A naturally occurring calcineurin variant inhibits FoxO activity and enhances skeletal muscle regeneration

The calcium-activated phosphatase calcineurin (Cn) transduces physiological signals through intracellular pathways to influence the expression of specific genes. Here, we characterize a naturally occurring splicing variant of the CnAβ catalytic subunit (CnAβ1) in which the autoinhibitory domain that controls enzyme activation is replaced with a unique C-terminal region. The CnAβ1 enzyme is constitutively active and dephosphorylates its NFAT target in a cyclosporine-resistant manner. CnAβ1 is highly expressed in proliferating myoblasts and regenerating skeletal muscle fibers. In myoblasts, CnAβ1 knockdown activates FoxO-regulated genes, reduces proliferation, and induces myoblast differentiation. Conversely, CnAβ1 overexpression inhibits FoxO and prevents myotube atrophy. Supplemental CnAβ1 transgene expression in skeletal muscle leads to enhanced regeneration, reduced scar formation, and accelerated resolution of inflammation. This unique mode of action distinguishes the CnAβ1 isoform as a candidate for interventional strategies in muscle wasting treatment.     

Isoform-specific downregulation of peroxiredoxin in human failing myocardium

Peroxiredoxins (Prx) are a family of antioxidant thioredoxin or glutathione dependent peroxidases. The major functions of Prx comprise modulation of signalling cascades that apply hydrogen peroxide (H(2)O(2)) and cellular protection against oxidative stress. Nothing is known about Prx isoforms in human myocardium. We investigated the protein expression of Prx isoforms 1-6 in human non-failing (NF, donor hearts, n=6, male, age: 53.3+/-2.1 years) and failing myocardium (DCM, orthotopic heart transplantation, dilated cardiomyopathy, n=15, male, 57.0+/-1.7 years). In addition, we performed immunohistochemical stainings and measured Prx 4 mRNA expression levels (RNAse protection assay). The protein expression of Prx 1-2 was similar in NF and DCM. The protein expression of Prx 3-6 and the mRNA-expression of Prx 4 were decreased in DCM. Immunohistochemical analyses provided evidence that all Prx isoforms are present in cardiomyocytes and endothelial cells. Whereas Prx 1-5 staining was more pronounced in endothelial cells, Prx6 staining was more evident in cardiomyocytes. This study provides evidence that Prx are differentially regulated in DCM. The selective downregulation of peroxiredoxin 3-6 isoforms may point towards a subcellular specific dysregulation of the antioxidative defence during the development of DCM.     

Temperature dependence of erythromelalgia mutation L858F in sodium channel Nav1.7

Background

The disabling chronic pain syndrome erythromelalgia (also termed erythermalgia) is characterized by attacks of burning pain in the extremities induced by warmth. Pharmacological treatment is often ineffective, but the pain can be alleviated by cooling of the limbs. Inherited erythromelalgia has recently been linked to mutations in the gene SCN9A, which encodes the voltage-gated sodium channel Nav1.7. Nav1.7 is preferentially expressed in most nociceptive DRG neurons and in sympathetic ganglion neurons. It has recently been shown that several disease-causing erythromelalgia mutations alter channel-gating behavior in a manner that increases DRG neuron excitability.

Results

Here we tested the effects of temperature on gating properties of wild type Nav1.7 and mutant L858F channels. Whole-cell voltage-clamp measurements on wild type or L858F channels expressed in HEK293 cells revealed that cooling decreases current density, slows deactivation and increases ramp currents for both mutant and wild type channels. However, cooling differentially shifts the midpoint of steady-state activation in a depolarizing direction for L858F but not for wild type channels.

Conclusion

The cooling-dependent shift of the activation midpoint of L858F to more positive potentials brings the threshold of activation of the mutant channels closer to that of wild type Nav1.7 at lower temperatures, and is likely to contribute to the alleviation of painful symptoms upon cooling in affected limbs in patients with this erythromelalgia mutation.

     

The analysis of the mechanosensory origin of the infrared sensilla in Melanophila acuminata (Coeloptera; Buprestidae) adduces new insight into the transduction mechanism

The thoracic infrared (IR) sensilla of the pyrophilous jewel beetle Melanophila acuminata most likely have evolved from hair mechanoreceptors (sensilla trichodea). To further elucidate the sensory transduction mechanism, the morphology of IR sensilla and of neighbouring hair mechanoreceptors was investigated by using conventional electron microscopical techniques (SEM, TEM) in combination with focused ion beam milling (FIB). It was assumed that any deviation from the bauplan of a sensillum trichodeum is of particular concern for the transduction of IR radiation into a mechanical stimulus. Thus, the structures supposed to be relevant for stimulus uptake and transduction were homologized. Compared to a hair mechanoreceptor, an IR sensillum shows the following special features: (i) the formation of a complex cuticular sphere instead of the bristle; the sphere consists of an outer exocuticular shell as well as of an inner porous mesocuticular part. (ii) The enclosure of the dendritic tip of the mechanosensitive neuron inside the sphere in a fluid-filled inner pressure chamber which is connected with a system of microcavities and nanocanals in the mesocuticular part. Hence we propose that an IR sensillum most probably acts as a microfluidic converter of infrared radiation into an increase in internal pressure inside the sphere which is measured by the mechanosensitive neuron.     

GrlJ, a Dictyostelium GABAB-like receptor with roles in post-aggregation development

Background

Term Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).

Results

The recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells.

Conclusion

The current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.     

Rapid control of protein level in the apicomplexan Toxoplasma gondii

Analysis of gene function in apicomplexan parasites is limited by the absence of reverse genetic tools that allow easy and rapid modulation of protein levels. The fusion of a ligand-controlled destabilization domain (ddFKBP) to a protein of interest enables rapid and reversible protein stabilization in T. gondii. This allows an efficient functional analysis of proteins that have a dual role during host cell invasion and/or intracellular growth of the parasite.     

Fermentation metabolism in roots of wheat seedlings after hypoxic pre-treatment in different anoxic incubation systems

A hypoxic pre-treatment (HPT) can improve the anoxic survival of flooding sensitive plants. Here, we tested whether a 4-d HPT of wheat plants (Triticum aestivum L.) would improve their anoxic resistance, and if so, why. We found that the metabolic adjustment during prolonged HPT involved an increased lactate excretion rate, the up-regulation of glycolytic and fermentative enzymes as well as the accumulation of various sugars. Therefore, HPT wheat roots could sustain a 3 times higher ethanolic fermentation rate during an anoxic period compared to non-pre-treated (NHPT) roots. Nevertheless, the enhanced fermentation rate provided temporary relief to the energy crisis only, and both NHPT and HPT plants died after 5d of anoxia in illumination. Comparison of different low oxygen incubation systems using excised roots or roots of intact plants revealed striking differences. The benefits of intact shoots, oxygen transport as well as additional sugar supply enabled a more stable energy supply of anoxia-treated NHPT and HPT roots. However, the height of the fermentation rate was correlated with a high ATP content during dark anoxic incubation, but not in illumination.