesis and shown previously to promote survival of postmitotic neurons. Likewise, the ability of FoxG1 to promote neuronal survival depends on TLE1. Taken together, our study demonstrates that TLE1 cooperates with FoxG1 to promote neuronal survival in a CK2- and PI3K-Akt-dependent manner.     

Expression and distribution of grp-78/bip in mineralizing tissues and mesenchymal cells

Glucose-regulated protein 78 (GRP-78) is one of the many endoplasmic reticulum chaperone proteins that have been shown to possess multifunctional roles. We have previously demonstrated that GRP-78 functions as a receptor for dentin matrix protein 1 (DMP1) and is required for DMP1-mediated calcium release; that it is a secreted protein and can bind to type I collagen and DMP1 extracellularly and aid in the nucleation of calcium phosphate. We provide evidence in this study that tyrosine phosphorylation is required for DMP1/GRP-78-mediated calcium release in mesenchymal cells. We further demonstrate that GRP-78 is localized in the nucleus of mesenchymal cells and that the cell surface GRP-78 is not associated with the G-protein Gαq in mesenchymal cells. Results from this study show that during development of mineralized tissues, increased expression of GRP-78 can be observed in condensing cartilage and mesenchymal cells of the alveolar bone, endochondral bone and dental pulp. Additionally, we show that GRP-78 is present in the mineralizing matrices of teeth, bone and in the extracellular matrix of differentiating human marrow stromal cells and dental pulp stem cells. Collectively, our observations provide a new perspective on GRP-78 with respect to mineralized matrix formation.     

Protein Secondary Structure Determination by Constrained Single-Particle Cryo-Electron Tomography

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Highlights? Efficient extraction of high-resolution information from cryo-EM tilt series ? Successful CTF correction strategy despite low-contrast and quality of tilted images ? Use of geometric constraints in refinement improves orientational accuracy of images ? Enforcement of tomographic constraints reduces model bias and overfitting artifacts     

Visualization by atomic force microscopy and FISH of the 45S rDNA gaps in mitotic chromosomes of Lolium perenne

The mitotic chromosome structure of 45S rDNA site gaps in Lolium perenne was studied by atomic force microscope (AFM) combining with fluorescence in situ hybridization (FISH) analysis in the present study. FISH on the mitotic chromosomes showed that 45S rDNA gaps were completely broken or local despiralizations of the chromatid which had the appearance of one or a few thin DNA fiber threads. Topography imaging using AFM confirmed these observations. In addition, AFM imaging showed that the broken end of the chromosome fragment lacking the 45S rDNA was sharper, suggesting high condensation. In contrast, the broken ends containing the 45S rDNA or thin 45S rDNA fibers exhibited lower density and were uncompacted. Higher magnification visualization by AFM of the terminals of decondensed 45S rDNA chromatin indicated that both ends containing the 45S rDNA also exhibited lower density zones. The measured height of a decondensed 45S rDNA chromatin as obtained from the AFM image was about 55–65 nm, composed of just two 30-nm single fibers of chromatin. FISH in flow-sorted G2 interphase nuclei showed that 45S rDNA was highly decondensed in more than 90% of the G2/M nuclei. Our results suggested that a failure of the complex folding of the chromatin fibers occurred at 45S rDNA sites, resulting in gap formation or break.     

Epigallocatechin-3-gallate exhibits anti-fibrotic effect by attenuating bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in rat model pulmonary fibrosis

Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix components in the alveolar space, which hampers normal respiration process. Pathophysiological enzymes, glycoprotein moieties and matrix degrading lysosomal hydrolases are key markers and play a crucial role in the progression of fibrosis. Bleomycin is an anti-neoplastic drug, used for the treatment of various types of cancers and induces pulmonary fibrosis due its deleterious side effect. Tea catechin epigallocatechin-3-gallate (EGCG) is known for its wide array of beneficial effects. The present study was intended to evaluate the beneficial efficacy of EGCG against bleomycin-induced glycoconjugates, lysosomal hydrolases and ultrastructural changes in the lungs of Wistar rats. Intratracheal instillation of bleomycin (6.5 U/kg body weight) to rats increased the activities of pathophysiological enzymes such as aspartate transaminase, alanine transaminase, lactate dehydrogenase and alkaline phosphatase, which were attenuated upon EGCG treatment. The increased level of hydroxyproline and histopathological parameters in bleomycin-induced rats were decreased upon EGCG treatment. Bleomycin-induced increase in the level of glycoconjugates was restored closer to normal levels on EGCG treatment. Furthermore, the increased activities of matrix degrading lysosomal enzymes in bleomycin-induced rats were reduced upon EGCG supplementation. Treatment with EGCG also attenuated bleomycin-induced ultrastructural changes as observed from transmission electron microscopy studies. The results of the present study put-forward EGCG as a potential anti-fibrotic agent due to its attenuating effect on potential fibrotic markers.     

High-level expression of non-glycosylated and active staphylokinase from <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Staphylokinase (SAK) is a promising thrombolytic agent for treating blood-clotting disorders. Recombinant SAK (rSAK) was produced after integration of the gene into Pichia pastoris genome. The recombinant Pichia carrying multiple insertions of the SAK gene yielded high-level (~1 g/l) of extracellular glycosylated rSAK (~18 kDa) with negligible plasminogen activation activity. Addition of tunicamycin during the induction phase resulted in expression of non-glycosylated and highly active rSAK (~15 kDa) from the same clone. Two simple steps of ion-exchange chromatography produced an homogenous rSAK of >95% purity which suitable for future structural and functional studies.     

Determination of biomass composition of Catharanthus roseus hairy roots for metabolic flux analysis

Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering, and determination of biomass composition is indispensable to accurate flux evaluation. We report the elemental and biomolecular composition of Catharanthus roseus hairy roots, a pharmaceutically significant plant system and an important metabolic engineering target. The molecular formula of the organic material in the hairy roots was C12.0H22.7N0.4O7.6 during mid-exponential growth. The abundances of lipids, lignin, cellulose, hemicellulose, starch, protein, proteinogenic amino acids, mineral ash, and moisture in the biomass were quantified. Analysis of water-soluble components of the biomass with 1-D 13C and 2-D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water-soluble components were composed almost entirely of -glucans. Agropine, a frequently reported hairy root biomass component, was not detected. Our measurements of the biomass components quantified 83.6 +/- 9.3% (w/w) of the biomass. Together with previously reported abundances of indole alkaloids, we accounted for at least 85.9 +/- 11.6% (w/w) of the carbon in the biomass, which enabled the precise determination of 12 biomass synthesis fluxes.     

Novel self-cleavage activity of Staphylokinase fusion proteins: An interesting finding and its possible applications

Staphylokinase (SAK) is reported to have a serine protease domain with no proteolytic activity unlike other plasminogen activators like tissue plasminogen activator (t-PA) and urokinase. A unique protease property of Staphylokinase was observed when SAK was expressed as a fusion protein in inducible Escherichia coli expression vectors. This finding was further investigated by cloning and expressing different SAK fusions, both native and N-terminal deletions, with fusion tags like glutathione S-transferase (GST) and signal sequence of SAK in bacterial system. While all the N-terminal SAK fusions were found to self-cleave in crude and purified preparations, the C-terminal SAK fusion was stable. The cleavage property of Staphylokinase fusion proteins, inhibited by reduced glutathione and PMSF, was independent of its thrombolytic activity and also independent on the type of host employed for its expression. The serine protease domain of the SAK gene possibly lies between 20th to 77th amino acid and serine 41 of this region appears critical for such a cleavage property.