Histological and transcriptome-wide level characteristics of fetal myofiber hyperplasia during the second half of gestation in Texel and Ujumqin sheep

<p>Abstract</p> <p>Background</p> <p>Whether myofibers increase with a pulsed-wave mode at particular developmental stages or whether they augment evenly across developmental stages in large mammals is unclear. Additionally, the molecular mechanisms of myostatin in myofiber hyperplasia at the fetal stage in sheep remain unknown. Using the first specialized transcriptome-wide sheep oligo DNA microarray and histological methods, we investigated the gene expression profile and histological characteristics of developing fetal ovine longissimus muscle in Texel sheep (high muscle and low fat), as a myostatin model of natural mutation, and Ujumqin sheep (low muscle and high fat). Fetal skeletal muscles were sampled at 70, 85, 100, 120, and 135 d of gestation.</p> <p>Results</p> <p>Myofiber number increased sharply with a pulsed-wave mode at certain developmental stages but was not augmented evenly across developmental stages in fetal sheep. The surges in myofiber hyperplasia occurred at 85 and 120 d in Texel sheep, whereas a unique proliferative surge appeared at 100 d in Ujumqin sheep. Analysis of the microarray demonstrated that immune and hematological systems' development and function, lipid metabolism, and cell communication were the biological functions that were most differentially expressed between Texel and Ujumqin sheep during muscle development. Pathways associated with myogenesis and the proliferation of myoblasts, such as calcium signaling, chemokine (C-X-C motif) receptor 4 signaling, and vascular endothelial growth factor signaling, were affected significantly at specific fetal stages, which underpinned fetal myofiber hyperplasia and postnatal muscle hypertrophy. Moreover, we identified some differentially expressed genes between the two breeds that could be potential myostatin targets for further investigation.</p> <p>Conclusions</p> <p>Proliferation of myofibers proceeded in a pulsed-wave mode at particular fetal stages in the sheep. The myostatin mutation changed the gene expression pattern in skeletal muscle at a transcriptome-wide level, resulting in variation in myofiber phenotype between Texel and Ujumqin sheep during the second half of gestation. Our findings provide a novel and dynamic description of the effect of myostatin on skeletal muscle development, which contributes to understanding the biology of muscle development in large mammals.</p

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The Atypical Calpains: Evolutionary Analyses and Roles in Caenorhabditis elegans Cellular Degeneration

The calpains are physiologically important Ca2+-activated regulatory proteases, which are divided into typical or atypical sub-families based on constituent domains. Both sub-families are present in mammals, but our understanding of calpain function is based primarily on typical sub-family members. Here, we take advantage of the model organism Caenorhabditis elegans, which expresses only atypical calpains, to extend our knowledge of the phylogenetic evolution and function of calpains. We provide evidence that a typical human calpain protein with a penta EF hand, detected using custom profile hidden Markov models, is conserved in ancient metazoans and a divergent clade. These analyses also provide evidence for the lineage-specific loss of typical calpain genes in C. elegans and Ciona, and they reveal that many calpain-like genes lack an intact catalytic triad. Given the association between the dysregulation of typical calpains and human degenerative pathologies, we explored the phenotypes, expression profiles, and consequences of inappropriate reduction or activation of C. elegans atypical calpains. These studies show that the atypical calpain gene, clp-1, contributes to muscle degeneration and reveal that clp-1 activity is sensitive to genetic manipulation of [Ca2+]i. We show that CLP-1 localizes to sarcomeric sub-structures, but is excluded from dense bodies (Z-disks). We find that the muscle degeneration observed in a C. elegans model of dystrophin-based muscular dystrophy can be suppressed by clp-1 inactivation and that nemadipine-A inhibition of the EGL-19 calcium channel reveals that Ca2+ dysfunction underlies the C. elegans MyoD model of myopathy. Taken together, our analyses highlight the roles of calcium dysregulation and CLP-1 in muscle myopathies and suggest that the atypical calpains could retain conserved roles in myofilament turnover

Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism.

In rat hepatocytes subjected to a fructose load, ATP content decreased from 3.8 to 2.6 micromol/g of cells. Under these conditions, the intracellular free Mg2+ ion concentration,as measured with mag-fura 2, increased from 0.25 to 0.43 micromol/g of cells and 0.35 micromol of Mg2+ ions were released per g of cells in the extracellular medium. Therefore the increase in the intracellular free Mg2+ ion concentration was less than expected from the decrease in ATP, indicating that approx. 80% of the Mg2+ ions released from MgATP2- were buffered inside the cells. When this buffer capacity was challenged with an extra Mg2+ ion load by blocking the fructose-induced Mg2+ efflux, again approx. 80% of the extra Mg2+ ion load was buffered. The remaining 20% appearing as free Mg2+ions in fructose-treated hepatocytes could act as second messenger for enzymes having a Km for Mg2+ in the millimolar range. Fructose activated glycogen synthase and glycogen phosphorylase, although both the time course and the dose-dependence of activation were different. This was reflected in a stimulation of glycogen synthesis with concentrations of fructose below 5 mM. Indeed, activation of glycogen synthase reached a maximum at 30 min of incubation and was observed with small (5 mM or less) concentrations of fructose, whereas the activation of glycogen phosphorylase was almost immediate (within 5 min) and maximal with large doses of fructose. The fructose-induced activation of glycogen phosphorylase, but not that of glycogen synthase, could be related to an increase in free Mg2+ ion concentration