GSK-3beta activity is increased in skeletal muscle after burn injury in rats

Previous reports suggest that burn-induced muscle proteolysis can be inhibited by treatment with GSK-3beta inhibitors, suggesting that burn injury may be associated with increased GSK-3beta activity. The influence of burn injury on muscle GSK-3beta activity, however, is not known. We determined the effect of a 30% total body surface full-thickness burn injury in rats on muscle GSK-3beta activity by measuring GSK-3beta activity and tissue levels of serine 9 phosphorylated GSK-3beta, p(Ser9)-GSK-3beta, by Western blot analysis and immunohistochemistry. Because burn-induced muscle wasting is, at least in part, mediated by glucocorticoids, we used dexamethasone-treated cultured muscle cells in which GSK-3beta expression was reduced with small interfering RNA (siRNA) to further assess the role of GSK-3beta in muscle atrophy. Burn injury resulted in a seven-fold increase in GSK-3beta activity in skeletal muscle. This effect of burn was accompanied by reduced tissue levels of p(Ser9)-GSK-3beta, suggesting that burn injury stimulates GSK-3beta in skeletal muscle secondary to inhibited phosphorylation of the enzyme. In addition, burn injury resulted in inhibited phosphorylation and activation of Akt, an upstream regulatory mechanism of GSK-3beta activity. Reducing the expression of GSK-3beta in cultured muscle cells with siRNA inhibited dexamethasone-induced protein degradation by approximately 50%. The results suggest that burn injury stimulates GSK-3beta activity in skeletal muscle and that GSK-3beta may, at least in part, regulate glucocorticoid-mediated muscle wasting.     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Molecular motors: the driving force behind mammalian left-right development

The molecular motors dynein and kinesin are large protein complexes that convert the energy generated by ATP hydrolysis into directional movement along the microtubule cytoskeleton. They are required for a myriad of cellular processes, including mitotic spindle movement, axonal and vesicular transport, and ciliary beating. Recently, it has been shown that, in addition, they have a unique role during embryonic patterning: they are required to orient and establish the left-right axis in early vertebrate development.     

Characterization of Hair Follicle Development in Engineered Skin Substitutes

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.     

Integrating community assembly and biodiversity to better understand ecosystem function: the Community Assembly and the Functioning of Ecosystems (CAFE) approach

The research of a generation of ecologists was catalysed by the recognition that the number and identity of species in communities influences the functioning of ecosystems. The relationship between biodiversity and ecosystem functioning (BEF) is most often examined by controlling species richness and randomising community composition. In natural systems, biodiversity changes are often part of a bigger community assembly dynamic. Therefore, focusing on community assembly and the functioning of ecosystems (CAFE), by integrating both species richness and composition through species gains, losses and changes in abundance, will better reveal how community changes affect ecosystem function. We synthesise the BEF and CAFE perspectives using an ecological application of the Price equation, which partitions the contributions of richness and composition to function. Using empirical examples, we show how the CAFE approach reveals important contributions of composition to function. These examples show how changes in species richness and composition driven by environmental perturbations can work in concert or antagonistically to influence ecosystem function. Considering how communities change in an integrative fashion, rather than focusing on one axis of community structure at a time, will improve our ability to anticipate and predict changes in ecosystem function.     

Increased expression of integrins and decreased apoptosis correlate with increased melanocyte retention in cultured skin substitutes

Losses of human melanocytes (HM) in transplantation of cultured skin substitutes (CSS) may result from poor cellular attachments. To test this hypothesis, HM integrin expression was measured in four culture media: (a) melanocyte growth medium (MGM), an HM proliferation medium; (b) UCMC 160, a CSS maturation medium; (c) mMGM, modified MGM with 1.8 mM calcium; and (d) modified UCMC 160 with HM supplements (mUCMC 160). HM grew well in all media except UCMC 160. Increased expression of beta1, beta4, alpha3beta1 and alpha5 integrins on HM cultured in MGM and mMGM versus UCMC 160 was found by flow cytometry. Annexin V-allophycocyanin (APC) labeled HM in apoptosis and increased significantly in UCMC 160 (31.1%) compared with MGM (11.9%) or mMGM (13.9%). CSS were incubated in UCMC 160, mMGM or mUCMC 160 media, and grafted to athymic mice. In the mMGM group, grafts were darker as measured with a chromameter through 6 weeks and the average number of basal HM per field was greater at 12 weeks post-grafting. Increased graft loss was observed in the mMGM group which corresponded with the poor epidermal morphology in vitro. Although HM retention improved in vivo using mMGM to culture the CSS, the stability of the epidermis decreased. These results indicate that expression of integrins on HM in vitro correlates with HM retention in CSS and short-term survival after transplantation, but that long-term survival depends also on stable epithelium.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Inhibition of phosphodiesterase 4 modulates cytokine induction from toll like receptor activated,but not rhinovirus infected,primary human airway smooth muscle

Background

Virus-induced exacerbations of Chronic Obstructive Pulmonary Disease (COPD) are a significant health burden and occur even in those receiving the best current therapies. Rhinovirus (RV) infections are responsible for half of all COPD exacerbations. The mechanism by which exacerbations occur remains undefined, however it is likely to be due to virus-induced inflammation. Given that phophodiesterase 4 (PDE₄) inhibitors have an anti-inflammatory effect in patients with COPD they present a potential therapy prior to, and during, these exacerbations.

Methods

In the present study we investigated whether the PDE₄ inhibitor piclamilast (10^-6 M) could alter RV or viral mimetic (5 μg/mL of imiquimod or poly I:C) induced inflammation and RV replication in primary human airway smooth muscle cells (ASMC) and bronchial epithelial cells (HBEC). The mediators IL-6, IL-8, prostaglandin E₂ and cAMP production were assayed by ELISA and RV replication was assayed by viral titration.

Results

We found that in ASMCs the TLR3 agonist poly I:C induced IL-8 release was reduced while induced IL-6 release by the TLR7/8 agonist imiquimod was further increased by the presence of piclamilast. However, in RV infected ASMCs, virus replication and induced mediator release were unaltered by piclamilast, as was also found in HBECs. The novel findings of this study reveal that although PDE inhibitors may not influence RV-induced cytokine production in ASMCs and replication in either ASMCs or HBECs, they have the capacity to be anti-inflammatory during TLR activation by modulating the induction of these chemotactic cytokines.

Conclusion

By extrapolating our in vitro findings to exacerbations of COPD in vivo this suggests that PDE₄ inhibitors may have beneficial anti-inflammatory properties when patients are infected with bacteria or viruses other than RV.