Workspace analysis and design improvement of a carotid flow measurement system.

Heart and cerebrovascular diseases such as arteriosclerosis and myocardial ischemia dysfunction are currently among the main causes of death in developed countries. Recently, wave intensity (WI), which is an index used to obtain the force of cardiac contraction, has been investigated as a method for early-stage diagnosis of the above-mentioned diseases. Nevertheless, experimental tests have proven that the manual measurements of WI by means of commercial ultrasonic diagnostic systems require too much time and can be affected by the operator's skills. For this purpose, the introduction of robotic-assisted technology has advantages in terms of repetitiveness and accuracy of the measurement procedure. Therefore, at Waseda University, the development of a carotid blood flow measurement system has been proposed to support doctors while using ultrasound diagnostic equipment to measure the WI. This robotic system is composed of a serial robot with a wrist having a six-degree-of-freedom (6-DOF) parallel mechanism. The main focus is to obtain a suitable workspace performance of the 6-DOF parallel mechanism wrist. In this paper, a workspace analysis is carried out on a wrist prototype built for the Waseda-Tokyo Women's Medical Aloka Blood Flow Measurement System No.1 Refined (WTA-1R). Then, mechanical design enhancements are proposed and validated to provide a suitable workspace performance both as reachable workspace and dexterity, and a refined prototype WTA-1RII has been built.

Cloning of genes of the aminopeptidase T family from Thermus thermophilus HB8 and Bacillus stearothermophilus NCIB8924: apparent similarity to the leucyl aminopeptidase family.

To obtain genes with sequence similarity to aminopeptidase T (AP-T) of Thermus aquaticus YT-1, we cloned the genes encoding aminopeptidase Th (AP-Th) from Thermus thermophilus HB8 and aminopeptidase II (APII) from Bacillus stearothermophilus NCIB8924. The AP-Th gene encoded a polypeptide of 408 amino acid residues and the deduced molecular weight of this subunit was 45,015. The APII gene encoded a polypeptide of 413 amino acid residues with a deduced molecular weight of 46,207. The extent of amino acid sequence similarity between AP-Th and AP-T was 86%, and that between APII and AP-T was 43%. The substrate specificities of these expressed enzymes were similar, and each efficiently hydrolyzed leucyl- or phenyl-peptide substrates. Since the deduced amino acid sequence of these enzymes show no similarity to other known aminopeptidases, they appear to comprise an independent family of peptidases, designated the AP-T family. However, a conserved region within the enzymes of the AP-T family shows similarity to the active site signature of the leucyl aminopeptidase family, suggesting that these enzymes may belong to the leucyl aminopeptidase superfamily.

The active site of carboxypeptidase Taq possesses the active-site motif His-Glu-X-X-His of zinc-dependent endopeptidases and aminopeptidases.

Carboxypeptidase (CPase) Taq possesses the His-Glu-X-X-His sequence, which is the consensus sequence in the active site of zinc-dependent endopeptidases and amino-peptidases, at positions 276-280. Amino acid replacement of the conserved His and Glu drastically diminished the activity of CPase Taq, and the zinc content of the enzyme was also greatly reduced when either of the two His residues was replaced with Arg or Tyr. The results indicate that this sequence actually functions as the active site in CPase Taq, showing that CPase Taq is a novel type of zinc-dependent CPase that possesses the His-Glu-X-X-His active-site motif.

Carboxypeptidase Taq, a thermostable zinc enzyme, from Thermus aquaticus YT-1: molecular cloning, sequencing, and expression of the encoding gene in Escherichia coli.

The gene for carboxypeptidase Taq, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced. The gene comprised an open reading frame of 1,536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56,210 Da consisting of 511 amino acid residues. The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli. The enzyme purified from E. coli cells showed the same properties as those of carboxypeptidase Taq prepared from T. aquaticus cells. Analysis for metal ions bound to the enzyme found that one molecule of the enzyme contains one tightly bound zinc ion. Comparison of the entire sequence showed that the enzyme has no obvious sequence similarity to any other metallo-peptidases. However, a His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent endopeptidases and aminopeptidases, was found at positions 276 to 280 of the enzyme. These findings suggest that carboxypeptidase Taq is a novel type of zinc-dependent metallocarboxypeptidase.

Purification and characterization of a thermostable carboxypeptidase (carboxypeptidase Taq) from Thermus aquaticus YT-1.

A thermostable carboxypeptidase, which we named carboxypeptidase Taq, was purified from Thermus aquaticus YT-1 and characterized. The molecular weight of the enzyme was estimated to be about 56,000 and 58,000 on SDS-polyacrylamide gel electrophoresis and gel filtration, respectively, indicating that the enzyme has a monomeric structure. The optimum pH of the enzyme was 8.0, and the optimum temperature for the reaction was 80 degrees C. The enzyme activity was dependent on cobalt ion and was inhibited by metal-chelating reagents, indicating that the enzyme is a metalloenzyme. The enzyme had high thermostability independent of cobalt ion; about 90% of its activity remained even after treatment at 80 degrees C for 5 h. The enzyme showed broad substrate specificity, although proline at the C-terminus of peptides was not cleaved. The enzyme released amino acids sequentially from the C-terminus.

Molecular cloning and nucleotide sequence of the aminopeptidase T gene of Thermus aquaticus YT-1 and its high-level expression in Escherichia coli.

Aminopeptidase T (AP-T) is a metallo-dependent dimeric enzyme of Thermus aquaticus YT-1, an extremely thermophilic bacterium. We cloned the AP-T gene from T. aquaticus YT-1 into Escherichia coli using a synthetic oligonucleotide as a hybridization probe. The nucleotide sequence of the AP-T gene was found to encode 408 amino acid residues with GTG as a start codon. The molecular weight was calculated to be 44,820. The AP-T was overproduced in E. coli (about 5% of total soluble protein) when the start codon of the gene was changed from GTG to ATG, and the gene was downstream from the tac promoter. The AP-T expressed in E. coli was heat stable and easily purified by heat treatment (80 degrees C, 30 min). The N-terminal amino acid sequence of AP-T showed similarity with that of aminopeptidase II from Bacillus stearothermophilus.

Exopeptidase profiles of bifidobacteria.

The exopeptidase activities of five different strains of bifidobacteria occurring habitually in healthy human intestinal canal were measured on 61 synthetic substrates. The cluster analysis, based on the results, indicates that four strains, with the exception of Bifidobacterium adolescentis a M101-4, have similar exopeptidase profiles. All CFE from these five strains contained at least three kinds of aminopeptidases (aminopeptidase with broad substrate specificity, aminopeptidase hydrolyzing selectively X-Pro type and aminopeptidase hydrolyzing selectively Pro-X type) and carboxypeptidase.