Smad3 signaling is required for satellite cell function and myogenic differentiation of myoblasts

TGF-β and myostatin are the two most important regulators of muscle growth. Both growth factors have been shown to signal through a Smad3-dependent pathway. However to date, the role of Smad3 in muscle growth and differentiation is not investigated. Here, we demonstrate that Smad3-null mice have decreased muscle mass and pronounced skeletal muscle atrophy. Consistent with this, we also find increased protein ubiquitination and elevated levels of the ubiquitin E3 ligase MuRF1 in muscle tissue isolated from Smad3-null mice. Loss of Smad3 also led to defective satellite cell (SC) functionality. Smad3-null SCs showed reduced propensity for self-renewal, which may lead to a progressive loss of SC number. Indeed, decreased SC number was observed in skeletal muscle from Smad3-null mice showing signs of severe muscle wasting. Further in vitro analysis of primary myoblast cultures identified that Smad3-null myoblasts exhibit impaired proliferation, differentiation and fusion, resulting in the formation of atrophied myotubes. A search for the molecular mechanism revealed that loss of Smad3 results in increased myostatin expression in Smad3-null muscle and myoblasts. Given that myostatin is a negative regulator, we hypothesize that increased myostatin levels are responsible for the atrophic phenotype in Smad3-null mice. Consistent with this theory, inactivation of myostatin in Smad3-null mice rescues the muscle atrophy phenotype.     

Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22

The Tom20 and Tom22 receptor subunits of the TOM (translocase of the outer mitochondrial membrane) complex recognize N-terminal presequences of proteins that are to be imported into the mitochondrion. In plants, Tom20 is C-terminally anchored in the mitochondrial membrane, whereas Tom20 is N-terminally anchored in animals and fungi. Furthermore, the cytosolic domain of Tom22 in plants is smaller than its animal/fungal counterpart and contains fewer acidic residues. Here, NMR spectroscopy was used to explore presequence interactions with the cytosolic regions of receptors from the plant Arabidopsis thaliana and the fungus Saccharomyces cerevisiae (i.e., AtTom20, AtTom22, and ScTom22). It was found that AtTom20 possesses a discontinuous bidentate hydrophobic binding site for presequences. The presequences on plant mitochondrial proteins comprise two or more hydrophobic binding regions to match this bidentate site. NMR data suggested that while these presequences bind to ScTom22, they do not bind to AtTom22. AtTom22, however, binds to AtTom20 at the same binding site as presequences, suggesting that this domain competes with the presequences of imported proteins, thereby enabling their progression along the import pathway.     

Ex vivo expansion of umbilical cord blood

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.     

Therapeutic angiogenesis for cardiovascular disease

The identification of angiogenic growth factors, such as vascular endothelial growth factor and fibroblast growth factor, has fueled interest in using such factors to induce therapeutic angiogenesis. The results of numerous animal studies and clinical trials have offered promise for new treatment strategies for various ischemic diseases. Increased understanding of the cellular and molecular biology of vessel growth has, however, prompted investigators and clinicians alike to reconsider the complexity of therapeutic angiogenesis. The realization that formation of a stable vessel is a complex, multistep process may provide useful insights into the design of the next generation of angiogenesis therapy.     

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation

The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.     

Leucine-rich repeat (LRR) proteins: integrators of pattern recognition and signaling in immunity

The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in plants, invertebrates and vertebrates. Serving as a first line of defense, the innate immune response is initiated through the sensing of pathogen-associated molecular patterns (PAMPs). In plants, NBS (nucleotide-binding site)-LRR proteins provide recognition of pathogen products of avirulence (AVR) genes. LRRs also promote interaction between LRR proteins as observed in receptor-coreceptor complexes. In mammals, toll-like receptors (TLRs) and NOD-like receptors (NLRs) through their LRR domain, sense molecular determinants from a structurally diverse set of bacterial, fungal, parasite and viral-derived components. In humans, at least 34 LRR proteins are implicated in diseases. Most LRR domains consist of 2-45 leucine-rich repeats, with each repeat about 20-30 residues long. Structurally, LRR domains adopt an arc or horseshoe shape, with the concave face consisting of parallel β-strands and the convex face representing a more variable region of secondary structures including helices. Apart from the TLRs and NLRs, most of the 375 human LRR proteins remain uncharacterized functionally. We incorporated computational and functional analyses to facilitate multifaceted insights into human LRR proteins and outline a few approaches here.     

A proteomic approach in investigating the hepatoprotective mechanism of Schisandrin B: role of raf kinase inhibitor protein

To identify key proteins involved in the hepatoprotection afforded by schisandrin B (Sch B), we used a proteomic approach to screen proteins that were specifically regulated by Sch B in mouse livers and to investigate the role of the proteins in hepatoprotection. Thirteen proteins were specifically activated or suppressed by Sch B treatment. Among the 13 proteins, Raf kinase inhibitor protein (RKIP) was postulated to be the key regulator involved in the development of hepatotoxin-induced cellular damage. The results indicated that the downregulation of RKIP by antisense RKIP vector transfection led to the activation of the Raf-1/MEK/ERK signaling pathway, as evidenced by increases in the level of MEK/ERK phosphorylation and the level of nuclear factor erythroid 2-related factor 2 in the nucleus. The signaling effect produced by RKIP downregulation resembled that triggered by Sch B, wherein both treatments resulted in a decrease in the extent of carbon tetrachloride-induced apoptotic cell death in AML12 hepatocytes. Overexpression of RKIP by the sense RKIP transfection vector or the inhibition of MEK kinase by PD98059 was able to abrogate the cytoprotective effect of Sch B in the hepatocytes. The results indicate that Sch B triggers the Raf/MEK/ERK signaling pathway, presumably by downregulating RKIP, thereby protecting against carbon tetrachloride-induced cytotoxicity.     

First report of a novel plant lysozyme with both antifungal and antibacterial activities

A novel lysozyme exhibiting antifungal activity and with a molecular mass of 14.4kDa in SDS-polyacrylamide gel electrophoresis was isolated from mung bean (Phaseolus mungo) seeds using a procedure that involved aqueous extraction, ammonium sulfate precipitation, ion exchange chromatography on CM-Sephadex, and high-performance liquid chromatography on POROS HS-20. Its N-terminal sequence was very different from that of hen egg white lysozyme. Its pI was estimated to be above 9.7. The specific activity of the lysozyme was 355U/mg at pH 5.5 and 30 degrees C. The lysozyme exhibited a pH optimum at pH 5.5 and a temperature optimum at 55 degrees C. It is reported herein, for the first time, that a novel plant lysozyme exerted an antifungal action toward Fusarium oxysporum, Fusarium solani, Pythium aphanidermatum, Sclerotium rolfsii, and Botrytis cinerea, in addition to an antibacterial action against Staphylococcus aureus.