Cyclic nucleotide specificity of the activator and catalytic sites of a cGMP-stimulated cGMP phosphodiesterase from Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum has an intracellular phosphodiesterase which specifically hydrolyzes cGMP. The enzyme is activated by low cGMP concentrations, and is involved in the reduction of chemoattractant-mediated elevations of cGMP levels. The interaction of 20 cGMP derivatives with the activator site and with the catalytic site of the enzyme has been investigated. Binding of cGMP to the activator site is strongly reduced (more than 80-fold) if cGMP is no longer able to form a hydrogen bond at N2H2 or O2’H. Modifications at N7, C8, O3’ and O5’ induce only a small reduction of binding affinity. A cyclic phosphate structure, as well as a negatively charged oxygen atom at phosphorus, are essential to obtain activation of the enzyme. Substitution of the axial exocyclic oxygen atom by sulphur is tolerated; modification of the equatorial oxygen atom reduces the binding activity of cGMP to the activator site by 90-fold. Binding of cGMP to the catalytic site is strongly reduced if cGMP is modified at N1H, C6O, C8 and O3’, while modifications at N2H2, N3, N7, O2’H, and O5’ have minor effects. Both exocyclic oxygen atoms are important to obtain binding of cGMP to the catalytic site. The results indicate that activation of the enzyme by cGMP and hydrolysis of cGMP occur at different sites of the enzyme. cGMP is recognized at these sites by different types of molecular interaction between cGMP and the protein. cGMP derivatives at concentrations which saturate the activator site do not induce the same degree of activation of the enzyme (activation 2.3-6.6-fold). The binding affinities of the analogues for the activator site and their maximal activation are not correlated. Our results suggest that the enzyme is activated because cGMP bound to the activator site stabilizes a state of the enzyme which has a higher affinity for cGMP at the catalytic site.

The Stat3-Fam3a axis promotes muscle stem cell myogenic lineage progression by inducing mitochondrial respiration.

Metabolic reprogramming is an active regulator of stem cell fate choices, and successful stem cell differentiation in different compartments requires the induction of oxidative phosphorylation. However, the mechanisms that promote mitochondrial respiration during stem cell differentiation are poorly understood. Here we demonstrate that Stat3 promotes muscle stem cell myogenic lineage progression by stimulating mitochondrial respiration in mice. We identify Fam3a, a cytokine-like protein, as a major Stat3 downstream effector in muscle stem cells. We demonstrate that Fam3a is required for muscle stem cell commitment and skeletal muscle development. We show that myogenic cells secrete Fam3a, and exposure of Stat3-ablated muscle stem cells to recombinant Fam3a in vitro and in vivo rescues their defects in mitochondrial respiration and myogenic commitment. Together, these findings indicate that Fam3a is a Stat3-regulated secreted factor that promotes muscle stem cell oxidative metabolism and differentiation, and suggests that Fam3a is a potential tool to modulate cell fate choices

Genetic Regulation of Myofiber Hypertrophy?

Introduction. Progressive, resistance exercise training (RT) induces skeletal muscle hypertrophy, increases strength, power, and quality of muscle, and is potentially the most promising method to regenerate and re-grow muscle in populations suffering from involuntary atrophy. However, we have previously shown that there is a large degree of intersubject variability for myofiber hypertrophy in response to RT with adults having no response [-16μm2 (mean myofiber growth), Non], a modest response (1111μm2, Mod), or an extreme hypertrophic response (2475μm2, Xtr). Underlying mechanisms for this differential growth response are largely unknown. Therefore, the purpose of this study was to determine whether differences in the skeletal muscle transcriptome exist among the three response clusters, prior to 16 weeks of RT. Methods. mRNA was isolated from muscle biopsies taken from the vastus lateralis of 44 previously clustered men and women (aged 19-75y). Agilent 4X44K single color genechips were used to determine differences in skeletal muscle gene expression among the three response clusters. Ingenuity Pathways Analysis (IPA) and available Gene Ontology were used for functional annotation of differentially expressed genes and identification of informative genes that may instigate the observed myofiber growth phenotypes. Results. After removing genes with low signal intensities and normalizing the data, we identified substantial differences in the transcript profile among the response clusters with the most notable differences between the Xtr- and Non-responders. 8026 differentially expressed genes were identified between Xtr vs. Non, 2463 between Xtr vs. Mod, and 1294 between Mod vs. Non. There were 1632 genes with expression specific to the Xtr (i.e. differences existed between Xtr vs. Non and Mod, but not between the Non vs. Mod) and 617 genes with expression specific to the Non. Functional classification, with IPA, identified Skeletal Muscle System Development and Function (SMSDF) as a top functional category containing a significant number of differentially expressed genes (p\u3c0.05) in all three comparisons. SMSDF was also a top five functional category for the genes specific to both Xtr and Non (p\u3c0.05). Within the broad SMSDF category, IPA defined sub-categories of functional annotation, which allowed us to further interpret the differentially expressed genes. We have highlighted several genes that primarily had expression specific to the Xtr or had increased expression from Non to Mod to Xtr. Highlighted genes are involved with satellite cell activation and function (SOX8, HGF, PAX7), differentiation (MYOD1, MYOG, APOE, TRIO, MSTN), skeletal muscle growth (DGKZ, ESR1, OXT, OXTR, UCN2, GREB1), modulation of inflammation and fuel utilization (PYY), and improved function (TFAM, UCN2, CRHR1, CRHR2). Additionally, there was a decrease in expression (Xtr vs. Non) for several genes involved with modulation of inflammation and fuel utilization (AEBP1, NFKB1, CD36, AIF1). Discussion. These results indicate that differences in gene expression do exist among the response clusters prior to mechanically induced hypertrophy and that the Xtr-responders were “primed” to respond. We identified several genes and signaling pathways that may promote or inhibit muscle growth and thus, initiate the three observed hypertrophic response phenotypes. Results from this study enabled us to identify distinctive molecular pathways, particularly between the Xtr- and Non-responders, for development of targeted interventions. Further research is necessary to determine which of these genes or networks of genes truly distinguish load mediated hypertrophy potential

The role of ribosome biogenesis in exercise-induced skeletal muscle anabolism in aging

Numerous chronic medical conditions, as well as normal aging result in a significant loss of skeletal muscle mass. This has profound effects on quality of life and can increase the risk of all-cause mortality. Currently, the most potent treatment for reversing the loss of muscle mass is resistance exercise training (RT); however, the human muscle fiber growth (hypertrophy) response to this treatment is quite variable, and older adults do not respond as favorably to this treatment as younger adults. The focus of this dissertation is to elucidate the role that ribosome biogenesis plays in regulating the RT-induced hypertrophic response. We show that markers of ribosome biogenesis are attenuated in older adult muscle following an acute, single bout of resistance exercise, indicating a possible mechanism regulating the age-induced blunted hypertrophic response to RT. Additionally, using a K-means cluster analysis, we show that those older individuals with a successful hypertrophic response to a standardized RT program significantly increase muscle rRNA production, and that this is associated with a marked induction of c-Myc expression following RT. A small minority of older individuals with the most extreme hypertrophic response also increase the number of myonuclei per myofiber following RT, likely to provide more rDNA template for rRNA transcription. Finally, we show that chemical inhibition of de novo Pol I-mediated rRNA transcription completely abolishes growth factor-induced increases in protein synthesis and myotube hypertrophy in vitro. Previously, it was thought that increased translational efficiency was the primary regulator of RT-induced muscle hypertrophy. The data presented here suggest that ribosome biogenesis is a novel regulatory mechanism that controls RT-induced human skeletal muscle growth

COVID-19 Resource: Tips and tricks: Keeping students engaged in an online environment

Online learning environments provide the unique benefit of extending educational access to students at any time and place. However, there are numerous challenges related to thoughtful engagement of students, assessment of learning, and more. Many educators are needing to transition to virtual learning. This presentation provides tips and tricks for delivering a high-quality online learning experience. Learning Outcomes Discuss educational theories related to online or virtual student learning Describe at least two ideas that can be implemented in online or virtual teaching Audience: Nurse educators, Facult

A Roadmap for Functional Structural Variants in the Soybean Genome

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits

A Roadmap for Functional Structural Variants in the Soybean Genome

Gene structural variation (SV) has recently emerged as a key genetic mechanism underlying several important phenotypic traits in crop species. We screened a panel of 41 soybean (Glycine max) accessions serving as parents in a soybean nested association mapping population for deletions and duplications in more than 53,000 gene models. Array hybridization and whole genome resequencing methods were used as complementary technologies to identify SV in 1528 genes, or approximately 2.8%, of the soybean gene models. Although SV occurs throughout the genome, SV enrichment was noted in families of biotic defense response genes. Among accessions, SV was nearly eightfold less frequent for gene models that have retained paralogs since the last whole genome duplication event, compared with genes that have not retained paralogs. Increases in gene copy number, similar to that described at the Rhg1 resistance locus, account for approximately one-fourth of the genic SV events. This assessment of soybean SV occurrence presents a target list of genes potentially responsible for rapidly evolving and/or adaptive traits