Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear: cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice. (C) 2009 Elsevier B.V. All rights reserved.

Intra- and interspecific variations in vacuolar flavonoids among Ficus species from the Budongo Forest, Uganda

Leaves of 14 species of Ficus growing in the Budongo Forest, Uganda, were analysed for vacuolar flavonoids. Three to six accessions were studied for each species to see whether there was intraspecific chemical variation. Thirty-nine phenolic compounds were identified or characterised, including 14 flavonol O-glycosides, six flavone O-glycosides and 15 flavone C-glycosides. In some species the flavonoid glycosides were acylated. Ficus thonningii contained in addition four stilbenes including glycosides. Most of the species could be distinguished from each other on the basis of their flavonoid profiles, apart from Ficus sansibarica and Ficus saussureana, which showed a very strong intraspecific variation. However, on the whole flavonoid profiles were sufficiently distinct to help in future identifications.

Extracellular disulfide exchange and the regulation of cellular function

An emerging concept is that disulfide bonds can act as a dynamic scaffold to present mature proteins in different conformational and functional states on the cell surface. Two examples are the conversion of the receptor, integrin a alpha(IIb)beta(3), from a low affinity to a high affinity state, and the interaction of CD4 receptor with the HIV-1 envelope glycoprotein gp120 to promote virus-cell fusion. In both of these cases there is a remodeling of the protein disulfide bonding pattern. The formation and rearrangement of disulfide bonds is modulated by a family of enzymes known as the thiol isomerases, which include protein disulfide isomerase (PDI), ERp5, ERp57, and ERp72. While these enzymes were reported originally to be restricted in location to the endoplasmic reticulum, in some cells thiol isomerases are found on the cell surface. This may indicate a wider role for these enzymes in cell function. In platelets it has been shown that reagents that react with cell surface sulfhydryl groups are capable of blocking a number of functional responses, including integrin-mediated aggregation, adhesion, and granule secretion. Furthermore, the use of function blocking antibodies to either PDI or ERp5 causes inhibition of these functional responses. This review summarizes current knowledge of the extracellular regulation of disulfide exchange and the implications of this in the regulation of cell function.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Intra-individual variation of serum prostate specific antigen levels in men with benign prostate biopsies

To determine the intra-individual (physiological) variation of prostate-specific antigen (PSA) measurements in men after a benign prostatic biopsy. Sixty-four men were prospectively assessed, all of whom had a benign prostatic biopsy within the preceding 13 months. The degree of intra-individual variability was established by calculating the coefficient of variation on four PSA levels obtained from each patient weekly over a month. Six patients were subsequently diagnosed with prostate cancer and their data are presented separately. In the remaining 58 patients the median (range) individual mean PSA value was 6.3 (0.5-34.1) ng/mL. The median (range) coefficient of variation within the group was 9.5 (2.4-76.1)%. There was a clear linear relationship between mean PSA level and the standard deviation. In 48 of the 63 patients analysed, the coefficient of variation for serum PSA values in the group as a whole was greater than the variation claimed for the assay technique. The significance of the linear relationship between PSA and the standard deviation is discussed, with particular reference to those men who had a benign prostate biopsy.

The structure of echovirus type 12 bound to a two-domain fragment of its cellular attachment protein decay-accelerating factor (CD 55)

Echovirus type 12 (EV12), an enterovirus of the Picornaviridae family, uses the complement regulator, decay-accelerating factor (DAF, CD55) as a cellular receptor. We have calculated a three-dimensional reconstruction of EV12 bound to a fragment of DAF, consisting of short consensus repeat domains 3 and 4, from cryo-negative stain electron microscopy data (EMD #1057). This shows that, as for an earlier reconstruction of the related echovirus type 7 bound to DAF, attachment is not within the viral canyon but occurs close to the two-fold symmetry axes. Despite this general similarity, our reconstruction reveals a receptor interaction that is quite different from that observed for EV7. Fitting of the crystallographic co-ordinates for DAF34 and EV11 into the reconstruction shows a close agreement between the crystal structure of the receptor fragment and the density for the virus-bound receptor, allowing unambiguous positioning of the receptor with respect to the virion (PDB #1UPN). Our finding that the mode of virus-receptor interaction in EV12 is distinct from that seen for EV7 raises interesting questions regarding the evolution and biological significance of the DAF-binding phenotype in these viruses.

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine ( DCVC) resulted in a > 1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.

4,4 '-Dipyridyl-N,N '-dioxide complexes of metal-thiocyanate/selenocyanate: pi-stacked molecular rods as three-dimensional support for two-dimensional polymeric sheets and intra/interchain S center dot center dot center dot S interaction dependent architecture of the R-2(2)(8) synthon driven assembly of one-dimensional polymeric chains

Three supramolecular complexes of Co(II) using SCN-/SeCN- in combination with 4,4'-dipyridyl-N,N'-dioxide (dpyo), i.e., {[Co(SCN)(2)(dpyo)(2)].(dpyo)}(n) ( 1), {[Co(SCN)(2)(dpyo)(H2O)(2)].(H2O)}(n) ( 2), {[Co(SeCN)(2)(dpyo)(H2O)(2)]center dot(H2O)}(n) ( 3), have been synthesized and characterized by single-crystal X-ray analysis. Complex 1 is a rare example of a dpyo bridged two-dimensional (2D) coordination polymer, and pi-stacked dpyo supramolecular rods are generated by the lattice dpyo, passing through the rhombic grid of stacked layers, resulting in a three-dimensional (3D) superstructure. Complexes 2 and 3 are isomorphous one-dimensional (1D) coordination polymers [-Co-dpyo-Co-] that undergo self-assembly leading to a bilayer architecture derived through an R-2(2)(8) H-bonding synthon between coordinated water and dpyo oxygen. A reinvestigation of coordination polymers [Mn(SCN)(2)(dpyo)( H2O)(MeOH)](n) ( 4) and {[Fe(SCN)(2)(dpyo)(H2O)(2)]center dot(H2O)}(n) ( 5) reported recently by our group [ Manna et al. Indian J. Chem. 2006, 45A, 1813] reveals brick wall topology rather than bilayer architecture is due to the decisive role of S center dot center dot center dot S/Se center dot center dot center dot Se interactions in determining the helical nature in 4 and 5 as compared to zigzag polymeric chains in 2 and 3, although the same R-2(2)(8) synthon is responsible for supramolecular assembly in these complexes.

Exploring context-awareness in the construction logistics services delivery

Managing a construction project supply chain effectively and efficiently is extremely difficult due to involvement of numerous sectors that are supported by ineffective communication system. An efficient construction supply chain system ensures the delivery of materials and other services to construction site while minimising costs and rewarding all sectors based on value added to the supply chain. The advancement of information, communication and wireless technologies is driving construction companies to deploy supply chain management strategies to seek better outputs. As part of the emerging wireless technologies, contextaware computing capability represents the next generation of ICT to the construction services. Conceptually, context-awareness could be integrated with Web Services in order to ensure the delivery of pertinent information to construction site and enhance construction supply chain collaboration. An initial study has indicated that this integrated system has the potential of serving and improving the construction services delivery through access to context-specific data, information and services on as-needed basis.

Intelligent wireless web services: context-aware computing in construction-logistics supply chain

The construction industry has incurred a considerable amount of waste as a result of poor logistics supply chain network management. Therefore, managing logistics in the construction industry is critical. An effective logistic system ensures delivery of the right products and services to the right players at the right time while minimising costs and rewarding all sectors based on value added to the supply chain. This paper reports on an on-going research study on the concept of context-aware services delivery in the construction project supply chain logistics. As part of the emerging wireless technologies, an Intelligent Wireless Web (IWW) using context-aware computing capability represents the next generation ICT application to construction-logistics management. This intelligent system has the potential of serving and improving the construction logistics through access to context-specific data, information and services. Existing mobile communication deployments in the construction industry rely on static modes of information delivery and do not take into account the worker’s changing context and dynamic project conditions. The major problems in these applications are lack of context-specificity in the distribution of information, services and other project resources, and lack of cohesion with the existing desktop based ICT infrastructure. The research works focus on identifying the context dimension such as user context, environmental context and project context, selection of technologies to capture context-parameters such wireless sensors and RFID, selection of supporting technologies such as wireless communication, Semantic Web, Web Services, agents, etc. The process of integration of Context-Aware Computing and Web-Services to facilitate the creation of intelligent collaboration environment for managing construction logistics will take into account all the necessary critical parameters such as storage, transportation, distribution, assembly, etc. within off and on-site project.

Do mitochondriotropic antioxidants prevent chlorinative stress-induced mitochondrial and cellular injury?

Reactive chlorine species such as hypochlorous acid ( HOCl) are cytotoxic oxidants generated by activated neutrophils at the sites of chronic inflammation. Since mitochondria are key mediators of apoptosis and necrosis, we hypothesized that mitochondriotropic antioxidants could limit HOCl-mediated intracellular oxidative injury to human fetal liver cells, preserve mitochondrial function, and prevent cell death. In this current study, we show that recently developed mitochondria-targeted antioxidants ( MitoQ and SS31) significantly protected against HOCl-induced mitochondrial damage and cell death at concentrations >= 25 nM. Our study highlights the potential application of mitochondria-specific targeted antioxidants for the prevention of cellular dysfunction and cell death under conditions of chlorinative stress, as occurs during chronic inflammation.

The pro-inflammatory oxidant hypochlorous acid induces Bax-dependent mitochondrial permeabilisation and cell death through AIF-/EndoG-dependent pathways

At sites of chronic inflammation, such as in the inflamed rheumatoid joint, activated neutrophils release hydrogen peroxide (H2O2) and the enzyme myeloperoxidase to catalyse the formation of hypochlorous acid (HOCl). 3-chlorotyrosine, a marker of HOCl in vivo, has been observed in synovial fluid proteins from rheumatoid arthritis patients. However the mechanisms of HOCl-induced cytotxicity are unknown. We determined the molecular mechanisms by which HOCl induced cell death in human mesenchymal progenitor cells (MPCs) differentiated into a chondrocytic phenotype as a model of human cartilage cells and show that HOCl induced rapid Bax conformational change, mitochondrial permeability and release of intra-mitochondrial pro-apoptotic proteins which resulted in nuclear translocation of AIF and EndoG. siRNA-mediated knockdown of Bax substantially prevented mitochondrial permeability, release of intra-mitochondrial pro-apoptotic proteins. Cell death was inhibited by siRNA-mediated knockdown of Bax, AIF or EndoG. Although we observed several biochemical markers of apoptosis, caspase activation was not detected either by western blotting, fluorescence activity assays or by using caspase inhibitors to inhibit cell death. This was further supported by findings that (1) in vitro exposure of recombinant human caspases to HOCl caused significant inhibition of caspase activity and (2) the addition of HOCl to staurosporine-treated MPCs inhibited the activity of cellular caspases. Our results show for the first time that HOCl induced Bax-dependent mitochondrial permeability which led to cell death without caspase activity by processes involving AIF/EndoG-dependent pathways. Our study provides a novel insight into the potential mechanisms of cell death in the inflamed human joint. (c) 2006 Elsevier Inc. All rights reserved.