Liver-spleen contrast standardization of gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid-enhanced magnetic resonance imaging based on cross-calibration.

Background: Liver-spleen contrast in the hepatobiliary phase highly depends on the devices used for liver function tests. This study aimed to develop and validate a method to convert liver-spleen contrast data acquired with another device to reference liver-spleen contrast data, using the regression line of phantom contrasts (i.e., cross-calibration). Methods: As cohort studies, two-dimensional gradient echo images of T1-weighted fat-suppression in the hepatobiliary phase were retrospectively obtained and analyzed for a total of 126 patients who underwent gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid-enhanced magnetic resonance imaging using four different magnetic resonance imaging scanner-coil combinations. The liver-spleen contrast measured from these images was converted into reference liver-spleen contrast using cross-calibration with purified water and gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid phantoms of 0.06, 0.14, 0.27, 0.63, 1.37, and 2.82 mM/L. At this point, the error of the regression lines with phantom contrasts was assessed and corrected. Lastly, the liver-spleen and converted liver-spleen contrasts, which are the values before and after cross-calibration respectively, were compared with reference liver-spleen contrast in three cases using different coils and magnetic resonance imaging scanners from a reference device. Results: Regarding the regression lines with phantom contrasts, the coefficient of determination was 0.99. Although regression lines with phantom contrasts tended to be 0.0105 lower in logarithmic contrasts than those with liver-spleen contrast, no significant difference was observed between the two lines (P=0.0612) by analysis of covariance. In the case of different coils, there was a significant difference between liver-spleen and reference liver-spleen contrasts (P<0.00001), but there was no significant difference between converted liver-spleen and reference liver-spleen contrasts (P=0.492). Moreover, the regression equation between converted liver-spleen and reference liver-spleen contrasts corresponded with an identity line. Likewise, in the two cases of different magnetic resonance imaging scanners, there was a significant difference between liver-spleen and reference liver-spleen contrast (both P<0.00001), but there was no significant difference between converted liver-spleen and reference liver-spleen contrast (P=0.923 and P=0.541). Conclusions: Cross-calibration using the precision and valid regression lines with phantom contrasts had high accuracy and utility.

[Influence of Different Contrast Agent Concentrations and Injection Protocols on Dynamic Contrast Enhanced MRI of Prostate: Equimolar Comparison of 1.0M Gadobutrol and 0.5M Gadolinium Chelates].

The purpose of this study was to evaluate the enhancement profile of 1.0M gadobutrol (high concentration: HC) in comparison to 0.5M gadopentetate dimeglumine and gadoterate meglumine (low concentration: LC) in dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of prostate. In total, 48 patients who were diagnosed with prostate cancer by radiologist were included. Each patient was examined after intravenous injection of 0.1 mmol/kg body weight contrast agent with flow rate of 1.5 (HC) or 3.0 mL/s (LC). Circular regions of interest were placed at prostate cancer (PCa) and normal peripheral zone (normal PZ) in DCE-MRI. The enhancement curves were calculated as a relative enhancement. Statistical analysis was performed by Mann-Whitney U test (p<0.05 were considered significant). As a result, the enhancement at first phase of HC was significantly lower compared with LC in PCa (HC, 0.47; LC, 0.85; p=0.029), and in normal PZ (HC, 0.12; LC, 0.22; p=0.033). The enhancement of HC in PCa was significantly higher compared with LC at late phase. Although not significant, a similar tendency was observed in normal PZ. The present study suggested that the enhancement profile with HC was higher at late phase but the rise of the enhancement curve with HC tended to be delayed.

[Examination of the means of measuring liver function in the hepatobiliary phase].

In a field of contrast-enhanced magnetic resonance imaging of the liver, attention has been focused on evaluation of liver function using gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid(EOB). In this study, we examined the possibility of obtaining liver function in only one hepatobiliary phase 60 minutes after injection. First, in regard to the difference between the signal intensity of two materials, we examined the effects of slice gap, surface coil intensity correction(SCIC), and others. Secondly, we compared the difference between liver and spleen signal intensity with biochemical laboratory tests, Child-Pugh class, liver damage class, and the two indices(HH(15) and LHL(15))calculated by 99mTc-DTPA-galactosyl-human serum albumin hepatic scintigraphy in patients with chronic liver diseases. Finally, we designated the "Liver EOB uptake index(L-EOB(60))" from those results, compared with HH(15) and LHL(15). The results demonstrated that the difference between the signal intensity of two materials increased in the lack of slice gap explained by cross talk, and decreased with SCIC. The difference between liver and spleen signal intensity decreased with worsened liver and kidney function. In the case of slice gap >20% and direct bilirubin <0.5 mg/dL without SCIC, the correlation coefficient between L-EOB(60) and LHL(15) was 0.97. L-EOB(60) was strongly proportional to LHL(15). We conclude that L-EOB(60) meeting the above conditions can be employed as a useful index to determine liver function.

Crystal structure of A3B3 complex of V-ATPase from Thermus thermophilus.

Vacuolar-type ATPases (V-ATPases) exist in various cellular membranes of many organisms to regulate physiological processes by controlling the acidic environment. Here, we have determined the crystal structure of the A(3)B(3) subcomplex of V-ATPase at 2.8 A resolution. The overall construction of the A(3)B(3) subcomplex is significantly different from that of the alpha(3)beta(3) sub-domain in F(o)F(1)-ATP synthase, because of the presence of a protruding 'bulge' domain feature in the catalytic A subunits. The A(3)B(3) subcomplex structure provides the first molecular insight at the catalytic and non-catalytic interfaces, which was not possible in the structures of the separate subunits alone. Specifically, in the non-catalytic interface, the B subunit seems to be incapable of binding ATP, which is a marked difference from the situation indicated by the structure of the F(o)F(1)-ATP synthase. In the catalytic interface, our mutational analysis, on the basis of the A(3)B(3) structure, has highlighted the presence of a cluster composed of key hydrophobic residues, which are essential for ATP hydrolysis by V-ATPases.

[Optimal scan timing for Gd-EOB-DTPA enhanced liver dynamic MR imaging].

Gadoxetate Sodium (Gd-EOB-DTPA, EOB) is a new contrast agent for magnetic resonance (MR) imaging that allows both vascular and hepatobiliary imaging in one examination. Often in the arterial phase, however, appropriate scan timing is missed and contrast enhancement is not enough. In addition, to shorten the complete examination, some studies have been conducted to examine scan timing at the hepatobiliary phase earlier than 20 min after injection. We studied the optimal scan timing both at the arterial and the hepatobiliary phase. It was appropriate that multiphase acquisition of MR imaging at the arterial phase should be aimed around 25 sec after injection. Moreover, the liver-spleen contrast ratio (C(L-S)) at the hepatobiliary phase was highest at 60 min after injection, and the acquisition of an image earlier than 20 minutes lowered the C(L-S). In the future, it is desirable to establish how to use Gd-EOB-DTPA (EOB) for hepatic MR imaging after taking the extent of liver damage into consideration.

Molecular mechanism of energy conservation in polysulfide respiration.

Bacterial polysulfide reductase (PsrABC) is an integral membrane protein complex responsible for quinone-coupled reduction of polysulfide, a process important in extreme environments such as deep-sea vents and hot springs. We determined the structure of polysulfide reductase from Thermus thermophilus at 2.4-A resolution, revealing how the PsrA subunit recognizes and reduces its unique polyanionic substrate. The integral membrane subunit PsrC was characterized using the natural substrate menaquinone-7 and inhibitors, providing a comprehensive representation of a quinone binding site and revealing the presence of a water-filled cavity connecting the quinone binding site on the periplasmic side to the cytoplasm. These results suggest that polysulfide reductase could be a key energy-conserving enzyme of the T. thermophilus respiratory chain, using polysulfide as the terminal electron acceptor and pumping protons across the membrane via a previously unknown mechanism.

Glutamates 99 and 107 in transmembrane helix III of subunit I of cytochrome bd are critical for binding of the heme b595-d binuclear center and enzyme activity.

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli.

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the DeltayhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in gamma-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.

Probing the ubiquinol-binding site in cytochrome bd by site-directed mutagenesis.

To probe the structure of the quinol oxidation site in loop VI/VII of the Escherichia coli cytochrome bd, we substituted three conserved residues (Gln249, Lys252, and Glu257) in the N-terminal region and three glutamates (Glu278, Glu279, and Glu280) in the first internal repeat. We found that substitutions of Glu257 by Ala or Gln, and Glu279 and Glu280 by Gln, severely reduced the oxidase activity and the expression level of cytochrome bd. In contrast, Lys252 mutations reduced only the oxidase activity. Blue shifts in the 440 and 630 nm peaks of the reduced Lys252 mutants and in the 561 nm peak of the reduced Glu257 mutants indicate the proximity of Lys252 to the heme b(595)-d binuclear center and Glu257 to heme b(558), respectively. Perturbations of reduced heme b(558) upon binding of aurachin D support structural changes in the quinol-binding site of the mutants. Substitutions of Lys252 and Glu257 caused large changes in kinetic parameters for the ubiquinol-1 oxidation. These results indicate that Lys252 and Glu257 in the N-terminal region of the Q-loop are involved in the quinol oxidation by bd-type terminal oxidase.

O2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form.

FixL, a rhizobial heme-based O2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low O2 tension, while the kinase activity is inhibited in the case of the O2-bound form. The present study unambiguously shows that the binding of CO and NO does not significantly inhibit the kinase activity of dithiothreitol (DTT)-reduced ferrous FixL from Sinorhizobium meliloti, which is inconsistent with the spin state mechanism previously reported. Kinase inactivation is caused by aberrant disulfide (S-S) bond formation at Cys301 in the ferric homodimer, which explains these contradictory observations. The addition of DTT cleaved the S-S bond, leading to restoration of kinase activity in the ferric form as well as heme reduction, but, sodium hydrosulfite treatment produced the kinase-inactive deoxy form without S-S cleavage. On the basis of these experimental results, it can be concluded that ferrous FixL discriminates O2 from CO and NO, and signals the O2-bound state by downregulating the phosphoryl transfer reaction.