Biomedical subjects

Hiroyuki Takashima

Publications and source records attributed to Hiroyuki Takashima.

15 recordsLinked to original sources

PubMed2006

Structure of cytochrome c552 from a moderate thermophilic bacterium, Hydrogenophilus thermoluteolus: comparative study on the thermostability of cytochrome c.

We have studied the structure-thermostability relationship using cytochromes c from mesophilic and thermophilic bacteria; Pseudomonas aeruginosa (PAc(551)) growing at 37 degrees C and Hydrogenobacter thermophilus (HTc(552)) at 72 degrees C and showed that only five residues primarily differentiate their stabilities. For a more comprehensive study, we found Hydrogenophilus thermoluteolus (Pseudomonas hydrogenothermophila) growing at 52 degrees C and showed the moderate stability of the cytochrome c from this bacterium (PHc(552)). To explore the stabilization mechanisms, the crystal structure of PHc(552) was determined by X-ray analysis. The solution structure of HTc(552) elucidated previously by NMR was refined using distributed computational implementation. Furthermore, the recently reported crystal structure of HTc(552) has become available [Travaglini-Allocatelli, C. et al. (2005) J. Biol. Chem. 280, 25729-25734]. When the structures of these three cytochromes c were combined, this revealed that the five residues, corresponding to those mentioned above, determine the difference of stabilities among them as well. These facts suggested the stabilization mechanisms as follows: (1) improved van der Waals interactions by packing optimization at the N-terminal helix, (2) attractive electrostatic interactions with the heme propionate group, and (3) favorable van der Waals interaction with the heme. This comparative study, by supplementing the structural information of PHc(552) with its complementary feature, demonstrates that just a small number of amino acid residues determine the overall molecular stability by means of additivity of the effects of their substitutions. It is interesting that, in naturally occurring proteins, these adaptation strategies are accommodated by these bacteria to survive in the wide range of thermal conditions.

Amino Acid Sequence↗

PubMed2006

The occurrence of systemic lupus erythematosus in an asymptomatic carrier of human T-cell lymphotropic virus type I.

A 63-year-old asymptomatic carrier of human T-cell lymphotropic virus type I (HTLV-1) infection was admitted because of chest oppression, a high-grade fever, polyarthralgia, and erythematous rashes. Laboratory examination revealed lymphocytopenia, proteinuria, and high titers of antinuclear antibodies and antidouble-stranded DNA antibody; thus, she was diagnosed as having systemic lupus erythematosus (SLE). This case indicates that HTLV-1 infection might be related with the pathogenesis of SLE.

Carrier State↗

PubMed2006

Relationship between renal function and plasma brain natriuretic peptide in patients with heart failure.

OBJECTIVES: This study sought to evaluate the relationship between brain natriuretic peptide (BNP), renal function, and the severity of congestive heart failure (CHF). BACKGROUND: Both BNP and renal function are prognostic predictors in CHF patients. METHODS: We measured the plasma BNP level in the aortic root and coronary sinus in 366 consecutive patients with CHF. Estimated glomerular filtration rate (eGFR) by the Cockcroft-Gault equation was used as an indicator of renal function. RESULTS: By stepwise multivariate analyses, hemodynamic parameters such as left ventricular ejection fraction (LVEF) and left ventricular end-diastolic pressure (LVEDP) but not eGFR were independent predictors of a transcardiac increase (coronary sinus-aortic root) in BNP. Regarding the plasma level of BNP in the aortic root, not only LVEF (p < 0.0001) and LVEDP (p < 0.0001) but also eGFR (p < 0.0001) were independent predictors. Patients were divided into two groups, patients with an eGFR > or =60 ml/min (group 1, n = 229) and patients with an eGFR <60 ml/min (group 2, n = 137). There was no difference in LVEF, LVEDP, or the transcardiac gradient of BNP between the two groups, but the plasma level of BNP in the aortic root was approximately two-fold greater in group 2 than in the group 1. CONCLUSIONS: These findings suggest that decreased clearance from the kidney contributes to the elevated BNP in CHF patients with renal dysfunction, especially in patients with an eGFR <60 ml/min.

Aged↗

PubMed2006

Conformational analysis of human calcitonin in solution.

The solution conformation of human calcitonin in a mixture of 60% water and 40% trifluoroethanol has been determined by the combined use of 1H NMR spectroscopy and distance geometry calculations with a distributed computing technique. 1H NMR spectroscopy provided 195 distance constraints and 13 hydrogen bond constraints. The 20 best converged structures exhibit atomic rmsd of 0.43 A for the backbone atoms from the averaged coordinate position in the region of Asn3-Phe22. The conformation is characterized by a nearly amphiphilic alpha-helix domain that extends from Leu4 in the cyclic region to His20. There are no significant differences observed among the overall structures of a series of calcitonins obtained from ultimobranchial bodies, including those that possess 20- to 50-fold greater activity. Three aromatic amino acid residues, Tyr12, Phe16 and Phe19, form a hydrophobic surface of human calcitonin. Bulky side chains on the surface could interfere with the ligand-receptor interaction thereby causing its low activity, relative to those of other species.

Animals↗

PubMed2006

Impact of paraoxonase polymorphism (Q192R) on endothelial function in intact coronary circulation.

Paraoxonase-1 (PON1) can protect endothelial function by preventing the oxidation of low-density lipoprotein (LDL) cholesterol and retarding the development of atherosclerosis. We examined whether PON1 polymorphism influences endothelium-dependent coronary vasomotor responses. Sixty-seven patients underwent diagnostic cardiac catheterization, but showed no significant coronary artery stenosis. In all patients, PON1 genotypes (Q/Q, Q/R and R/R) were determined, and provocative testing was performed by the intracoronary administration of graded doses of bradykinin (BK; 0.2, 0.6 and 2.0 mug/min) and acetylcholine (ACh; 3, 10 and 30 mug/min). Coronary blood flow (CBF) was evaluated by a Doppler guide wire. The patients were divided into 2 groups on the basis of ACh testing: one with coronary spastic angina (CSA) and one with non-CSA. The frequencies of the PON1 genotype in the CSA group did not differ significantly from those in the non-CSA group. In the non-CSA group, the patients were subdivided into 2 groups: a group with the Q/Q or Q/R genotypes and a group with the R/R genotype. The vasoconstrictive responses of the epicardial coronary artery to ACh were comparable between the Q/Q + Q/R and R/R groups. Also, the coronary vasodilations induced by BK in the R/R group were similar to those in the QR + QQ group. There were no significant differences in the CBF responses induced by BK or ACh between the Q/Q + Q/R and R/R groups. In conclusion, as estimated by BK and ACh testing, our findings suggest that PON1 genotypes may not play a critical role in the modulation of endothelial vasomotor function in the intact coronary circulation.

Acetylcholine↗

PubMed2005

Solution NMR structure investigation for releasing mechanism of neocarzinostatin chromophore from the holoprotein.

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulin-like seven-stranded anti-parallel beta-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the beta-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.

Crystallography, X-Ray↗

PubMed2005

Brachial artery flow-mediated vasodilation is correlated with coronary vasomotor and fibrinolytic responses induced by bradykinin.

Endothelium plays a key role in the regulation of not only vascular tone but also thrombosis and fibrinolysis. Brachial flow-mediated vasodilation (FMD) provides a noninvasive method of assessing coronary endothelial dysfunction. However, no data are available on the relationship between brachial FMD and coronary fibrinolytic activity. Thus, we examined the relationship between brachial FMD and coronary vasomotor and fibrinolytic function. Brachial FMD by reactive hyperemia was defined as a change in diameter relative to the baseline as measured using high-resolution ultrasound. Coronary blood flow (CBF) responses to bradykinin (BK) were analyzed using Doppler flow velocity measurement. Coronary release of tissue-type plasminogen activator (tPA) antigen was determined as the transcardiac tPA gradient x [CBF x (100 - hematocrity 100)]. In 77 patients with normal coronary arteries, BK caused dose-dependent increases in CBF, transcardiac tPA gradient, and coronary tPA release. Among them, brachial FMD, the BK-induced CBF increase, and the coronary tPA release induced by BK in 14 diabetic subjects were lower than those in 63 non-diabetic subjects (p < 0.05, respectively). Brachial FMD correlated with the CBF increase, transcardiac tPA gradient (0.2 microg/min: r = 0.25; 0.6 microg/min: r = 0.43; 2.0 microg/min: r = 0.34; p < 0.05, respectively), and coronary tPA release (0.2 microg/min: r = 0.24; 0.6 microg/min: r = 0.44; 2.0 microg/min: r = 0.32; p < 0.05, respectively) in response to BK. Brachial FMD correlated significantly with coronary endothelial function and fibrinolytic activity in response to BK. Type 2 diabetes impaired coronary and brachial endothelium-dependent vasodilation and coronary fibrinolytic activity.

Adult↗

PubMed2004

Hydrophobic core around tyrosine for human endothelin-1 investigated by photochemically induced dynamic nuclear polarization nuclear magnetic resonance and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Human endothelin-1 (ET-1) is a potent cardiovascular bioactive peptide. Its activity is based on the C-terminal residues, e.g., Trp 21 in particular. Recently, we reported an NMR solution structure of ET-1, which has a C-terminal hydrophobic core around Tyr 13. This C-terminal conformation does not agree with a previously reported X-ray crystal structure. To clarify the discrepancy, we performed photo-CIDNP NMR in combination with MALDI-TOF MS. The photo-CIDNP results revealed that the Tyr 13 aromatic ring is concealed in a hydrophobic interaction. MALDI-TOF MS experiments showed this is an intramolecular interaction in monomeric form, which is also supported by sedimentation analysis and two-dimensional NMR cross-peak line shapes. Thus, we confirmed the intramolecular hydrophobic core around Tyr 13 in aqueous solution, which agrees with the solution structure. The C-terminal conformational discrepancy between the solution and crystal was caused by the intermolecular hydrogen bond between Tyr 13 of one molecule and Asp 8 of the other in a dimer-like formation of crystalline ET-1. On the other hand, we indicated that endothelin-3, another isoform of the endothelin, has an apparent self-association equilibrium under the same condition in which three tyrosines participate.

Amino Acid Sequence↗

PubMed2004

Plasma level of oxidized low-density lipoprotein is an independent determinant of coronary macrovasomotor and microvasomotor responses induced by bradykinin.

OBJECTIVES: We examined the relationship between coronary endothelium-dependent vasodilation in response to bradykinin (BK) and plasma levels of oxidized low-density lipoprotein (oxLDL) in subjects with normal coronary arteries. BACKGROUND: It is unclear whether the plasma oxLDL level is a determinant of coronary endothelial function. Bradykinin plays an important role in regulating resting coronary tone and flow-mediated coronary vasomotion. METHODS: Coronary blood flow (CBF) in the left anterior descending (LAD) coronary artery was assessed by quantitative angiography and a Doppler flow wire in 94 consecutive subjects with normal coronary arteries. The plasma oxLDL level was measured by enzyme-linked immunosorbent assay using DLH3R, a specific antibody against oxLDL. RESULTS: Plasma levels of oxLDL in diabetic subjects (n = 13) were higher than those in non-diabetic subjects (n = 81). Plasma levels of oxLDL correlated with body mass index (BMI). Bradykinin at doses of 0.2, 0.6, and 2.0 microg/min caused dose-dependent increases in diameter and CBF in the LAD coronary artery. By a univariate analysis, oxLDL levels significantly correlated with epicardial (r = -0.30, p < 0.0001) and resistant (r = -0.36, p = 0.003) coronary vasodilator responses to BK at 2.0 microg/min, whereas total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and triglycerides were not associated with these coronary responses. In a stepwise multivariate analysis, oxLDL levels were significantly correlated with epicardial and resistant coronary vasomotor responses to BK, independent of age, gender, smoking status, other lipid levels, BMI, hypertension, and diabetes. CONCLUSIONS: The plasma level of oxLDL is an appropriate surrogate for assessing coronary endothelial-dependent vasomotor function as estimated by responses to BK compared with conventional risk factors for atherosclerosis.

Adult↗

PubMed2004

Distributed computing and NMR constraint-based high-resolution structure determination: applied for bioactive Peptide endothelin-1 to determine C-terminal folding.

Distributed computing has been implemented to the solution structure determination of endothelin-1 to evaluate efficiency of the method for NMR constraint-based structure calculations. A key target of the investigation was determination of the C-terminal folding of the peptide, which had been dispersed in previous studies of NMR, despite its pharmacological significances. With use of tens of thousands of random initial structures to explore the conformational space comprehensively, we determined high-resolution structures with good convergences of C-terminal as well as previously defined N-terminal structures. The previous studies had missed the C-terminal convergence because of initial structure dependencies trapped in localized folding of the N-terminal region, which are strongly constricted by two disulfide bonds.

Endothelin-1↗

PubMed2004

The relationship between flow-mediated brachial artery vasodilation and coronary vasomotor responses to bradykinin: comparison with those to acetylcholine.

Flow-mediated dilation (FMD) of brachial artery provides a noninvasive assessment of coronary endothelial dysfunction. Acetylcholine (ACh) has been used as an agent for estimating coronary endothelial function. In contrast to ACh, there is no evidence for a relationship between FMD and coronary vasodilation to bradykinin (BK). The aim of this study was to compare the flow-mediated vasodilation of brachial artery with coronary vasomotor responses to intracoronary ACh or BK in patients with an angiographically normal left anterior descending coronary artery. Ninety-one patients underwent the cardiac catheterization examination with coronary endothelial function testing and the brachial ultrasound study. BK (0.2, 0.6, 2.0 microg/min) and ACh (3, 10, 30 microg/min) were administered into the left coronary artery in a stepwise manner. Coronary blood flow was evaluated by the Doppler flow velocity measurement. Coronary diameters were measured by the quantitative coronary angiography. The assessment of endothelial function in the brachial artery was made in response to reactive hyperemia with high-resolution ultrasound. Bradykinin induced dose-dependent increases in epicardial coronary diameter and blood flow. There was a significant positive correlation between FMD- and BK-induced vasodilations of epicardial coronary arteries (0.2 microg/min: r = 0.30; 0.6 microg/min: r = 0.42; 2.0 microg/min: r = 0.44, P < 0.01, respectively) and resistance coronary arteries (0.2 microg/min: r = 0.40; 0.6 microg/min: r = 0.56; 2.0 microg/min: r = 0.59, P < 0.0001, respectively). FMD correlated with ACh-induced vasomotions of resistance but not epicardial coronary arteries. No correlation was seen between nitroglycerin-induced brachial artery vasodilation and BK-induced coronary vasodilation. The endothelial dysfunction of peripheral arteries correlated well with that of the coronary arteries especially vasomotor responses to BK.

Acetylcholine↗

PubMed2004

Angiotensin-converting enzyme insertion/deletion polymorphism modulates coronary release of tissue plasminogen activator in response to bradykinin.

The aim of this study was to assess the relationship between the angiotensin converting enzyme gene (ACE) genotype and endothelium-dependent coronary vasomotor and fibrinolytic activity. The ACE DD genotype has been reported to be a risk factor for myocardial infarction. However, the mechanism is unknown. The fibrinolytic and renin-angiotensin systems are linked via ACE at the vascular beds. We studied 73 patients (II: n=24; ID: n=37; DD: n=12) who underwent diagnostic cardiac catheterization. Graded doses of bradykinin (BK) (0.2, 0.6, 2.0 microg/min) and acetylcholine (30,100 microg/min) were administered into the left coronary artery. Coronary blood flow (CBF) was evaluated by measuring Doppler flow velocity. Blood samples were taken from the aorta (Ao) and the coronary sinus (CS). Coronary release of tPA antigen was determined as a CS-Ao gradientXCBFX[(100-hematocrit) / 100]. ACE genotypes were determined using polymerase chain reaction. The ACE genotype did not appear to affect coronary macro- and microvascular responses induced by BK or acetylcholine. Coronary tissue plasminogen activator (tPA) release induced by BK was depressed in subjects with the ACE DD genotype. ACE levels in the DD genotype were significantly higher than those in the ID or II genotype. In all of the subjects, there was a significant negative correlation between the serum level of ACE activity and net coronary tPA release in response to BK at 0.6 microg/min. In conclusions, the DD genotype of the ACE gene impairs the coronary release of tPA induced by BK.

Aged↗

PubMed2003

Endothelial modulation and tolerance development in the vasorelaxant responses to nitrate of rabbit aorta.

We investigated the endothelial modulations in nitrate tolerance in isolated rabbit aorta. Nitrate tolerance was induced by a 72-h treatment with transdermal nitroglycerin (NTG, 0.4 mg/h) in conscious rabbits, which was verified by a 20-fold increase in the EC50 values [NTG tolerance (6.1 +/- 0.8) x 10(-7) M vs control (3.0 +/- 0.6) x 10(-8) M]. The relaxations to NTG in tolerant and nontolerant aortic strips were enhanced when their endothelia were denuded [E(-)]. In the presence of endothelium [E(+)], NTG-tolerant vessels were not tolerant to acetylcholine (ACh), which can release endothelial nitric oxide (NO), exogenous NO or 8-bromo (Br)-cGMP. In NTG-tolerant and nontolerant vessels with endothelium, concentration-response curves for NO were the same as those in endothelium-absent tolerant vessels. In both NTG-tolerant and nontolerant vessels, treatment with superoxide dismutase (SOD, 20 units/ml), an O2-. scavenger, unaffected the responses to NTG reduced in the presence of endothelium, but treatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), an NO synthase (NOS) inhibitor, reversed these reductions. Thus, our data did not indicate that an increased endothelial superoxide O2-. production contributes to nitrate tolerance. Our study suggested that (i) an impaired biotransformation process from NTG to NO is responsible for the occurrence of nitrate tolerance and (ii) vascular response to NTG enhanced by endothelial removal is related to blocked endothelial NO release.

Acetylcholine↗

PubMed2003

Angiotensin-converting enzyme inhibition but not angiotensin II type 1 receptor antagonism augments coronary release of tissue plasminogen activator in hypertensive patients.

OBJECTIVES: We compared the effects of perindopril and losartan on endothelium-dependent coronary vasomotor and fibrinolytic function. BACKGROUND: The renin-angiotensin system regulates the vascular fibrinolytic balance. However, the effects of angiotensin-converting enzyme inhibitors and angiotensin II type 1 receptor antagonists on coronary fibrinolytic function have not been compared in hypertensive patients. METHODS: Forty-five patients with hypertension were randomly assigned to three groups: 16 patients were treated with perindopril (4 mg/day) for four weeks; 15 were treated with losartan (50 mg/day) for four weeks; and 14 were not treated with either perindopril or losartan (control group). Graded doses of bradykinin (BK) (0.2, 0.6, and 2.0 microg/min) were administered into the left coronary artery. Coronary blood flow (CBF) was evaluated by Doppler flow velocity measurement. RESULTS: Bradykinin induced dose-dependent increases in CBF in all groups. The increases in CBF induced by BK in the perindopril and losartan groups were significantly greater than those in the control group. Net coronary tissue-type plasminogen activator (t-PA) release was enhanced by BK in all groups, and the increase in the perindopril group was greater than that in the losartan and control groups. Bradykinin did not alter plasminogen activator inhibitor type 1 levels in any of the groups. CONCLUSIONS: Perindopril and losartan similarly augment BK-induced coronary vasodilation. Perindopril may have a greater potential to enhance the BK-induced coronary release of t-PA than losartan.

Aged↗

PubMed2002

Noninvasive assessment of coronary flow velocity and coronary flow velocity reserve in the right coronary artery by transthoracic Doppler echocardiography: comparison with intracoronary Doppler guidewire.

The aim of this study was to evaluate whether coronary flow velocity (CFV) and coronary flow velocity reserve (CFVR) in the posterior descending right coronary artery can be reliably measured by transthoracic Doppler echocardiography (TTDE). In 17 patients, CFV in the posterior descending right coronary artery was measured with TTDE at the time of Doppler guidewire examination. CFV was measured by both methods at baseline and under hyperemic conditions. TTDE data were obtained for 12 patients. CFV and CFVR by TTDE show a good correlation with those obtained by the Doppler guidewire method (average diastolic peak velocity: r = 0.98, y = 0.85x + 5.26; diastolic peak velocity: r = 0.97, y = 0.94x + 3.39; CFVR: r = 0.97, y = 0.87x + 0.56). CFV and CFVR in the posterior descending right coronary artery obtained noninvasively by TTDE accurately reflect these values obtained by the invasive Doppler guidewire method.

Aged↗