Mechanical stretch activates signaling events for protein translation initiation and elongation in C2C12 myoblasts

It has been proposed that mechanically induced tension is the critical factor in the induction of muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, the effect of mechanical stretch on intracellular signaling for protein translation initiation and elongation was studied in C2C12 myoblasts. Cells were grown on a silicone elastomer chamber and subjected to 30-min of 5 or 15% constant static or cyclic (60 cycles/min) uniaxial stretch. Western blot analyses revealed that p70 S6 kinase (p70S6K) and eukaryotic elongation factor 2 (eEF2), which are the markers for translation initiation and peptide chain elongation, respectively, were activated by both static and cyclic stretch. The magnitude of activation was greater in response to the 15% cyclic stretch. Cyclic stretch also increased the phosphorylation of MAP kinases (p38 MAPK, ERK1/2 and JNK). However, the pharmacological inhibition of MAP kinases did not block the stretch-induced activation of p70S6K and eEF2. An inhibitor of the mammalian target of rapamycin (mTOR) blocked the stretch-induced phosphorylation of p70S6K but did not affect the eEF2 activation. A broad-range tyrosine kinase inhibitor, genistein, blocked the stretch-induced activation of p70S6K and eEF2, whereas Src tyrosine kinase and Janus kinase (JAK) inhibitors did not. These results suggest that the stretch-induced activation of protein translation initiation and elongation in mouse myoblast cell lines is mediated by tyrosine kinase(s), except for Src kinase or JAK.     

Citral,a component of lemongrass oil,activates PPARα and γ and suppresses COX-2 expression

Lemongrass is a widely used herb as a food flavoring, as a perfume, and for its analgesic and anti-inflammatory purposes; however, the molecular mechanisms of these effects have not been elucidated. Previously, we identified carvacrol from the essential oil of thyme as a suppressor of cyclooxygenase (COX)-2, a key enzyme for prostaglandin synthesis, and also an activator of peroxisome proliferator-activated receptor (PPAR), a molecular target for “lifestyle-related” diseases. In this study, we evaluated the essential oil of lemongrass using our established assays for COX-2 and PPARs. We found that COX-2 promoter activity was suppressed by lemongrass oil in cell-based transfection assays, and we identified citral as a major component in the suppression of COX-2 expression and as an activator of PPARα and γ. PPARγ-dependent suppression of COX-2 promoter activity was observed in response to citral treatment. In human macrophage-like U937 cells, citral suppressed both LPS-induced COX-2 mRNA and protein expression, dose-dependently. Moreover, citral induced the mRNA expression of the PPARα-responsive carnitine palmitoyltransferase 1 gene and the PPARγ-responsive fatty acid binding protein 4 gene, suggesting that citral activates PPARα and γ, and regulates COX-2 expression. These results are important for understanding the anti-inflammatory and anti-lifestyle-related disease properties of lemongrass.     

Mdm10 as a dynamic constituent of the TOB/SAM complex directs coordinated assembly of Tom40

The mitochondrial outer membrane contains two protein translocators: the TOM40 and TOB/SAM complexes. Mdm10 is distributed in the TOB complex for β‐barrel protein assembly and in the MMM1 complex for tethering of the endoplasmic reticulum and mitochondria. Here, we establish a system in which the Mdm10 level in the TOB complex—but not in the MMM1 complex—is altered to analyse its part in β‐barrel protein assembly. A decrease in the Mdm10 level results in accumulation of in vitro imported Tom40, which is a β‐barrel protein, at the level of the TOB complex. An increase in the Mdm10 level inhibits association not only of Tom40 but also of other β‐barrel proteins with the TOB complex. These results show that Mdm10 regulates the timing of release of unassembled Tom40 from the TOB complex, to facilitate its coordinated assembly into the TOM40 complex.     

Therapeutic angiogenesis by transplantation of induced pluripotent stem cell-derived Flk-1 positive cells

Background

The ubiquitously expressed POU homeodomain protein Oct-1 serves as a sensor for stress induced by irradiation. We found recently that in pancreatic and intestinal endocrine cells, Oct-1 also functions as a sensor for cyclic AMP (cAMP). The caudal homeobox gene Cdx-2 is a transactivator of proglucagon (gcg) and pro-insulin genes. Oct-1 binds to Cdx-2 promoter and represses its expression. cAMP elevation leads to increased nuclear exclusion of Oct-1, associated with reduced recruitment of nuclear co-repressors to the Cdx-2 promoter and increased Cdx-2 expression.

Results

We show in this study that inducing oxidative stress by hydrogen peroxide (H₂O₂) increased nuclear Oct-1 content in both pancreatic α and β cell lines, as well as in a battery of other cells. This increase was then attributed to accelerated nuclear import of Oct-1, assessed by Fluorescence Recovery After Photobleaching (FRAP) using green fluorescence protein (EGFP) tagged Oct-1 molecule. H₂O₂ treatment was then shown to stimulate the activities of DNA-dependent protein kinase (DNA-PK) and c-jun N-terminal kinase (JNK). Finally, increased Oct-1 nuclear content upon H₂O₂ treatment in a pancreatic α cell line was associated with reduced Cdx-2 and gcg mRNA expression.

Conclusion

These observations suggest that Oct-1 functions as a sensor for both metabolic and stress/survival signaling pathways via altering its nuclear-cytoplasmic shuttling.     

Codon optimization of human parvovirus B19 capsid genes greatly increases their expression in nonpermissive cells

Parvovirus B19 (B19V) is pathogenic for humans and has an extreme tropism for human erythroid progenitors. We report cell type-specific expression of the B19V capsid genes (VP1 and VP2) and greatly increased B19V capsid protein production in nonpermissive cells by codon optimization. Codon usage limitation, rather than promoter type and the 3' untranslated region of the capsid genes, appears to be a key factor in capsid protein production in nonpermissive cells. Moreover, B19 virus-like particles were successfully generated in nonpermissive cells by transient transfection of a plasmid carrying both codon-optimized VP1 and VP2 genes.     

Mice Deficient in N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase Are Unable to Synthesize Chondroitin/Dermatan Sulfate containing N-Acetylgalactosamine 4,6-Bissulfate Residues and Exhibit Decreased Protease Activity in Bone Marrow-derived Mast Cells

Chondroitin sulfate (CS) and dermatan sulfate (DS) containing N-acetylgalactosamine 4,6-bissulfate (GalNAc(4,6-SO₄)) show various physiological activities through interacting with numerous functional proteins. N-Acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate from 3′-phosphoadenosine 5′-phosphosulfate to position 6 of N-acetylgalactosamine 4-sulfate in CS or DS to yield GalNAc(4,6-SO₄) residues. We here report generation of transgenic mice that lack GalNAc4S-6ST. GalNAc4S-6ST-null mice were born normally and fertile. In GalNAc4S-6ST-null mice, GalNAc(4,6-SO₄) residues in CS and DS disappeared completely, indicating that GalNAc4S-6ST should be a sole enzyme responsible for the synthesis of GalNAc(4,6-SO₄) residues in both CS and DS. IdoA-GalNAc(4,6-SO₄) units that account for ∼40% of total disaccharide units of DS in the liver of the wild-type mice disappeared in the liver DS of GalNAc4S-6ST-null mice without reduction of IdoA content. Bone marrow-derived mast cells (BMMCs) derived from GalNAc4S-6ST-null mice contained CS without GlcA-GalNAc(4,6-SO₄) units. Tryptase and carboxypeptidase A activities of BMMCs derived from GalNAc4S-6ST-null mice were lower than those activities of BMMCs derived from wild-type mice, although mRNA expression of these mast cell proteases was not altered. Disaccharide compositions of heparan sulfate/heparin contained in the mast cells derived from BMMCs in the presence of stem cell factor were much different from those of heparan sulfate/heparin in BMMCs but did not differ significantly between wild-type mice and GalNAc4S-6ST-null mice. These observations suggest that CS containing GalNAc(4,6-SO₄) residues in BMMCs may contribute to retain the active proteases in the granules of BMMCs but not for the maturation of BMMCs into connective tissue-type mast cells.     

Hormone-sensitive lipase is critical mediators of acute exercise-induced regulation of lipolysis in rat adipocytes

The purpose of the present study was to investigate the effect of acute exercise on lipolysis via coordination of hormone-sensitive lipase (HSL) and scaffold proteins, i.e., perilipin A and comparative gene identification-58 (CGI-58), in rat primary adipocytes. Glycerol release was significantly elevated immediately (0 h) and three hours (3 h) after exercise. Both activity and localization to the pellet of HSL were significantly greater in the pellet fraction, which is included in lipid droplet associated-proteins, than in the supernatant fraction. In the pellet fraction, although neither perilipin A nor CGI-58 protein level changed, level of perilipin A/CGI-58 complex was significantly reduced, accompanied by up-regulated association of perilipin A/HSL at 0 h and 3 h after exercise. On the other hand, there were no changes in these molecules at 24 h after exercise, despite a significant decrease in lipolysis that was observed in response to isoproterenol. These findings suggest that acute exercise enhances lipolysis up to at least 3 h after exercise in a manner dependent on modification of HSL and its association with and alteration in scaffold protein.     

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial

Background

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes.

Results

The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans.

Conclusion

Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.     

Carbon nanotube reinforced Bombyx mori silk nanofibers by the electrospinning process

Nanocomposite fibers of Bombyx mori silk and single wall carbon nanotubes (SWNT) were produced by the electrospinning process. Regenerated silk fibroin dissolved in a dispersion of carbon nanotubes in formic acid was electrospun into nanofibers. The morphology, structure, and mechanical properties of the electrospun nanofibers were examined by field emission environmental scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, and microtensile testing. TEM of the reinforced fibers shows that the single wall carbon nanotubes are embedded in the fibers. The mechanical properties of the SWNT reinforced fiber show an increase in Young's modulus up to 460% in comparison with the un-reinforced aligned fiber, but at the expense of the strength and strain to failure.