Increasing myosin light chain 3f (MLC3f) protects against a decline in contractile velocity

Disuse induces adaptations in skeletal muscle, which lead to muscle deterioration. Hindlimb-unloading (HU) is a well-established model to investigate cellular mechanisms responsible for disuse-induced skeletal muscle dysfunction. In myosin heavy chain (MHC) type IIB fibers HU induces a reduction in contraction speed (Vo) and a reduction in the relative myosin light chain 3f (MLC3f) protein content compared with myosin light chain 1f (MLC1f) protein. This study tested the hypothesis that increasing the relative MLC3f protein content via rAd-MLC3f vector delivery would attenuate the HU-induced decline in Vo in single MHC type IIB fibers. Fischer-344 rats were randomly assigned to one of three groups: control, HU for 7 days, and HU for 7 days plus rAd-MLC3f. The semimembranosus muscles were injected with rAd-MLC3f (3.75 x 1011-5 x 1011 ifu/ml) at four days after the initiation of HU. In single MHC type IIB fibers the relative MLC3f content decreased by 25% (12.00±0.60% to 9.06±0.66%) and Vo was reduced by 29% (3.22±0.14fl/s vs. 2.27±0.08fl/s) with HU compared to the control group. The rAd-MLC3f injection resulted in an increase in the relative MLC3f content (12.26±1.19%) and a concomitant increase in Vo (2.90±0.15fl/s) of MHC type IIB fibers. A positive relationship was observed between the percent of MLC3f content and Vo. Maximal isometric force and specific tension were reduced with HU by 49% (741.45±44.24μN to 379.09±23.77μN) and 33% (97.58±4.25kN/m2 to 65.05±2.71kN/m2), respectively compared to the control group. The rAd-MLC3f injection did not change the HU-induced decline in force or specific tension. Collectively, these results indicate that rAd-MLC3f injection rescues hindlimb unloading-induced decline in Vo in MHC type IIB single muscle fibers.Published versio

Myogenic specification of side population cells in skeletal muscle

Skeletal muscle contains myogenic progenitors called satellite cells and muscle-derived stem cells that have been suggested to be pluripotent. We further investigated the differentiation potential of muscle-derived stem cells and satellite cells to elucidate relationships between these two populations of cells. FACS® analysis of muscle side population (SP) cells, a fraction of muscle-derived stem cells, revealed expression of hematopoietic stem cell marker Sca-1 but did not reveal expression of any satellite cell markers. Muscle SP cells were greatly enriched for cells competent to form hematopoietic colonies. Moreover, muscle SP cells with hematopoietic potential were CD45 positive. However, muscle SP cells did not differentiate into myocytes in vitro. By contrast, satellite cells gave rise to myocytes but did not express Sca-1 or CD45 and never formed hematopoietic colonies. Importantly, muscle SP cells exhibited the potential to give rise to both myocytes and satellite cells after intramuscular transplantation. In addition, muscle SP cells underwent myogenic specification after co-culture with myoblasts. Co-culture with myoblasts or forced expression of MyoD also induced muscle differentiation of muscle SP cells prepared from mice lacking Pax7 gene, an essential gene for satellite cell development. Therefore, these data document that satellite cells and muscle-derived stem cells represent distinct populations and demonstrate that muscle-derived stem cells have the potential to give rise to myogenic cells via a myocyte-mediated inductive interaction

MyoD induces myogenic differentiation through cooperation of its NH2- and COOH-terminal regions

MyoD and Myf5 are basic helix-loop-helix transcription factors that play key but redundant roles in specifying myogenic progenitors during embryogenesis. However, there are functional differences between the two transcription factors that impact myoblast proliferation and differentiation. Target gene activation could be one such difference. We have used microarray and polymerase chain reaction approaches to measure the induction of muscle gene expression by MyoD and Myf5 in an in vitro model. In proliferating cells, MyoD and Myf5 function very similarly to activate the expression of likely growth phase target genes such as L-myc, m-cadherin, Mcpt8, Runx1, Spp1, Six1, IGFBP5, and Chrnβ1. MyoD, however, is strikingly more effective than Myf5 at inducing differentiation-phase target genes. This distinction between MyoD and Myf5 results from a novel and unanticipated cooperation between the MyoD NH2- and COOH-terminal regions. Together, these results support the notion that Myf5 functions toward myoblast proliferation, whereas MyoD prepares myoblasts for efficient differentiation

授業研究を通した教師の実践力の向上 : 授業観の変容に着目して

本稿の目的は，広島大学附属東雲中学校で展開されている授業研究を通して，その研究に携わる教師の実践力が向上する様相を考察することである。そのために，まず，東雲中学校で展開されている授業研究は，本時の目標に迫る要因を複数特定する研究であることなどを述べた。そして，教師の実践力のうち，授業を前提とした教材に関する知識に視点をあてて，エピソード分析により８つの要素を抽出・構造化して，東雲中学校のＡ教諭の授業観が変容する様相を示すことにより，授業研究を通して教師の実践力が向上したことを明らかにした

Loss of MyoD Promotes Fate Transdifferentiation of Myoblasts Into Brown Adipocytes

Brown adipose tissue (BAT) represents a promising agent to ameliorate obesity and other metabolic disorders. How- ever, the abundance of BAT decreases with age and BAT paucity is a common feature of obese subjects. As brown adipocytes and myoblasts share a common Myf5 lineage origin, elucidating the molecular mechanisms underlying the fate choices of brown adipocytes versus myoblasts may lead to novel approaches to expand BAT mass. Here we identify MyoD as a key negative regulator of brown adipocyte development. CRISPR/CAS9-mediated deletion of MyoD in C2C12 myoblasts facilitates their adipogenic transdifferentiation. MyoD knockout downregulates miR- 133 and upregulates the miR-133 target Igf1r, leading to amplification of PI3K–Akt signaling. Accordingly, inhibition of PI3K or Akt abolishes the adipogenic gene expression of MyoD null myoblasts. Strikingly, loss of MyoD converts satellite cell-derived primary myoblasts to brown adipocytes through upregulation of Prdm16, a target of miR-133 and key determinant of brown adipocyte fate. Conversely, forced expression of MyoD in brown preadipocytes blocks brown adipogenesis and upregulates the expression of myogenic genes. Importantly, miR-133a knockout signifi- cantly blunts the inhibitory effect of MyoD on brown adipogenesis. Our results establish MyoD as a negative regu- lator of brown adipocyte development by upregulating miR-133 to suppress Akt signaling and Prdm16

Evaluation of microRNA expression in patient bone marrow aspirate slides

<div><p>Like formalin fixed paraffin embedded (FFPE) tissues, archived bone marrow aspirate slides are an abundant and untapped resource of biospecimens that could enable retrospective molecular studies of disease. Historically, RNA obtained from slides is limited in utility because of their low quality and highly fragmented nature. MicroRNAs are small (≈22 nt) non-coding RNA that regulate gene expression, and are speculated to preserve well in FFPE tissue. Here we investigate the use of archived bone marrow aspirate slides for miRNA expression analysis in paediatric leukaemia. After determining the optimal method of miRNA extraction, we used TaqMan qRT-PCR to identify reference miRNA for normalisation of other miRNA species. We found hsa-miR-16 and hsa-miR-26b to be the most stably expressed between lymphoblastoid cell lines, primary bone marrow aspirates and archived samples. We found the average fold change in expression of hsa-miR-26b and two miRNA reportedly dysregulated in leukaemia (hsa-miR-128a, hsa-miR-223) was <0.5 between matching archived slide and bone marrow aspirates. Differential expression of hsa-miR-128a and hsa-miR-223 was observed between leukaemic and non-leukaemic bone marrow from archived slides or flash frozen bone marrow. The demonstration that archived bone marrow aspirate slides can be utilized for miRNA expression studies offers tremendous potential for future investigations into the role miRNA play in the development and long term outcome of hematologic, as well as non-hematologic, diseases.</p> </div

Slr0967 and Sll0939 induced by the SphR response regulator in Synechocystis sp. PCC 6803 are essential for growth under acid stress conditions

AbstractTwo-component signal transduction is the primary signaling mechanism for global regulation of the cellular response to environmental changes. We used DNA microarray analysis to identify genes that were upregulated by acid stress in the cyanobacterium Synechocystis sp. PCC 6803. Several of these genes may be response regulators that are directly involved in this type of stress response. We constructed deletion mutants for the response regulator genes and compared the growth rates of cells transfected with mutant and wild-type genes in a low pH medium. Of these mutants, deletion of sphR affected the growth rate under acid stress (pH 6.0) conditions. We examined genome-wide expression in ΔsphR mutant cells using DNA microarray to determine whether SphR was involved in the regulation of other acid stress responsive genes. Microarray and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses of wild-type cells showed that the expression of phoA, pstS1, and pstS2, which are upregulated under phosphate-limiting conditions, increased (2.48-, 1.88-, and 5.07-fold, respectively) after acid stress treatment for 0.5h. In contrast, pstS2 expression did not increase in the ΔsphR mutant cells after acid stress, whereas the phoA and sphX mRNA levels increased. Furthermore, qRT-PCR and northern blot analysis indicated that downregulation of the acid-responsive genes slr0967 and sll0939 occurred with the deletion of sphR. Indeed, mutants of these genes were more sensitive to acid stress than the wild-type cells. Thus, induction of Slr0967 and Sll0939 by SphR may be essential for growth under acid stress conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial