回復期での介入量は脳卒中患者のADL自立を予測できるか？ : 後ろ向きコホート研究

広島大学(Hiroshima University)博士(保健学)Doctor of Philosophy in Health Sciencedoctora

Pyrrolysyl-tRNA synthetase variants reveal ancestral aminoacylation function

AbstractPyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS

Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis

Development of a Functionally Minimized Mutant of the R3C Ligase Ribozyme Offers Insight into the Plausibility of the RNA World Hypothesis

The R3C ligase ribozyme is an artificial ligase ribozyme produced by modification of the ribozyme that lacks cytidine. Here, we attempted to modify the original R3C ribozyme (73 nucleotides) by reducing the number of nucleotides while maintaining the maximum possible catalytic efficiency. By partially deleting both the “grip” (P4 + P5) and “hammer” (P3) stem-loops, we found the critical border to retain activity comparable to that of full-length R3C. The three-way junction structure was necessary to maintain enzymatic function and the stability of the “grip” (P4 + P5) stem had a large influence on the catalytic activity of R3C. The final minimized ribozyme we obtained comprised ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Our findings suggest that the autocatalytic function in ribozymes is indeed possible to obtain using sequence lengths achievable with prebiotic synthesis

The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life

R3C ligase ribozyme catalyzes the nucleophilic attack by a 3′-hydroxyl on a 5′-α-phosphorus of triphosphates to form a 3′-5′-phosphodiester bond. In the present study, although the truncation of R3C ribozyme was accompanied by a large reduction in ligation activity (decrease by two orders of magnitude compared to that of the ligated product of full-length R3C ribozyme after 18.5 h at 23 °C), the introduction of complementary seven-membered kissing-loops served as a “switch” to reactivate the truncated R3C ribozyme with approximately one-fifth of the activity of the full-length R3C ribozyme. This reactivation occurred in a trans-manner, and the grip region and substrate-binding site of the truncated R3C ribozyme were necessary to locate the substrate in the proper position for ligation with the other molecule. Reactivation resulted from complex tertiary interactions between two ribozymes, including kissing-loop interaction-induced annealing and the formation of a stable duplex. The drastic increase of the activity of poorly active ribozymes through the kissing-loop interaction may provide an important clue into the acquisition of substantial activity during the evolution of the RNA world

Phase Angle Is Associated With Handgrip Strength in Older Patients With Heart Failure

Objective To assess the relationships between phase angle and muscle mass, strength, and physical function in patients with heart failure. Methods This study used a cross-sectional design. The analysis included 51 patients with heart failure. The Short Physical Performance Battery, one-leg standing time, handgrip strength, phase angle, and skeletal muscle index were measured. To identify explanatory variables of phase angle, hierarchical multiple regression analysis was performed. Results Handgrip strength was found to be an explanatory variable of phase angle independent of age, sex, and body mass index. This model was able to explain 30.4% of the model variance for phase angle. Conclusion In patients with heart failure, improving muscle strength rather than muscle mass or physical function might be more important for improving phase angle. Handgrip strength is an important outcome for improving prognosis in patients with heart failure

Peptide Bond Formation between Aminoacyl-Minihelices by a Scaffold Derived from the Peptidyl Transferase Center

The peptidyl transferase center (PTC) in the ribosome is composed of two symmetrically arranged tRNA-like units that contribute to peptide bond formation. We prepared units of the PTC components with putative tRNA-like structure and attempted to obtain peptide bond formation between aminoacyl-minihelices (primordial tRNAs, the structures composed of a coaxial stack of the acceptor stem on the T-stem of tRNA). One of the components of the PTC, P1c2UGGU (74-mer), formed a dimer and a peptide bond was formed between two aminoacyl-minihelices tethered by the dimeric P1c2UGGU. Peptide synthesis depended on both the existence of the dimeric P1c2UGGU and the sequence complementarity between the ACCA-3′ sequence of the minihelix. Thus, the tRNA-like structures derived from the PTC could have originated as a scaffold of aminoacyl-minihelices for peptide bond formation through an interaction of the CCA sequence of minihelices. Moreover, with the same origin, some would have evolved to constitute the present PTC of the ribosome, and others to function as present tRNAs