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Biomedical subjects

S Taguchi

Publications and source records attributed to S Taguchi.

At least 19 recordsLinked to original sources
PubMed2001

Analysis of mutational effects of a polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system.

Polymerase is a central enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbC(Re)) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of a polymerase chain reaction-mediated random mutagenesis and two assay procedures, a plate assay using a PHB-staining dye and a high-pressure liquid chromatographic assay based on the converting reaction from PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbC(Re) is high and well-optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an 'alpha/beta hydrolase fold' which is commonly found among hydrolytic enzymes. From a good correlation with the level of PHB accumulation, an activity estimation of the PhbC(Re) would be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here.

Acyltransferases↗
PubMed2001

Photodegradation of chlorinated hydrocarbons in the presence and absence of dissolved oxygen in water.

The photodegradation rates of seven chlorinated hydrocarbons; C2Cl4, C2HCl3, C2H4Cl2, 1,1,1-C2H3Cl3, 1,1,2-C2H3Cl3, CHCl3, CCl4 were investigated under the UV bandwidths of 185 and 254 nm in the presence and absence of dissolved oxygen (DO) in water. These hydrocarbons are possible contaminants of groundwater. This study confirms that the degradation rates of all chlorinated hydrocarbons are elevated in the absence of DO. This was especially apparent for chlorinated methane and ethane. Tetrachloroethylene's rate was the highest among the seven hydrocarbons regardless of the DO levels. It was clear that the concentration of intermediate trichloroethylene produced by photodegradation of tetrachloroethylene in the absence of DO was 1/50th of that in the presence of DO. Photodegradation in the presence of DO resulted in the formation of O3 and hydroxyl radicals. Alternatively, the photodegradation in the absence of DO resulted in the formation of organic radicals and a dissociation of the bond. It is discussed that DO acts as an "inner filter" or "scavenger" that reduces the UV light intensity in the photoreactor. Molecular O2 has absorption bands at 185 and 254 nm, the former being stronger. The processes of degradation depend on the degradation rate relative to the presence and absence of DO.

Chemistry, Organic↗
PubMed2001

Effects of exercise training on myocardial fatty acid metabolism in rats with depressed cardiac function induced by transient ischemia.

The effects of exercise training on metabolic and functional recovery after myocardial transient ischemia were investigated in a rat model. Male Wistar Kyoto rats were subjected either to a 30-min left coronary artery occlusion followed by reperfusion or to a sham operation. At 4 weeks after operation, the rats were randomly assigned either to sedentary conditions or to exercise training for 6 weeks. In the ischemic rats, pinhole SPECT (single photon emission computed tomography) imaging with thallium-201 (201Tl) and 123I-(rho-iodophenyl)-3-R,S-methylpentadecanoic acid (BMIPP) showed a reduction of both myocardial perfusion and fatty acid metabolism in the risk zone of the left ventricle (LV). The LV was dilated and the ejection fraction was decreased after ischemic injury. The severity score showed a significant decrease on both 201Tl and BMIPP (201Tl, from 19.9+/-2.7 to 17.0+/-2.2, p<0.05; BMIPP, from 21.5+/-2.4 to 18.6+/-1.9, p<0.05) after exercise training in the ischemic trained rats, but did not change significantly in their sedentary counterparts. Plasma levels of free fatty acids normalized in the ischemic trained rats, but elevated in the ischemic sedentary rats (0.53+/-0.05 vs 0.73+/-0.06 mmol/L, p<0.05). Furthermore, the trained rats had a significant increase in LV stroke volume (0.25+/-0.02 vs 0.21+/-0.01 ml/beat, p<0.05) and adaptive cardiac hypertrophy. These findings demonstrate that adaptive improvements in myocardial perfusion, fatty-acid metabolism and LV function were induced by exercise training after transient ischemia.

Animals↗
PubMed2001

[Drying ability of anhydrous sodium sulfate on wet organic solvents after liquid-liquid partition].

Water concentration in organic solvents after liquid-liquid partition was determined by the Karl Fischer titration method. n-Hexane and petroleum ether showed quite low levels of water, such as 0.1 mg/mL. The water concentration in wet ethyl acetate was about 20-30 mg/mL and that in diethyl ether was about 8-10 mg/mL. Anhydrous sodium sulfate absorbed about 20-25% of the water after vigorous mixing with wet ethyl acetate or diethyl ether. Wet acetonitrile extract from wet food, which contained about 60 mg/mL water after salting out with sodium chloride, was not dried at all with anhyfrous sodium sulfate treatment. Spiking n-hexane into wet ethyl acetate or wet diethyl ether was effective to exclude water. Spiking toluene into salted acetonitrile drove out water and dissolved sodium chloride. It can be concluded that the drying ability of anhydrous sodium sulfate towards wet organic solvents is poor, but it is effective in removing suspended water in solvents.

Desiccation↗
PubMed2000

Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis.

Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.

Acyltransferases↗
PubMed2000

Developmental expression of the hemichordate otx ortholog.

The phylogenetic location of hemichordates is unique because they seem to fill an evolutionary gap between echinoderms and chordates. We report here characterization of Pf-otx, a hemichordate ortholog of otx, with its embryonic and larval expression pattern. Pf-otx is initially expressed in the vegetal plate of the blastula. Expression remains evident in the archenteron through gastrulation and then disappears. A new expression domain appears near the mouth along the preoral and postoral ciliated bands in the early tornaria larva.

Amino Acid Sequence↗
PubMed2000

Characterization of a hemichordate fork head/HNF-3 gene expression.

Based on anatomical and developmental similarities, hemichordates are thought to be most closely related to chordates. However, so far very few developmental genes have been characterized from hemichordates. To gain molecular insight into the developmental mechanisms involved in the origin and evolution of chordates, we investigated the expression of a fork head/HNF-3 (PfHNF3) gene in the acorn worm embryo. Chordate fork head genes are implicated in the formation of endoderm, notochord and floor plate. We found that a PfHNF3 transcript was first detected at the early blastula stage; the signal of in situ hybridization was found in the vegetal plate cells, invaginating endoderm and then in the archenteron. By the late gastrula and into the early tornaria larva stages, an intense signal remained in the anterior region of the archenteron, while the expression in the other regions of archenteron decreased. The intense signal was retained in the pharynx of the tornaria larva. A comparison of the pattern of PfHNF3 with that of HNF-3 genes of sea urchin, ascidian, amphioxus and vertebrate suggests a possible acquisition of new functions of the gene during deuterostome evolution.

Amino Acid Sequence↗
PubMed2000

Use of three-dimensional computed tomography images in deciding the approach for ministernotomy operations.

BACKGROUND: In ministernotomy operations for valvular and congenital heart diseases, several approaches exist. Because topographical relationships of the sternum, ribs, heart, and great vessels differ slightly with each patient, three-dimensional (3D) computed tomography (CT) images are valuable in deciding the best approach for each patient. METHODS: Ministernotomy patients were studied preoperatively using 3D-CT images. By subtracting some parts of the sternum and the ribs from the 3D images, the topographical relationships of the structures were revealed. RESULTS: Different methods of approach (ie, the site, shape, and length of partial sternotomy) were compared on the 3D images of each patient to decide the most appropriate incision. The images were shown to resemble closely the actual operative field. CONCLUSIONS: Three-dimensional CT images are valuable in preoperative planning of ministernotomy, and are essential to individualized planning for each operation.

Humans↗
PubMed2000

Substrate specificity analysis of microbial transglutaminase using proteinaceous protease inhibitors as natural model substrates.

The substrate specificity of microbial transglutaminase (MTG) from Streptomyces mobaraensis (formerly categorized Streptoverticillium) was studied using a Streptomyces proteinaceous protease inhibitor, STI2, as a model amine-donor substrate. Chemical modification and mutational analysis to address the substrate requirements for MTG were carried out around the putative reactive site region of STI2 on the basis of the highly refined tertiary structure and the solvent accessibility index of Streptomyces subtilisin inhibitor, SSI, a homolog of STI2. The results suggest that the P1 reactive center site (position 70 of STI2) for protease subtilisin BPN' or trypsin may be the prime Lys residue that can be recognized by MTG, when succinylated beta-casein was used as a partner Gln-substrate. It is characteristic in that the same primary enzyme contact region of STI2 is shared by both enzymes, MTG and proteases. For quantitative analysis of the TG reaction, we established an ELISA-based monitoring assay system using an anti-SSI polyclonal antibody highly cross-reactive with STI2. Site-specific STI2 mutants were prepared by an Escherichia coli expression-secretion vector system and subjected to the assay system. We reached several conclusions concerning the nature of the flanking amino acid residues affecting the MTG reactivity of the substrate Lys residue: (i) site-specific mutations from Asn to Lys or Arg at position 69 preceding the amine-donor 70Lys, led to enhanced substrate reactivity; (ii) amino acid replacement at 67Ile with Ser led to higher substrate reactivity, (iii) additive effects were obtained by a combination of the positive mutations at positions 67 and 69 as described above, and (iv) Gly at position 65 might be essential for MTG reaction. Moreover, the substrate specificity of guinea pig liver tissue transglutaminase (GTG) was compared with that of MTG using STI2 and its mutants. In contrast to MTG, replacement of Gly by Asp at position 65 was the most favorable for substrate reactivity. Also, 70Lys appeared not to be a prime amine-donor site for GTG-mediated cross-linking, suggesting a difference in substrate recognition between MTG and GTG.

Amino Acid Sequence↗
PubMed2000

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

Amino Acid Sequence↗
PubMed2000

The complete amino acid substitutions at position 131 that are positively involved in cold adaptation of subtilisin BPN'.

To ascertain whether position 131 of a mesophilic protease, subtilisin BPN', is a potential critical site for cold adaptation as screened by evolutionary engineering (S. Taguchi, A. Ozaki, and H. Momose, Appl. Environ. Microbiol. 64:492-495, 1998), a full set of subtilisin BPN' mutants with mutations at position 131 was constructed by site-saturation mutagenesis. All mutated enzymes were measured for specific activity at 10 degrees C by the quantitative titer microplate assay system using polyclonal antibody against subtilisin BPN' and a synthetic chromogenic substrate. All the mutants exhibited proteolytic activities almost the same as or higher than that of the wild-type enzyme, suggesting that position 131 may be important for cold adaptation. In comparison with the wild type, purified mutants G131F, G131R, G131M, and G131W were found to acquire proteolytic activities (k(cat)/K(m)) at 10 degrees C that were 150, 94, 84, and 50% higher, respectively. In particular, for the G131F mutant, temperature dependency in enzyme activity was shown by an increase in k(cat) and a decrease in K(m). All of these amino acid substitution mutants, G131F, G131R, G131M, and G131W, acquired increased proteolytic activities at 10 degrees C for three different synthetic peptide substrates but no increase in caseinolytic activity. Furthermore, they all conferred thermolability on the enzyme to differing extents in terms of the half-life of enzyme inactivation at 60 degrees C. No significant correlation was found between the amino acids preferred for cold adaptation surveyed here and those present at position 131 of subtilisin of psychrophilic cells naturally occurring in cold environments. Based on these findings, position 131 is a contributor in artificial evolution for acquiring a cold-active character and may not be related to physiological requirements for subtilisin-producing cells living in cold environments. Therefore, saturation mutagenesis would be effective in achieving rapid improvement in protein properties via evolutionary engineering.

Adaptation, Physiological↗
PubMed2000

Variability of bio-optical factors influencing the seasonal attenuation of ultraviolet radiation in temperate coastal waters of Japan

The study identifies the relative contribution of various bio-optical factors to the total attenuation of ultraviolet radiation (UVR) wavelengths and photosynthetically active radiation (PAR) in temperate coastal waters of Japan by surveying the physical properties of the water column, UVR and PAR penetration, and the absorption characteristics of dissolved and particulate material. Spectral absorbance properties of pigment (aph), detritus (ad) and chromophoric dissolved organic material (aCDOM) displayed both seasonal and wavelength specific variability. On an annual basis, absorbance by aCDOM was the highest absorbing fraction (47-59%) for the UVR wavelengths measured (305, 320, 340 and 380 nm) but decreased (32%) at 450 nm. Contribution of pigments to total absorbance was highest (40-60%) during a spring bloom for both UVR and PAR. A large variability (C.V. > 42%) for annual average attenuation coefficients (Kd[lambda]) at respective wavelengths observed suggests that the spectral composition of the water column changes throughout the year in this region. A significant relationship was observed between Kd(lambda) and aCDOM at 305, 320, 340 and 380 nm only (P < 0.01) but not for 450 nm (PAR) indicating the role of CDOM in regulating variations in Kd(lambda), particularly in the UVR range. The slope S, obtained from a natural-log plot of the absorption coefficient of CDOM against wavelength, ranged between 0.014 and 0.036 nm-1 annually (average = 0.020 +/- 0.007, C.V. = 35%) and suggests seasonal changes in the origin of CDOM between terrestrial (low S) and biogenous (high S) CDOM.

Journal Article↗
PubMed2000

A comparative study of cultured smooth muscle cell proliferation and injury, utilizing glycated low density lipoproteins with slight oxidation, auto-oxidation, or extensive oxidation.

We investigated the influence of glycated low density lipoprotein (LDL) for vascular smooth muscle cell (SMC) proliferation or injury. We utilized glycated, slightly oxidized LDL (GLDL-LOX), glycated, auto-oxidized LDL (GLDL) and glycated, metal-induced extensively oxidized LDL (GLDL-OX) to examine the effect of glycation itself or combined glycation and oxidation on SMC. GLDL-LOX induced SMC proliferation and migration, and increased the number of platelet-derived growth factor receptor, beta subunits, (PDGF-R) positive SMC. Also, GLDL-LOX promoted protease activity, compared with the other groups including native LDL (control). GLDL and GLDL-OX demonstrated SMC injury with apoptosis and Bax protein expression, compared with native LDL and GLDL-LOX. These results suggested that LDL glycation contributed to the progression of atherosclerosis by promoting SMC migration and proliferation, with little dependence on oxidative modification. Secondary auto-oxidation adding to glycation induced SMC apoptosis, and SMC injury occurred in the state of strong oxidation with glycation. We concluded that LDL glycation might play a key role in the progression of atherosclerosis in diabetes, and glycated LDL promoted atherosclerosis, even with little assistance from oxidation.

Apoptosis↗
PubMed2000

[Aortocaval fistula with acute right heart failure during surgery: case report].

A 71-year-old man was transferred to our institution with suspected of impending rupture of the abdominal aorta. The clinical manifestations included abdominal pain, pulsatile mass, and abdominal bruit. We made on early diagnosis of aortocaval fistula based on enhanced computed tomography and color Doppler flow imaging. Surgical repair was performed 24 hours after the onset of symptoms. At the introduction of anesthesia, cardiac arrest with high central venous pressure occurred. After resuscitation and rapid clamping of the proximal aorta, surgical closure of the fistula with graft replacement was performed successfully.

Aged↗
PubMed2000

[A surgical case of abdominal aortic aneurysm accompanied by hamartomatous changes in the vascular wall].

The current patient was a 22-year-old woman with dyspnea on exertion that had exacerbated over the past two to three years. Chest X-ray indicated pneumothorax in the right lung, and chest computed tomography showed diffuse microcysts in both lungs. The patient was diagnosed with lymphangiomyomatosis based on pleural biopsy by intrathoracic endoscopy. Systemic testing revealed complications of tuberous sclerosis and abdominal aortic aneyrysm, and a prosthetic graft replacement was perfomed following pneumothorax treatment. Aortic wall tissue images showed hamartomatous changes in smooth muscle tissue, similar to the pulmonary lesion, and this was thought to have caused vascular fragility and aneurysmal change.

Adult↗
PubMed1999

Esophageal carcinoma with high serum parathyroid hormone-related protein (PTHrP) level.

We report two patients with esophageal carcinoma with high levels of serum parathyroid hormone-related protein (PTHrP). Patient 1 was a 66-year-old man in whom the serum calcium level was also high, and patient 2 was an 81-year-old woman. The serum PTHrP level was 411 pM (normal range, 13.8-55.3pM) in patient 1 and 94.5 pM in patient 2 (in whom the serum granulocyte colony-stimulating factor level was also high). We demonstrated PTHrP immunohistologically in esophageal carcinoma cells in both patients. After admission, patient 1 died of pneumonia on the 17th day of hospitalization (the 48th day after he had had an episode of frequent hiccuping) and patient 2 died of acute circulatory failure on the 12th day of hospitalization (the 25th day after she had vomited after a meal). Neither of these patients died of cancer. Pneumonia in patient 1 was believed to be due to weakened body defenses, while the acute circulatory failure in patient 2 was due to emaciation. Since esophageal carcinoma with humoral hypercalcemia of malignancy and leukocytosis is characterized by rapid progression and metastasis, early diagnosis and treatment are mandatory.

Aged↗
PubMed1999

Obesity affects expression of progesterone receptors and node metastasis of mammary carcinomas in postmenopausal women without a family history.

Possible relationships between risk factors, such as obesity and a family history of breast cancer, and prognostic factors of mammary carcinomas were investigated by examining the body mass index of patients and the expression of estrogen (ER) and progesterone receptors (PgR), c-erbB-2 and p53, grade of histology, size of tumors and nodal status of mammary carcinomas. There was no significant difference in the body mass index of premenopausal patients either with or without a family history. For postmenopausal patients, the body mass index was significantly low in patients with a family history compared with patients without a family history. In premenopausal patients with or without a family history and in postmenopausal patients with a family history, there was no significant difference in the body mass index regardless of the mammary carcinoma prognostic factor, such as expression of ER, PgR, c-erbB-2 and p53, grade of histology, size of tumors and nodal status. However, in postmenopausal patients without a family history, body mass index was significantly high for patients with mammary carcinomas that had PgR expression and node metastasis. These results suggest that obesity may affect the PgR status and nodal status of mammary carcinomas in postmenopausal patients without a family history.

Body Mass Index↗