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Nobuaki Suzuki

Publications and source records attributed to Nobuaki Suzuki.

At least 19 recordsLinked to original sources
PubMed2007

Random segment deletion based on IS31831 and Cre/loxP excision system in Corynebacterium glutamicum.

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2-186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene-gene interactions can be easily and efficiently determined.

Blotting, Southern↗
PubMed2006

Molecular species identification of spiny lobster phyllosoma larvae of the genus Panulirus from the northwestern Pacific.

To identify lobster phyllosoma larvae of the genus Panulirus occurring in waters adjacent to Japan, genetic variation within and between 10 Indo-Pacific lobster species was investigated using restriction fragment length polymorphism (RFLP) analysis for the 1300-base pair mitochondrial cytochrome oxidase I (COI) gene. RFLP analysis using two endonucleases (AluI and TaqI) enabled discrimination of all species, including the P. longipes complex. The diagnostic DNA markers, supplemented with nucleotide sequence analysis, were applied to 44 mid- to late-stage phyllosoma larvae (7.4 to 27.7 mm in body length) collected in the northwestern Pacific. These samples were unexpectedly variable in species composition, comprising P. japonicus (n = 16), P. longipes bispinosus (21), P. longipes longipes (1), P. "aka" (1), and P. penicillatus (5). Comparison of larval size at similar stages revealed that P. l. bispinosus larvae were significantly larger than P. japonicus.

Animals↗
PubMed2006

High-throughput transposon mutagenesis of Corynebacterium glutamicum and construction of a single-gene disruptant mutant library.

A simple and high-throughput transposon-mediated mutagenesis system employing two different types of transposons in combination with direct genomic DNA amplification and thermal asymmetric interlaced PCR (TAIL-PCR) was developed. Each of the two minitransposons based on IS31831 (ISL3 family) and Tn5 (IS4 family) was integrated into the Corynebacterium glutamicum R genome. By using BLAST and Perl, transposon insertion locations were automatically identified based on the sequences of TAIL-PCR products of mutant cells. Insertion locations of 18,000 mutants were analyzed, and a comprehensive insertion library covering nearly 80% of the 2,990 open reading frames of C. glutamicum R was generated. Eight thousand of the mutants, exhibiting disruption in 2,330 genes, survived on complex medium under normal laboratory conditions, indicating that the genes were not essential for cell survival. Of the 2,330 genes, 30 exhibited high similarity to essential genes of Escherichia coli or Bacillus subtilis. This approach could be useful in furthering genetic understanding of cellular life and facilitating the functional analysis of microorganisms.

Chromosome Mapping↗
PubMed2005

Multiple large segment deletion method for Corynebacterium glutamicum.

A precise and scarless genome excision method, employing the Cre/loxP system in concert with double-strand break (DSB)-stimulated intramolecular recombination was developed. The DSBs were mediated by the restriction endonuclease, I-SceI. It permitted multiple deletions of independent 14-, 43-, and 10-kb-long genomic regions on the Corynebacterium glutamicum genome. Accuracy of deletion was confirmed by the loss of marker genes, PCR, and sequencing of new genome joints. Eleven, 58, and 4 genes were predicted on the 14-, 43-, and 10-kb deleted regions, respectively. Although the resultant mutant lost a total of 67 kb encoding 73 genes, it still exhibited normal growth under standard laboratory conditions. Such a large segment deletion method in which multiple, successive deletions are possible is useful for genome engineering.

Chromosome Deletion↗
PubMed2005

Ultrastructural stability under high temperature or intensive light stress conferred by a small heat shock protein in cyanobacteria.

The role and sub-cellular localization of the small heat shock protein HspA under stress conditions was investigated comparing the cyanobacterium Synechococcus strain ECT16-1, which constitutively expresses HspA, with the reference strain ECT. The ultrastructure of ECT cells under elevated temperature or intensive light stress exhibited severe damage including aggregation of cytosol and disordered thylakoid membranes, but in ECT16-1 cells these ultrastructural changes were much less conspicuous. Immunocytochemical studies showed that the main localization of HspA in the ECT16-1 cells shifted from the thylakoid area to the cytoplasm, then back to thylakoid area during the heat stress. Expression of HspA stabilized the morphology of nucleoids. The results are discussed, in particular with respect to the unique property of HspA to associate with thylakoid membranes.

Bacterial Proteins↗
PubMed2005

A new insertion sequence, IS14999, from Corynebacterium glutamicum.

A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence. This indicated that IS14999 belonged to the IS630/Tc1 superfamily, which was first identified in C. glutamicum. IS14999 has a unique distance of 38 amino acid residues between the second and third amino acids in the DDE motif, which is well known as the catalytic centre of transposase. This suggested that IS14999 constituted a new subfamily of the IS630/Tc1 superfamily. A phylogenetic tree constructed on the basis of amino acid sequences of transposases revealed that this new transposable element was more similar to eukaryotic Tc1/mariner family elements than to prokaryotic IS630 family elements. Added to the fact that IS14999 was present in only a few C. glutamicum strains, this implies that IS14999 was probably acquired by a recent lateral transfer event from eukaryotic cells. Analysis of the insertion site in C. glutamicum R revealed that IS14999 appeared to transpose at random and always caused a target duplication of a 5'-TA-3' dinucleotide upon insertion, like the other IS630/Tc1 family elements. These findings indicated that IS14999 could be a powerful tool for genetic manipulation of corynebacteria and related species.

Amino Acid Sequence↗
PubMed2005

Isolation and characterization of a native composite transposon, Tn14751, carrying 17.4 kilobases of Corynebacterium glutamicum chromosomal DNA.

A native composite transposon was isolated from Corynebacterium glutamicum ATCC 14751. This transposon comprises two functional copies of a corynebacterial IS31831-like insertion sequence organized as converging terminal inverted repeats. This novel 20.3-kb element, Tn14751, carries 17.4 kb of C. glutamicum chromosomal DNA containing various genes, including genes involved in purine biosynthesis but not genes related to bacterial warfare, such as genes encoding mediators of antibiotic resistance or extracellular toxins. A derivative of this element carrying a kanamycin resistance cassette, minicomposite Tn14751, transposed into the genome of C. glutamicum at an efficiency of 1.8 x 10(2) transformants per mug of DNA. Random insertion of the Tn14751 derivative carrying the kanamycin resistance cassette into the chromosome was verified by Southern hybridization. This work paves the way for realization of the concept of minimum genome factories in the search for metabolic engineering via genome-scale directed evolution through a combination of random and directed approaches.

Bacterial Proteins↗
PubMed2005

New multiple-deletion method for the Corynebacterium glutamicum genome, using a mutant lox sequence.

Due to the difficulty of multiple deletions using the Cre/loxP system, a simple, markerless multiple-deletion method based on a Cre/mutant lox system combining a right-element (RE) mutant lox site with a left-element (LE) mutant lox site was employed for large-scale genome rearrangements in Corynebacterium glutamicum. Eight distinct genomic regions that had been identified previously by comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes were targeted for deletion. By homologous recombination, LE and RE mutant lox sites were integrated at each end of a target region. Highly efficient and accurate deletions between the two chromosomal mutant lox sites in the presence of Cre recombinase were realized. A deletion mutant lacking 190 kb of chromosomal regions, encoding a total of 188 open reading frames (ORFs), was obtained. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of numerous predicted ORFs, the mutant exhibited normal growth under standard laboratory conditions. The Cre/loxP system using a pair of mutant lox sites provides a new, efficient genome rearrangement technique for C. glutamicum. It should facilitate the understanding of genome functions of microorganisms.

Base Sequence↗
PubMed2005

Large-scale engineering of the Corynebacterium glutamicum genome.

The engineering of Corynebacterium glutamicum is important for enhanced production of biochemicals. To construct an improved C. glutamicum genome, we developed a precise genome excision method based on the Cre/loxP recombination system and successfully deleted 11 distinct genomic regions identified by comparative analysis of C. glutamicum genomes. Despite the loss of several predicted open reading frames, the mutant cells exhibited normal growth under standard laboratory conditions. With a total of 250 kb (7.5% of the genome), the 11 genomic regions were loaded with cryptic prophages, transposons, and genes of unknown function which were dispensable for cell growth, indicating recent horizontal acquisitions to the genome. This provides an interesting background for functional genomic studies and can be used in the improvement of cell traits.

Corynebacterium glutamicum↗
PubMed2005

Immunological evaluation of personalized peptide vaccination for patients with pancreatic cancer.

The prognosis of pancreatic cancer is extremely poor, and development of new treatment modalities is needed. One such treatment could be specific immunotherapy. To evaluate safety and immunological responses, we conducted a phase I study of personalized peptide vaccination for pancreatic cancer patients (n=11). Namely, pre-vaccination peripheral blood mononuclear cells were screened for their reactivity in vitro to each of 14 or 16 peptides in HLA-A24(+) or -A2(+) patients, and only the reactive peptides (maximum: 4) were vaccinated in vivo. This regimen was generally well tolerated, although inflammatory reactions at the injection site were observed in 7 patients. Delayed-type hypersensitivity to peptides used for vaccination was observed in 7 patients. Increased cellular and humoral immune responses to at least one of peptides used for vaccination were observed in the post-vaccination PBMCs and sera from 4 of 8 patients and 4 of 10 patients tested, respectively. The 6- and 12-month survival rates for patients who received >3 vaccinations (n=10) were 80% and 20%, respectively. Due to tolerability and capability of inducing specific immunity, further development of personalized peptide-based immunotherapy for pancreatic cancer patients is warranted.

Aged↗
PubMed2005

[Cardiac rupture caused by myocardial infarction in the diagonal branch area: evaluation by cardiac multislice computed tomography: a case report].

A 75-year-old woman was admitted to the emergency room because of hypotension and loss of consciousness induced by cardiac tamponade. Electrocardiography revealed ST elevation and laboratory data showed elevation of serum creatine kinase and troponin I. The patient was referred to the cardiology department 5 days later. Cardiac catheterization revealed ventricular aneurysm in the anterior wall, significant stenosis (75%) in the left anterior descending coronary artery and subtotal stenosis (99%) in the diagonal branch. Cardiac multislice computed tomography suggested that the ventricular pseudoaneurysm was probably due to cardiac rupture caused by myocardial infarction in the diagonal area. Subsequently, aneurysmectomy and coronary artery bypass graft surgery were performed. Cardiac multislice computed tomography is useful for evaluating coronary artery and cardiac rupture.

Aged↗
PubMed2004

Cre/loxP-mediated deletion system for large genome rearrangements in Corynebacterium glutamicum.

Genome rearrangement is an increasingly important technique to facilitate the understanding of genome functions. A Cre/loxP-mediated deletion system for large-scale genome rearrangements in Corynebacterium glutamicum was developed. By comparative analysis of C. glutamicum R and C. glutamicum 13032 genomes, distinct 14.5-kb and 56-kb regions not essential for cell survival were identified and targeted for deletion. By homologous recombination, loxP sites were integrated at each end of the target region. Deletions between the two chromosomal loxP sites in the presence of Cre recombinase were highly efficient. Accurate deletion was observed in all 96 Cre-expressing strains tested. These deletions represent the largest genomic excisions in C. glutamicum reported to date. Despite the loss of 11 and 58 predicted ORF(s), respectively, upon the deletion of the 14.5-kb and 56-kb regions, the cells still exhibited normal growth under standard laboratory conditions. Based on the precision of its deletion, the Cre/loxP system provides a new, efficient genome rearrangement technique for studying C. glutamicum.

Corynebacterium glutamicum↗
PubMed2004

Development and implementation of a database system to manage a large-scale mouse ENU-mutagenesis program.

A mouse ENU-mutagenesis program at RIKEN GSC has been initiated to conduct a large-scale, genome-wide, early- and late-onset phenotypic screen of mutant mice. We screened about a hundred mice every week with a comprehensive set of phenotype assays including behavioral tests based on a modified SHIRPA protocol, blood tests (both clinical biochemical testing and hemogram), and measurement of locomotor activity in their home cages. To manage the entire program, we developed a client/server architecture database system and named it MUSDB (Mutagenesis Universal Support DataBase). It manages mouse husbandry, mating protocols, procedures for ENU injection and phenotypic screens, phenotype inheritance tests, preservation of sperm and organs, and other materials generated during the program. We have implemented MUSDB in quite a large-scale system that includes 150 client computers. It has, helped reduce typographical errors and provided simple and efficient operation via its front-end user interface. It significantly contributed to the communication within and between workgroups in the program and in the accumulation of various phenotypic and inheritance data.

Animals↗
PubMed2004

[Relationship between brain natriuretic peptide and coronary stenosis: influence of aging].

OBJECTIVES: The relationship between brain natriuretic peptide (BNP) and coronary stenosis, and the utility of BNP for the prediction of coronary stenosis were investigated. METHODS: This study included 100 consecutive patients (48 men, 52 women, mean age 65.8 +/- 9.9 years) who underwent elective cardiac catheterization for the diagnosis of coronary stenosis without other heart diseases (heart failure, valvular heart disease, cardiomyopathy, sick sinus syndrome), and E/A was recorded by echocardiography. The relationship between coronary stenosis, left ventricular ejection fraction by left ventriculography, left ventricular end-diastolic pressure, E/A, left ventricular stroke volume index by echocardiography and BNP were investigated. RESULTS: Thirty-nine patients revealed coronary stenosis > or = 75% (CS), and 6 patients had chronic total occlusion. In the CS(+) group, BNP and left ventricular end-diastolic pressure were elevated significantly (50.7 +/- 48.5 vs 22.1 +/- 21.6 pg/ml, p < 0.05; 9.2 +/- 5.3 vs 6.4 +/- 3.4 mmHg, p < 0.05). In logistic regression analysis, BNP and left ventricular end-diastolic pressure had significant correlations with CS(+), independent of age, systolic blood pressure, E/A and left ventricular ejection fraction (p = 0.007, p = 0.05, respectively). Prognostic values of BNP (> 20 pg/ml) for the diagnosis of CS(+) were sensitivity of 79%, and specificity of 61% (p < 0.005). In the CS(-) group, the patients showing BNP > 20 pg/ml were older than the patients showing BNP < or = 20 pg/ml (68.4 +/- 8.5 vs 60.0 +/- 9.4, p < 0.05). Therefore, the prognostic values were reduced (sensitivity 78%, specificity 82%, p < 0.005) in the younger group (age < 65, n = 42). Even in patients with coronary stenosis but without chronic total occlusion, both BNP and left ventricular end-diastolic pressure were elevated significantly compared with the patients without coronary stenosis (22.1 +/- 21.6 vs 44.0 +/- 43.2 pg/ml, p < 0.01; 6.4 +/- 3.4 vs 9.1 +/- 5.2 mmHg, p < 0.01). CONCLUSIONS: Plasma BNP levels are useful markers for detecting coronary stenosis, especially in younger patients.

Aged↗
PubMed2004

[Anesthetic management for removal of pheochromocytoma in a patient after repair of tetralogy of Fallot].

A 45-year-old woman after repair of tetralogy of Fallot at the age of 19, was admitted to the hospital for treatment of paroxysmal hypertension. She was diagnosed with pheochromocytoma of the left adrenal gland, and was scheduled for removal of pheochromocytoma. She received doxazosin for preoperative preparation. Anesthesia was induced with propofol and vecuronium, and maintained with intermittent fentanyl and sevoflurane in nitrous oxide and oxygen. Before induction of anesthesia, magnesium sulfate 40 mg x kg(-1) was injected, and followed by continuous infusion of 40 mg x kg(-1) x hr(-1) until the removal of pheochromocytoma. Under neuromuscular monitoring, vecuronium was administered prudently. Cardiac output was continuously monitored with pulmonary arterial catheter and transesophageal aortic blood flow monitor. Blood pressure and heart rate were stable even though the tumor was handled, and no additional vasodilator therapy was needed. After surgery, prolonged neuromuscular blockade caused by magnesium sulfate was not observed. We consider that successful management was feasible by administration of magnesium sulfate and preoperative administration of alpha1 blocker, under adequate perioperative monitoring.

Adrenal Gland Neoplasms↗
PubMed2003

Multifunctional Alloys Obtained via a Dislocation-Free Plastic Deformation Mechanism.

We describe a group of alloys that exhibit "super" properties, such as ultralow elastic modulus, ultrahigh strength, super elasticity, and super plasticity, at room temperature and that show Elinvar and Invar behavior. These "super" properties are attributable to a dislocation-free plastic deformation mechanism. In cold-worked alloys, this mechanism forms elastic strain fields of hierarchical structure that range in size from the nanometer scale to several tens of micrometers. The resultant elastic strain energy leads to a number of enhanced material properties.

Journal Article↗
PubMed2003

Measles infection in pregnancy.

OBJECTIVES: Measles during pregnancy has deleterious effects on both the perinatal outcome and the mother. However, in-depth knowledge about gestational measles is lacking. The objectives of this study were to describe the clinical course of eight cases of gestational measles and to study the effect of measles and pregnancy on each other. METHODS: From late 2000 to early 2001, we experienced a measles outbreak with eight infected pregnant women. The clinical course of each case is described in detail. RESULT: Three of the four cases before 24 weeks of gestation ended in spontaneous abortion or stillbirth. The clinical course of the three abortions and stillbirth were singular because of the sudden onset of the abortion and the spontaneous pregnancy termination. In contrast, the four pregnancies after 25 weeks of gestation ended in live-term delivery and two out of the four neonates were diagnosed with congenital measles. There was no maternal death, instead two pneumonia cases and one hemorrhagic shock case. CONCLUSIONS: Gestational measles may potentially damage the fetus and is one of the serious complications that can occur during pregnancy.

Abortion, Spontaneous↗