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Biomedical subjects

Yoshihiro Shimizu

Publications and source records attributed to Yoshihiro Shimizu.

At least 19 recordsLinked to original sources
PubMed2006

Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog.

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched beta-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.

Amino Acid Sequence↗
PubMed2006

Efficient protein selection based on ribosome display system with purified components.

Using the PURE (Protein synthesis Using Recombinant Elements) system, we developed an efficient and highly controllable ribosome display method for selection of functional protein. The PURE system is composed of purified factors and enzymes that are responsible for gene expression in Escherichia coli. We performed the detailed analyses and optimization of the ribosome display system and demonstrated the formation of stable mRNA/ribosome/polypeptide ternary complexes. As complex formation is fundamental to successful ribosome display, these improvements resulted in a dramatic increase in the mRNA recovery rate. As a result, a approximately 12,000-fold enrichment of single-chain antibody (scFv) cDNA was achieved in a single round of selection. Specific selection of scFv mRNA from a 1:10(10) dilution in competitor mRNA was achieved with only three rounds of affinity selection. These findings, together with the results in the accompanying paper [T. Matsuura, H. Yanagida, J. Ushioda, I. Urabe, T. Yomo, Nascent chain, RNA, and ribosome complexes generated by pure translation system (see the accompanying paper).], demonstrate that the PURE system can provide a basis for reliable and reproducible ribosome display.

Cell-Free System↗
PubMed2006

Cell-free translation systems for protein engineering.

Cell-free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell-free translation systems might be used as the fundamental basis for cell-like systems. We review recent progress in the field of cell-free translation systems and describe their use as tools for protein production and engineering.

Animals↗
PubMed2006

SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans-translation initiation.

Bacterial tmRNA rescues ribosomes that stall because of defective mRNAs via the trans-translation process. Although entry of the charged transfer messenger RNA (tmRNA) into the ribosome proceeded in the absence of elongation factor (EF-Tu) and in the presence of EF-Tu and the antibiotic kirromycin, evidence was found for the involvement of EF-Tu in trans-translation initiation. The polyalanine synthesis system attained by using a tmRNA variant consisting of only the tRNA-like domain revealed that it was completely dependent on the presence of SmpB and greatly enhanced by EF-Tu and EF-G. Actually, ribosome-dependent GTPase activity of EF-Tu was stimulated by the addition of SmpB and tmRNA but independently of template mRNA, demonstrating that SmpB compensates for the lack of codon-anticodon interaction during the first step of the trans-translation initiation. Based on these results, we suggest that SmpB structurally mimics the anticodon arm of tRNA and elicits GTP hydrolysis of EF-Tu upon tmRNA accommodation in the A site of the ribosome.

Anticodon↗
PubMed2006

Kinetic analysis of ribosome binding process onto mRNA using a quartz-crystal microbalance.

Translation initiation is the most dynamic and important step along a series of protein synthesis processes. In bacteria, it is generally accepted that the 70S ribosome initially dissociates into the 30S and 50S subunit, and then, the 30S ribosomal subunit binds to the Shine-Dargalno (SD) sequence of mRNA. We analyzed binding kinetics of 70S, 50S and 30S ribosomes to the SD sequence by using a mRNA-immobilized 27 MHz quartz-crystal microbalance (QCM). The 70S ribosome was found to bind strongly to the SD sequence as a ratio of 1:1 without dissociation to each subunit from the lateral side, as well as the 30S subunit. The binding constant for 70S increased in the presence of the initiator tRNA, which suggests that the SD and initiator codon of AUG could be also recognized precisely with 70S.

Base Sequence↗
PubMed2005

Persistent left superior vena cava draining to left atrium with normal coronary sinus.

In embryology, a persistent connection of the left superior vena cava to the left atrium rarely coexists with a coronary sinus. We herein report an unusual case of persistent left superior vena cava terminating in a left atrium with normal coronary sinus, which was revealed at the time of permanent pacemaker implantation after a second operation for recurrent left atrioventricular valve regurgitation. Because this anomaly had gone undiagnosed at the first operation, we were unable to diagnose it prior to the second operation, because the preoperative coronary angiogram clearly demonstrated a coronary sinus that was not dilated. We would have repaired the anomaly using a patch or other procedure had it been diagnosed before the second operation in order to prevent cyanosis or brain complications secondary to right-to-left shunting. One should always be on guard for this rare condition.

Aortic Valve Insufficiency↗
PubMed2005

Optimal method of coronary endarterectomy for diffusely diseased coronary arteries.

BACKGROUND: Although several techniques for coronary artery bypass grafting have been introduced that incorporate coronary endarterectomy (CE), there is little information about late patency among the various CE methods. To clarify the quality of CE, we assessed clinical and angiographic results of our experience with this procedure on 127 patients who had diffusely diseased coronary arteries. METHODS: Between January 1994 and December 2002, 127 patients underwent coronary artery bypass grafting with CE. Sixty-eight patients undergoing CE with long arteriotomies and on-lay patch bypass grafting (group O) were compared with 59 patients undergoing CE with the conventional pull-out method (group P). RESULTS: Thirty-day mortality was 2.9% in group O and 6.8% in group P. The early angiographic results of 115 patients revealed a patency rate of 92.1% (grade A: 79.4%) in group O and 88.6% (grade A: 68.6%) in group P. Actuarial survival at 5 years was 90.7% in group O and 74.0% in group P (p = 0.1). Angiograms performed on 78 patients after a mean period of 21 +/- 16 months showed a patency rate of 89.1% (grade A:76.1%) in group O and 81.0% (grade A: 38.1%) in group P (grade A: p < 0.001). CONCLUSIONS: The midterm angiographic results of CE with long arteriotomies and on-lay patch bypass grafting were better than the results obtained with the conventional pull-out method. This procedure was found to be safe and effective for complete revascularization in patients with a severely and diffusely diseased coronary artery.

Aged↗
PubMed2005

Protein synthesis by pure translation systems.

We have developed a partially recombinant, cell-free, protein-synthesis system reconstituted solely from those essential elements of the Escherichia coli translation system, termed protein synthesis using recombinant elements (PURE). It provides higher reaction controllability in comparison to crude cell-free protein-synthesis systems for translation studies and biotechnology applications. The PURE system stands out among translation methods in that it provides not only a simple and unique "reverse" purification method of separating the synthesized protein from reaction mixture, but also that the system can be tailor-made according to individual protein requirements. In this paper, two new approaches to obtaining active proteins are described: the use of molecular chaperones, and modification of the reaction conditions. Several possible applications of the PURE system are also discussed.

Escherichia coli↗
PubMed2005

Esterification of Escherichia coli tRNAs with D-histidine and D-lysine by aminoacyl-tRNA synthetases.

It is generally believed that only L-amino acids are acceptable in protein synthesis, though some D-amino acids, including D-tyrosine, D-aspartate, and D-tryptophan are known to be bound enzymatically to tRNAs. In this report, we newly show that D-histidine and D-lysine are also able to be the substrates of respective Escherichia coli aminoacyl-tRNA synthetases.

Amino Acyl-tRNA Synthetases↗
PubMed2004

Extensive thoracoabdominal aortic aneurysm repair using deep hypothermic bypass and circulatory arrest.

We sought to evaluate the safety and usefulness of deep hypothermic cardiopulmonary bypass with intervals of circulatory arrest for extensive thoracoabdominal aortic aneurysms. Between March 1994 and December 2002, 17 patients with Crawford type I and II were reviewed retrospectively. The patients were divided into two groups: group H (hypothermic circulatory arrest, n = 8) and group N (normothermic cardiopulmonary bypass, n = 9). In group H, in-hospital mortality was 12.5%, and that in group N was 11.1%. Operation times were similar between the two groups though the cardiopulmonary bypass time was significantly shorter in group N than in group H (p < 0.05). Postoperative paraplegia occurred in 1 patient of group N. Postoperative renal dysfunction occurred in none of group H except in 1 preoperative dialysis case, whilst it occurred in 6 patients of group N. Postoperative creatinine levels were significantly higher in group N than in group H. Three cases in group H required tracheostomy. Our experience with hypothermic cardiopulmonary bypass and circulatory arrest for diffuse type thoracoabdominal aortic aneurysm confirms the safety and efficacy of this technique. Although respiratory complications remain a problem, the technique is considered to be effective for renal protection.

Analysis of Variance↗
PubMed2003

Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria.

In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA.70 S.fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors (IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.

Escherichia coli↗
PubMed2003

Intramolecular cycloaddition reactions of silyl nitronate tethered to vinylsilyl group: 2-nitroalkanols as precursors for amino polyols.

[reaction: see text] A method for converting 2-nitroalkanols to precursors for stereodefined amino polyols is described. Diphenylvinylsilylation of the 2-nitroalkanols' hydroxy groups and subsequent silyl nitronate generation by using TMS-Cl and Et(3)N in CH(3)CN at 0 degrees C to room temperature led to fused-bicyclic heterocycles through stereoselective intramolecular nitronate-olefin [3 + 2] cycloaddition reaction. Some examples for transforming the cycloadducts to amino polyols are also presented.

Journal Article↗
PubMed2003

Conversion of D-glucose to cyclitol with hydroxymethyl substituent via intramolecular silyl nitronate cycloaddition reaction: application to total synthesis of (+)-cyclophellitol.

[reaction: see text] A diastereoisomeric mixture of 1-nitro-6-heptene-2,3,4,5-tetraol derivative (A) was prepared by Henry reaction between d-glucose-based aldehyde and nitromethane. Only the (2S)-isomer of A led to cyclitol (B) via nitronate-olefin cycloaddition on treatment with TMS-Cl and Et(3)N in the presence of catalytic DMAP followed by acid treatment. (+)-Cyclophellitol (1) was synthesized from B in eight steps.

Journal Article↗
PubMed2003

A point mutation in ribosomal protein L7/L12 reduces its ability to form a compact dimer structure and to assemble into the GTPase center.

An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.

Amino Acid Sequence↗
PubMed2003

Off-pump long onlay bypass grafting using left internal mammary artery for diffusely diseased coronary artery.

Surgical treatment of a diffusely diseased coronary artery has been considered a relative contraindication for off-pump coronary artery bypass grafting. To our knowledge, long onlay-patch grafting with off-pump coronary artery bypass grafting has not been described. Two sets of Octopus 3 tissue stabilizers were placed longitudinally along the target coronary artery. This allowed us to perform surgical angioplasty and bypass grafting without cardiopulmonary bypass support (double Octopus technique). We report our early experience with off-pump long onlay bypass grafting in patients with a diffusely diseased coronary artery using double Octopus tissue stabilizers. Diffusely diseased coronary artery; off-pump coronary artery bypass grafting; coronary artery reconstruction; coronary artery bypass grafting; onlay patch anastomosis

Aged↗
PubMed2003

A novel screening system for self-mRNA targeting proteins.

Here we describe the application of an in vitro translation system for genetic screening, to identify RNA-binding proteins that bind to their own mRNAs. It is a relatively novel system designed using an advanced cell-free translation system reconstructed with purified translational components. Due to the absence of nucleases and proteases, the complex of mRNA and nascent polypeptide synthesized in this system is expected to exhibit high stability ensuring the following efficient selection toward the protein. Escherichia coli ribosomal protein S15, which is known to bind to its own mRNA, was employed as a model molecule to evaluate the system. Wild-type S15 mRNA specifically isolated from a mutant mRNA lacking the secondary structure responsible for binding the S15 protein accumulated markedly after several rounds of selection-amplification. The success of this selection demonstrates the potentiality of the systematic screening of self-mRNA targeting proteins through direct and functional selection. This strategy as a method to identify peptides or proteins that bind to their own mRNAs, is of general interest and has different potential applications, such as, the identification of new regulatory proteins or peptide motifs for RNA recognition, the study of self-mRNA-protein interactions, etc.

Amino Acid Motifs↗