Ligands for FKBP12 Increase Ca2+ Influx and Protein Synthesis to Improve Skeletal Muscle Function

Rapamycin at high doses (2–10 mg/kg body weight) inhibits mammalian target of rapamycin complex 1 (mTORC1) and protein synthesis in mice. In contrast, low doses of rapamycin (10 μg/kg) increase mTORC1 activity and protein synthesis in skeletal muscle. Similar changes are found with SLF (synthetic ligand for FKBP12, which does not inhibit mTORC1) and in mice with a skeletal muscle-specific FKBP12 deficiency. These interventions also increase Ca²⁺ influx to enhance refilling of sarcoplasmic reticulum Ca²⁺ stores, slow muscle fatigue, and increase running endurance without negatively impacting cardiac function. FKBP12 deficiency or longer treatments with low dose rapamycin or SLF increase the percentage of type I fibers, further adding to fatigue resistance. We demonstrate that FKBP12 and its ligands impact multiple aspects of muscle function.     

Experimental evidence for a 9-binding subsite of Bacillus licheniformis thermostable α-amylase

The action pattern of Bacillus licheniformis thermostable α-amylase (BLA) was analyzed using a series of ¹⁴C-labeled and non-labeled maltooligosaccharides from maltose (G2) to maltododecaose (G12). Maltononaose (G9) was the preferred substrate, and yielded the smallest K_m = 0.36 mM, the highest k_cat = 12.86 s⁻¹, and a k_cat/K_m value of 35.72 s⁻¹ mM⁻¹, producing maltotriose (G3) and maltohexaose (G6) as the major product pair. Maltooctaose (G8) was hydrolyzed into two pairs of products: G3 and maltopentaose (G5), and G2 and G6 with cleavage frequencies of 0.45 and 0.30, respectively. Therefore, we propose a model with nine subsites: six in the terminal non-reducing end-binding site and three at the reducing end-binding site in the binding region of BLA.     

Biochemical Characterization of Mutants in Chaperonin Proteins CCT4 and CCT5 Associated with Hereditary Sensory Neuropathy

Hereditary sensory neuropathies are a class of disorders marked by degeneration of the nerve fibers in the sensory periphery neurons. Recently, two mutations were identified in the subunits of the eukaryotic cytosolic chaperonin TRiC, a protein machine responsible for folding actin and tubulin in the cell. C450Y CCT4 was identified in a stock of Sprague-Dawley rats, whereas H147R CCT5 was found in a human Moroccan family. As with many genetically identified mutations associated with neuropathies, the underlying molecular basis of the mutants was not defined. We investigated the biochemical properties of these mutants using an expression system in Escherichia coli that produces homo-oligomeric rings of CCT4 and CCT5. Full-length versions of both mutant protein chains were expressed in E. coli at levels approaching that of the WT chains. Sucrose gradient centrifugation revealed chaperonin-sized complexes of both WT and mutant chaperonins, but with reduced recovery of C450Y CCT4 soluble subunits. Electron microscopy of negatively stained samples of C450Y CCT4 revealed few ring-shaped species, whereas WT CCT4, H147R CCT5, and WT CCT5 revealed similar ring structures. CCT5 complexes were assayed for their ability to suppress aggregation of and refold the model substrate γd-crystallin, suppress aggregation of mutant huntingtin, and refold the physiological substrate β-actin in vitro. H147R CCT5 was not as efficient in chaperoning these substrates as WT CCT5. The subtle effects of these mutations are consistent with the homozygous disease phenotype, in which most functions are carried out during development and adulthood, but some selective function is lost or reduced.     

Global Ablation of the Mouse Rab11a Gene Impairs Early Embryogenesis and Matrix Metalloproteinase Secretion

Rab11a has been conceived as a prominent regulatory component of the recycling endosome, which acts as a nexus in the endo- and exocytotic networks. The precise in vivo role of Rab11a in mouse embryonic development is unknown. We globally ablated Rab11a and examined the phenotypic and molecular outcomes in Rab11a^null blastocysts and mouse embryonic fibroblasts. Using multiple trafficking assays and complementation analyses, we determined, among multiple important membrane-associated and soluble cargos, the critical contribution of Rab11a vesicular traffic to the secretion of multiple soluble MMPs. Rab11a^null embryos were able to properly form normal blastocysts but died at peri-implantation stages. Our data suggest that Rab11a critically controls mouse blastocyst development and soluble matrix metalloproteinase secretion.     

Nitrogen application enhanced the expression of developmental plasticity of root systems triggered by mild drought stress in rice

Background

The promoted root growth under developmental plasticity triggered specifically by mild drought stress (MDS) is known to contribute to maintained water uptake and dry matter production (DMP).

Aims

To examine whether the expression of developmental plasticity of root systems and its contribution to DMP would be affected by the levels of nitrogen (N) application.

Methods

Two genotypes (CSSL50 derived from Nipponbare/Kasalath cross and Nipponbare) were grown under soil moisture gradients with a line source sprinkler system. Three N fertilizer treatments were used; 25 (low), 75 (standard) and 150 kg N ha^?1 (high) in 2009 and 60 (low), 120 (standard) and 180 kg N ha^?1 (high) in 2011.

Results

Across varying N level treatments, there were no significant differences in any of the traits examined between the two genotypes under well-watered and severe drought stress conditions. In contrast, under MDS conditions (15–25 % w/w of soil moisture content (SMC) in 2009 and 17–25 % w/w of SMC in 2011), CSSL50 showed greater DMP than Nipponbare. The difference, however, varied with N level treatments since CSSL50’s greater root system development under MDS, was more pronounced at standard and high N levels than at low N level than it was for Nipponbare.

Conclusions

N application enhanced the expression of plasticity in root system development at standard and high N levels as compared with low N level under MDS conditions, which contributed to the maintenance of DMP.