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T Asakura

Publications and source records attributed to T Asakura.

At least 55 records · Page 3Linked to original sources
PubMed2000

Fluid viscosity of Aureobasidium pullulans cultures obtained at two different initial pH values.

Aureobasidium pullulans IAM 5060 grown in media with initial pHs of 6 and 7 differed in its production of exopolysaccharides (EPS). Under the latter condition, EPS of a high molecular weight was produced in an especially large amount. The viscosity of the culture broths and the EPS solutions obtained by removing the microbial cells exhibited a marked non-Newtonian and complex behavior depending on the measurement method. The nature of the viscosity of the fluids is explained by means of several structural models of their constitutive particles.

Journal Article↗
PubMed2000

Structure and dynamics of photosynthetic membrane-bound proteins in Rhodobacter Sphaeroides, studied with solid-state NMR spectroscopy.

The photosynthetic purple bacteria such as Rb. sphaeroides possesses an intracytoplasmic membrane (ICM) and a variety of pigment-binding membrane proteins located in the ICM, acting as photoreceptor. Such photosynthetic apparatus is concentrated in the ICM. It is composed of three multimeric membrane-bound proteins; light-harvesting complexes (LH 1, LH 2), a reaction center (RC) and a cytochrome b/c1 complex. We have purified these membranes, which are called chromatophores, and characterized the structure and dynamics of the photosynthetic membrane-bound proteins by means of multi-nuclear solid state NMR. First, the isotropic chemical shift of carbonyl carbons in natural abundance and [1-(13)C] Phe labeled chromatophores indicates that the membrane-bound proteins take mainly the helical conformation. Second, the chemical shifts of side-chain resonances of uniformly (15)N-labeled chromatophores indicate the side-chain histidine residue is mainly hydrogen bonded, whereas structural heterogeneity of arginine and lysine side-chains are probed by those wide distribution of (15)N shifts. Thirdly, the [beta-(2)H(3)]Ala and [epsilon-(2)H(2)]Tyr labeling of the chromatophores are performed and dynamics of the [beta-(2)H]Ala and the [epsilon-(2)H(2)]Tyr labeled chromatophores are studied by means of (2)H solid state NMR. The dynamics of [beta-(2)H(3)]Ala is found to be a 10(8)Hz three-site jump motion with 10 degrees liberation along the Calpha-Cbeta bond axis. The (2)H-NMR powder pattern spectrum of [epsilon-(2)H(2)] Tyr labeled chromatophores was interpreted with an averaged correlation time of 5x10(5) Hz with 180 degrees two-fold flips, the result of the averaging of two kinds of split spectra in terms of motional time scale.

Journal Article↗
PubMed2000

Functional effects of replacing human alpha- and beta-globins with their embryonic globin homologues in defined haemoglobin heterotetramers.

Embryonic- and adult-stage globin subunits assemble into haemoglobin (Hb) heterotetramers that are expressed at low levels throughout human intrauterine development. These haemoglobins differ from adult Hb A (alpha2beta2) by the substitution of embryonic zeta for adult alpha globin (Hb zeta2beta2), or embryonic epsilon for adult beta globin (Hb alpha2epsilon2). Several key physiological properties of these 'semiembryonic' haemoglobins remain undefined, as ethical and methodological considerations have limited their availability from both human sources and conventional expression systems. The current study attempts to estimate how the physiological properties of semiembryonic and adult haemoglobins may differ, by determining whether the O2-binding characteristics of hybrid human/mouse haemoglobins change when human alpha- or beta-globin subunits are replaced by human embryonic zeta- or epsilon-globin subunits respectively. Each of the four human globins is expressed in transgenic mice that are nullizygous for either the endogenous mouse alpha- or beta-globin genes, resulting in the high-level expression of haemoglobins that can be studied either in situ in intact erythrocytes or in vitro. We showed that the exchange of human zeta-globin for human alpha-globin chains increased haemoglobin O2 affinity, both in the presence and in the absence of 2, 3-bisphosphoglycerate (2,3-BPG), and reduced the pH-dependent shift in its oxygen equilibrium curve (Bohr effect). By comparison, hybrid haemoglobins containing either human epsilon-globin or human beta-globin exhibited nearly identical O2-binding properties, both in situ and in vitro, regardless of 2,3-BPG levels or ambient pH. Neither the zeta-for-alpha nor the epsilon-for-beta substitutions substantially altered binding affinity for 2,3-BPG or cooperativity between globin subunits. These studies suggest that semiembryonic haemoglobins that assemble entirely from human subunits may exhibit properties that are similar to those of human Hb A.

Adult↗
PubMed2000

Structure determination of [Arg8]vasopressin methylenedithioether in dimethylsulfoxide using NMR.

The structure of [Arg8]vasopressin methylenedithioether ([AVP]CH2) has been determined in dimethylsulfoxide-d6. Two-dimensional DQF-COSY and NOESY spectra were measured and used to derive angle and distance constraints for restrained molecular dynamics (MD) calculations. In the MD trajectory, two types of beta-turn structure were found in the region from Tyr2 to Asn5, suggesting an equilibrium between type-I and type-II' beta-turn structures. When Halpha chemical shifts were used as an additional constraint, the type-I turn was favoured. To validate this result, an independent energy minimization procedure was used, using differences between calculated and observed chemical shifts. The two approaches gave essentially identical results. It is therefore concluded that the type-I turn predominates in solution. Analysis of calculated chemical shift contributions suggests that the beta-turn structure found in AVP is well preserved in [AVP]CH2, although the pressin ring size is expanded.

Amino Acid Sequence↗
PubMed2000

The plant aspartic proteinase-specific polypeptide insert is not directly related to the activity of oryzasin 1.

Many plant aspartic proteinases (APs) are different from animal and microbial APs in that they contain a polypeptide insert, approximately 100 amino acids in length, in the C-terminal region. To interpret the significance of this insert, we constructed an expression system for rice AP oryzasin 1 by linking a pro-oryzasin 1 downstream of glutathione S-transferase (GST). GST-proOS1 expressed the highest degree of hemoglobin-hydrolytic activity when treated at pH 3.3 and incubated for 24 h at room temperature. We carried out a similar experiment using an insert-lacking proOS1 mutant, GST-DeltaproOS1, as the fusion protein, and found it to show similar activity. This result indicates that the insert is not involved in the production of AP activity. We then investigated the autolysis of the two proteins by Western blot analysis. GST-proOS1 was autolyzed into 67- and 64-kDa fragments, while GST-DeltaproOS1 autolyzed to 54- and 52-kDa products. GST-DeltaproOS1 clearly produced two molecular species early in the autolytic process, and not later than 3 h from the start, but no such clear result was observed in the case of GST-proOS1. This suggests that, although the presence of the plant AP-specific insert does not influence the enzyme activity by itself, it apparently has an effect on the autolysis of OS1.

Amino Acid Sequence↗
PubMed2000

Analysis of conformational changes at the unique loop adjacent to the ATP binding site of smooth muscle myosin using a fluorescent probe.

Recent crystallographic studies have shown that smooth muscle myosin has three highly conserved unique loops, loop B (320-327), loop M (687-699), and loop N (125-134), similar to other myosins, skeletal muscle and dictyostelium myosins. We previously demonstrated that the effect of actin is mediated by a conformational change in one of the loops, loop M comprising amino acids 677 to 689 of skeletal muscle myosin [Maruta and Homma (1998) J. Biochem. 124, 528-533]. In the present study, in order to clarify the role of these smooth muscle myosin loops in energy transduction, we specifically labeled the loops with a fluorescent photoreactive ADP analogue, 3'-O-(N-methylanthraniloyl)-8-azido-ADP (Mant-8-N(3)-ADP), and then measured the fluorescent polarization. When Mant-8-N(3)-ADP was trapped by aluminium fluoride or vanadate into the ATPase site, Mant-8-N(3)-ADP was covalently incorporated into loop N (125-134). In contrast, Mant-8-N(3)-ADP trapped by beryllium fluoride was covalently incorporated into both loop M (687-699) and loop N (125-134) at an almost equimolar ratio. Actin binding to smooth muscle myosin S1 (SMO-S1) labeled at only loop N (125-134) increased the polarization due to the viscosity of actin. In contrast, S1 labeled at both loops N and M showed a much smaller increase in polarization. Our results indicate that the probe at loop M (687-699) of smooth muscle myosin moved to a less hindered region, suggesting that actin binding induces conformational changes at loop M (687-699) similar to those of the corresponding loop (677-689) in skeletal muscle myosin, as previously demonstrated in our laboratory.

Adenosine Diphosphate↗
PubMed2000

Bond strength of resin to acid-etched dentin studied by 13C NMR: interaction between N-methacryloyl-omega-amino acid primer and dentinal collagen.

The application of the hydrophilic methacrylate primer to acid-etched dentin increases the bond strength of the resin to the acid-etched dentin. However, the mechanism for the bonding of the resin to dentinal collagen through the primer remains to be determined. Before a more effective adhesive primer can be developed, we must understand the adsorption behavior of the primer to the dentinal collagen. The purpose of this study was to determine how 5 derivatives of N-methacryloyl-omega-amino acid (NMomegaA) primers enhance the bonding of the resin to acid-etched dentin. The interaction between the NMomegaA primers and dentinal collagen was studied by the 13C NMR technique, including the observation of spin-lattice relaxation times, T1. When the dentinal collagen was dispersed into the NMomegaA solution, the T1 values of the two carbonyl carbons attributed to the amide and the carboxylic acid in the NMomegaA molecule decreased dramatically. This result was due to the interaction between the amide group and the carboxylic acid group in the NMomegaA molecule and the dentinal collagen molecule. The T1 values of these carbonyl carbons decreased when the number of methylene groups in the NMomegaA molecule increased. The interaction became stronger as the number of methylene groups in the NMomegaA molecule was increased. Further, the bond strength of the resin to the acid-etched dentin primed with NMomegaA increased with a decrease in the T1 value of the amide carbonyl carbon. The strength of the interaction of the NMomegaA primer to the dentinal collagen molecule, determined by the 13C NMR technique, showed a direct correlation with the bond strength of the resin to acid-etched dentin that was treated with the NMomegaA primer.

Acid Etching, Dental↗
PubMed2000

Prediction of embolism in atrial fibrillation: classification of left atrial thrombi by transesophageal echocardiography.

The current study was undertaken to clarify the relationship between cerebral/arterial embolism and the morphology of left atrial thrombi. Forty-one patients with atrial fibrillation and left atrial thrombi were followed for 1 year, using transesophageal echocardiography (TEE) to study how the shape, site, movability, number and maximum dimension of left atrial thrombi are related to embolism. Left atrial thrombi were classified by their shape and movability into movable ball (MB) type (n=13), fixed ball (FB) type (n=17) and mountain (MN) type (n=11). The thrombi were also classified by location into the interior section (n=3), middle section (n=8), and the entrance section (n=19) of the left atrial appendage, and the section outside of the left atrial appendage (n=11). The rate of embolism in the MB-type group was significantly higher than that in the other groups (ie, MB 76.9% vs FB 17.6% (p<0.01) vs MN 9.1%; p<0.01), which indicates that the MB-type thrombus is an important risk factor for cerebral/arterial embolism.

Adult↗
PubMed2000

[Study of free to total prostate specific antigen ratio in the detection of patients with prostate cancer].

BACKGROUND: We investigated the clinical usefulness of free to total serum prostate specific antigen (PSA) ratio (F/T ratio) in order to improve the specificity of total PSA measurement for detecting prostate cancer. METHOD: In this study 129 patients with total PSA level 4-20 ng/ml underwent transrectal ultrasound guided sextant biopsy. Serum samples were assessed for total PSA, free PSA and the F/T ratio calculated. All patients were pathologically diagnosed as benign prostatic hyperplasia or prostate cancer. RESULTS: Of 129 patients 21 had prostate carcinoma (PCa) and 108 had benign prostatic hyperplasia (BPH) from the results of prostate biopsies. The mean of total PSA were not significantly different between men with PCa and with BPH. The mean of free PSA for PCa was significantly lower than that for BPH (p = 0.043). Furthermore, the mean of F/T ratio was significantly different between PCa and BPH group (p = 0.0014). The F/T ratio had a higher specificity than total PSA at all levels of sensitivity in detecting prostate cancers. Sensitivity, specificity and accuracy for cancer detection at a cut off 0.12 was 90.4%, 51.8% and 58.1%, respectively. Also, free PSA was as useful as F/T ratio for cancer detection when analyzed in receiver operating characteristic curves analysis. When determined the cut off number of free PSA at 0.78 ng/ml, the sensitivity, specificity and accuracy for cancer detection were 61.9%, 66.7% and 65.9%, respectively. CONCLUSION: This study indicated that the F/T ratio and free PSA could improve the specificity without impairing the sensitivity for detecting PCa in patients with 4-20 ng/ml of total PSA.

Aged↗
PubMed2000

Physical aging by soft ultrasonic wave enhances ethanol metabolism: metabolic process of wine as followed by 400 mhz 1H-NMR spectroscopy.

In natural aging of spirits or wine, the dynamic structure of ethanol-water clusters changes to a smaller and more uniform state. Through experience we know that naturally aged ones have higher metabolism than the non-aged ones. Also, the same effect as natural aging can be obtained in various types of spirits or wines by the treatment for a period of time with soft ultrasonic wave (US). In this study, we compared ethanol metabolism in human subjects dosed with non-treated white wine (control = CON) and with US treated wine. Ethanol levels in human sera were followed by 400 MHz 1H-NMR spectroscopy after administration of wine doses. Experimental results indicated that ethanol metabolism was enhanced 18% in subjects when US treated wine was used rather than when non-treated (CON) was used. Other experiments using rabbits showed that a 20% ethanol-aqueous solution was absorbed 18% more rapidly by the group dosed with US wine than by the CON group. From these experimental facts, it was theorized that ethanol metabolism depends on the rapidity of ethanol absorption in the human body. And it can be concluded that US treatment brings about the same effect on spirits or wines as natural aging.

Animals↗
PubMed2000

[Continuous hepatic arterial infusion of 5-fluorouracil with leucovorin for unresectable liver metastases from colorectal cancer].

Twenty-three patients with liver metastases from colorectal cancer were treated by continuous hepatic arterial infusion chemotherapy with 5-FU and Leucovorin. The regimen was that 500 mg/body of 5-FU with 30 mg/body of Leucovorin was administered continuously for 5 days, followed by no medication for 16 days. The effect of this therapy was evaluated and the relationship between this therapy and the overexpression of vascular endothelial growth factor (VEGF) or microvessel density (MVD) was also studied. Complete response was obtained in 4 patients and partial response in 3 patients; the response rate was 32%. The response rate was 60% in patients who underwent more than 7 courses. The response rate was 44% in patients with positive VEGF and 33% in patients with negative VEGF. The response rate was 50% in patients with an MVD of more than 30 and 33% in patients with an MVD of less than 30. The 3-year survival rate for patients who underwent more than 7 courses was 37.5%. This therapy had to be abandoned in 6 patients due to occlusion of the catheter. Skillful maintenance of the catheter is necessary for a high response rate and satisfactory prognosis using this therapy.

Aged↗
PubMed1999

Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR.

Solid state 31P NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The 31P chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group. Macroscopically oriented MP-lipid samples between glass plates were studied by 31P NMR as a function of tilt angle. These spectra showed the coexistence of orientation-dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non-lamellar phases in DMPC/DMPG membranes.

Amino Acid Sequence↗
PubMed1999

Solid phase synthesis and biological activities of [Arg8]-vasopressin methylenedithioether.

Solid phase synthesis of [Arg8]-vasopressin methylenedithioether, an analog of vasopressin which contains an extra methylene group between the two sulfur atoms of Cys1 and Cys6, is described. Methylene insertion occurred easily when the thiol free peptide on a solid support was treated with tetrabutylammonium fluoride in dichloromethane at room temperature for 3 h. The uterotonic in vitro, pressor, and antidiuretic activities of the compound were reduced in comparison to [Arg8]-vasopressin by one order of magnitude.

Animals↗
PubMed1999

Elimination of Kupffer cells and nafamostat mesilate rinse prevent reperfusion injury in liver grafts from agonal non-heart-beating donors.

BACKGROUND: We hypothesized that microcirculatory disturbance was an obstacle to liver transplantation (LTx) from non-heart-beating donors (NHBDs) and that it was attributed mainly to a deterioration of sinusoidal endothelial cells (SECs) and sinusoidal narrowing. This study was designed to examine porcine orthotopic LTx using livers obtained from pretreated agonal NHBDs, and to determine whether the maintenance of the liver microcirculation would result in successful LTx from agonal NHBDs. METHODS: Pigs were allocated to five groups: (i) control group; (ii) NM group, in which grafts were rinsed with nafamostat mesilate (NM) rinse; (iii) LD group, in which Kupffer cells in grafts were eliminated by liposome-encapsulated dichloromethylene diphosphonate (L-DMDP); (iv) LDNM group, in which grafts pretreated with L-DMDP were rinsed with NM rinse; (v) heart-beating donor (HBD) group. In all groups, but the HBD group, the livers were pretreated with FK506 and prostaglandin I2 analogue, and were preserved in University of Wisconsin solution after cardiac arrest. Thereafter orthotopic LTx was performed. RESULTS: After reperfusion, it was histologically demonstrated that elimination of Kupffer cells prevented SECs deterioration and NM rinse prevented sinusoidal narrowing. The hepatic energy charge recovered in all groups except the control group. In the LDNM group, three of four recipients survived more than 7 days. CONCLUSIONS: For a successful LTx from agonal NHBDs, it is important to prevent microcirculatory disturbance caused by SEC deterioration and sinusoidal narrowing after reperfusion. Combination therapy consisting in the elimination of Kupffer cells and NM rinse prevented primary graft non-function in liver grafts from agonal NHBDs.

Adenosine Triphosphate↗
PubMed1999

A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions.

The first study by nmr of the integral membrane protein, the bacterial light-harvesting (LH) antenna protein LH1 beta, is reported. The photosynthetic apparatus of purple bacteria contains two different kinds of antenna complexes (LH1 and LH2), which consist of two small integral membrane proteins alpha and beta, each of approximately 6 kDa, and bacteriochlorophyll and carotenoid pigments. We have purified the antenna polypeptide LH1 beta from Rhodobacter sphaeroides, and have recorded CD spectra and a series of two-dimensional nmr spectra. A comparison of CD spectra of LH1 beta observed in organic solvents and detergent micelles shows that the helical character of the peptide does not change appreciably between the two milieus. A significantly high-field shifted methyl signal was observed both in organic solvents and in detergent micelles, implying that a similar three-dimensional structure is present in each case. However, the 1H-nmr signals observed in organic solvents had a narrower line width and better resolution, and it is shown that in this case organic solvents provide a better medium for nmr studies than detergent micelles. A sequential assignment has been carried out on the C-terminal transmembrane region, which is the region in which the pigment is bound. The region is shown to have a helical structure by the chemical shift values of the alpha-CH protons and the presence of nuclear Overhauser effects characteristic of helices. An analysis of the amide proton chemical shifts of the residues surrounding the histidine chlorophyll ligand suggests that the local structure is well ordered even in the absence of protein-lipid and protein-pigment interactions. Its structure was determined from 348 nmr-derived constraints by using distance geometry calculations. The polypeptide contains an alpha-helix extending from Leu19 (position of cytoplasmic surface) to Trp44 (position of periplasmic surface). The helix is bent, as expected from the amide proton chemical shifts, and it is similar to the polypeptide fold of the previously determined crystal structure of Rhodopseudomonas acidophila Ac10050 LH2 beta (S. M. Prince et al., Journal of Molecular Biology, 1997, Vol. 268, pp. 412-423). It is concluded that the polypeptide conformation of this region may facilitate assembly of the LH complex.

Amino Acid Sequence↗
PubMed1999

Use of 13C conformation-dependent chemical shifts to elucidate the local structure of a large protein with homologous domains in solution and solid state.

In order to clarify the difference between solution NMR and X-ray diffraction analyses concerning the presence of alpha-helical structure in protein A, the 13C conformation-dependent chemical shifts of the 13C-labeled carbonyl carbons for selectively labeled protein A were used. In the 13C CP/MAS NMR spectra, the higher-field shifts of the carbonyl carbons of 13C-labeled Thr and Val residues compared with the random coil chemical shifts both in solution and solid state imply the presence of the third helix in the polypeptide chain, in contrast to the crystal structure of Fc-bound B-domain. Thus, a combination of selective isotope labeling and conformation-dependent chemical shifts will be a good Indicator to monitor the local structure of homologous protein in solution and solid state.

Amino Acid Sequence↗
PubMed1999

Spectroscopic investigation of tertiary fold of staphylococcal protein A to explore its engineering application.

Staphylococcal protein A is a cell wall constituent of most strains of Staphylococcus aureus, and it is characterized by its binding affinity to some immunological classes. A mutated low molecular weight type protein A (LPA; Mwt = 27 kDa) which consists of the domains, E, D, A, B and 13 residues of the C-domain was prepared in this study. Since LPA does not possess a cell wall-bound region in contrast to wild-type protein A (WPA; Mwt = 42 kDa), we have established a methodology of large scale purification of LPA without using any extracellular expression systems such as Escherichia coli. Using this relatively abundant protein, the immobilization of the LPA with silk fibroin of Bombyx mori was performed. Thermal stability of LPA immobilized with silk fibroin is higher than that of free LPA at high temperature judging from the immunoglobulin G (IgG)-binding affinity. However, the apparent value of its affinity decreased relative to that of immobilized WPA. These results indicate that structural information is essential to explore improvement of IgG-binding affinity of immobilized LPA. Therefore, secondary structure of free LPA was detected by its characteristic helical pattern in circular dichroism (CD) in aqueous solution. In addition to this, tertiary fold of four IgG-binding domains were investigated by two-dimensional 1H-NMR spectra. Four significantly high-field shifted cross-peaks attributed to methyl signals of alanine residues suggest that all four domains pack into a three helix bundle motif in solution. These structural data and properties of IgG-binding affinity suggest that spatial arrangement of four IgG-binding domains are packed into a compact globular molecular shape. This causes a certain active site of immobilized LPA to be buried in the silk fibroin fiber.

Alanine↗