A Dye-Decolorizing Peroxidase from <i>Vibrio cholerae</i>

The dye-decolorizing peroxidase (DyP) protein from <i>Vibrio cholerae</i> (<i>Vc</i>DyP) was expressed in <i>Escherichia coli</i>, and its DyP activity was assayed by monitoring degradation of a typical anthraquinone dye, reactive blue 19 (RB19). Its kinetic activity was obtained by fitting the data to the Michaelis–Menten equation, giving <i>k</i>_cat and <i>K</i>_m values of 1.3 ± 0.3 s^–1 and 50 ± 20 μM, respectively, which are comparable to those of other DyP enzymes. The enzymatic activity of <i>Vc</i>DyP was highest at pH 4. A mutational study showed that two distal residues, Asp144 and Arg230, which are conserved in a DyP family, are essential for the DyP reaction. The crystal structure and resonance Raman spectra of <i>Vc</i>DyP indicate the transfer of a radical from heme to the protein surface, which was supported by the formation of the intermolecular covalent bond in the reaction with H₂O₂. To identify the radical site, each of nine tyrosine or two tryptophan residues was substituted. It was clarified that Tyr129 and Tyr235 are in the active site of the dye degradation reaction at lower pH, while Tyr109 and Tyr133 are the sites of an intermolecular covalent bond at higher pH. <i>Vc</i>DyP degrades RB19 at lower pH, while it loses activity under neutral or alkaline conditions because of a change in the radical transfer pathway. This finding suggests the presence of a pH-dependent switch of the radical transfer pathway, probably including His178. Although the physiological function of the DyP reaction is unclear, our findings suggest that <i>Vc</i>DyP enhances the DyP activity to survive only when it is placed under a severe condition such as being in gastric acid

Stepwise differentiation between pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors using D, f, and D* or P values that were determined by geometric (Geo) or least-squares (Fit) method.

<p>Plot graphs show 2D distributions of Geo P and GeoD (<b>a</b>), Geo f and Geo D (<b>b</b>), Fit D* and Fit D (<b>C</b>), or Fit f and Fit D (<b>d</b>). Open triangles, open squares, and closed circles indicate pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors, respectively. In combinations of Geo D and Geo P (a), Geo D and Geo f, or Fit D and Fit D*, stepwise approach diagnosed 21 of 23 salivary gland tumors correctly; in these approaches, the same Warthin tumor was incorrectly diagnosed as a malignant salivary gland tumor owing to having a large Geo D ( = 1.11×10⁻³ mm²/s) or Fit D values ( = 1.11×10⁻³ mm²/s); or incorrectly diagnosed as a pleomorphic adenoma owing to having a large Geo D ( = 1.24×10⁻³ mm²/s) or Fit D ( = 1.23×10⁻³ mm²/s) and small Geo P ( = 0.36×10⁻³ mm²/s) or Fit D* ( = 7.90×10⁻³ mm²/s) values. The diagnostic accuracy with stepwise approach using Fit D and Fit f was lower than that using the corresponding geometric parameters (<b>b, d</b>). Diagnostic accuracy was provided for the respective classifications at the bottom of each diagram.</p

Stepwise differentiation between pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors using D, f, and D* or P values that were determined by geometric (Geo) method using 4 b-values (0, 100, 400, and 800 s/mm²).

<p>Plot graphs show 2D distributions of Geo P and GeoD (<b>a</b>), or Geo f and Geo D (<b>b</b>). Open triangles, open squares, and closed circles indicate pleomorphic adenomas, Warthin tumors, and malignant salivary gland tumors, respectively. Diagnostic accuracy was provided for the respective classifications at the bottom of each diagram.</p

Samtykke som opphevelsesgrunn av advokaters taushetsplikt

Oppgaven tar for seg problemstillinger som kan oppstå i situasjoner der det blir aktuelt å samtykke til opphevelse av advokaters taushetsplikt. Oppgaven behandler hensynene bak taushetsplikten og avveier disse mot hensyn til klienten, advokaten og samfunnet

IVIM parameter determination by least-squares or geometric method.

<p><b>a</b>, Least-squares method. Upper panel shows a representative signal decay curve obtained by using 11 b-values (0, 10, 20, 30, 50, 80, 100, 200, 300, 400, 800 s/mm²). At first step, D (Fit D) can be obtained by least-squares method using ln S₂₀₀, ln S₃₀₀, ln S₄₀₀, and ln S₈₀₀, and initial f value is calculated as , where S_inter is the interception of the logarithmic regression line obtained by using b-values of 200, 300, 400, and 800 s/mm² with the y-axis. Right panel shows relationship between S_b/S₀ and varying b-values. Given D and initial f and D* values, f (Fit f) and D* (Fit D*) values can be obtained by least-squares method based on the equation: . <b>b</b>, Geometric method. Graph shows geometric determination of IVIM parameters using 3 (0, 200, and 800 s/mm²) of the 11 b-values. D is calculated by the equation . f is estimated by the equation , and P is estimated by the equation Geo P = (ln S₀–In S_inter)/200. <b>c</b>, Geometric method based on 4-b-value data. Graph shows geometric determination of IVIM parameters using 4 (0, 100, 400, and 800 s/mm²) of the 11 b-values. D is calculated by the equation , f is estimated by the equation , and P is estimated by the equation .</p

IVIM parameters of 23 salivary gland tumors that were determined by least squares, 3b-geometrical, or 4b-geometrical methods.

<p>IVIM, intravoxel incoherent motion; Fit, least squares method; 3b-Geo, geometric method using 3 b-values; 4b-Geo, geometric method using 4 b-values. P/D*, Geo P/Fit D*.</p>a, b<p>significant differences (p<0.05) (Steel-Dwass test).</p><p>IVIM parameters of 23 salivary gland tumors that were determined by least squares, 3b-geometrical, or 4b-geometrical methods.</p

IVIM parameters of normal structures and tumors in the head and neck region.

<p>Plot graphs show D (Geo D), f (Geo f), and P (Geo P) values that were determined by geometric method; and D (Fit D), f (Fit f), and D* (Fit D*) values that were determined by least-squares method of normal structures (parotid glands, open circles; and masseter muscles, open squares) and head and neck tumors (closed circles). Broken white contours indicate tumor areas. Parotid gland: Geo D, Geo f and Geo P = 0.76±0.17×10⁻³ mm²/s, 0.20±0.04, and 1.12±0.27×10⁻³ mm²/s, respectively; and Fit D, Fit f, and Fit D* = 0.75±0.16×10⁻³ mm²/s, 0.20±0.05, and 62.96±46.78×10⁻³ mm²/s, respectively. Masseter muscle: Geo D, Geo f, and Geo P = 0.99±0.51×10⁻³ mm²/s, 0.24±0.10, and 1.41±0.71×10⁻³ mm²/s, respectively; and Fit D, Fit f, and Fit D* = 0.96±0.51×10⁻³ mm²/s, 0.25±0.10, and 40.50±30.13×10⁻³ mm²/s, respectively. Tumors: Geo D, Geo f, and Geo P = 1.00±0.38×10⁻³ mm²/s, 0.11±0.08, and 0.61±0.48×10⁻³ mm²/s, respectively; Fit D, Fit f, and Fit D* = 0.99±0.37×10⁻³ mm²/s, 0.12±0.08, and 24.14±21.15×10⁻³ mm²/s, respectively. Insert, Geo P distribution on a small scale. The values are the results of integrated signal intensities within the ROIs. *, p<0.001 (Wilcoxon signed-rank test).</p

105 head and neck tumors.

<p>SCC, squamous cell carcinoma.</p><p>Of 105 tumors, 18 were excluded from the study owing to measurement errors, including 6 pleomorphic adenomas, 2 lymphomas, 2 SCCs (oropharynx and hypopharynx), 1 SCC node, 1 adenocarcinoma, 1 metastatic node from papillary thyroid carcinoma, 1 neuroendocrine carcinoma, 1 ameloblastic carcinoma, 1 hemangioma, 1 keratocystic odontogenic tumor, 1 myxoma.</p><p>105 head and neck tumors.</p

IVIM maps of SCC and lymphoma.

<p><b>a–d</b>, Axial fat-suppressed T2-weighted MR image (<b>a</b>), and Geo D (<b>b</b>), Geo f (<b>c</b>), and Geo P (<b>d</b>) maps of 72-year-old man with SCC in oropharynx show tumor with homogeneous T2-signals and IVIM parameter values of Geo D, Geo f, and Geo P = 1.16×10⁻³ mm²/s, 0.14, and 0.76×10⁻³ mm²/s, respectively; and Fit D, Fit f, and Fit D* = 1.14×10⁻³ mm²/s, 0.18, and 8.50×10⁻³ mm²/s, respectively. <b>e–h</b>, Axial fat-suppressed T2-weighted MR image (e), and Geo D (f), Geo f (g), and Geo P (h) maps of 79-year-old man with lymphoma in nasopharynx show tumor with homogeneous T2 signals and IVIM parameter values of Geo D, Geo f, and Geo P = 0.59×10⁻³ mm²/s, 0.08, and 0.41×10⁻³ mm²/s, respectively; and Fit D, Fit f, and Fit D* = 0.60×10⁻³ mm²/s, 0.07, and 17.01×10⁻³ mm²/s, respectively. The values are the results of integrated signal intensities within the ROIs.</p

IVIM parameters of SCCs and lymphomas.

<p>IVIM, intravoxel incoherent motion; SCC, squamous cell carcinoma; Geo, IVIM parameters determined by geometric method; Fit, IVIM parameters determined by least squares method. P/D*, Geo P/Fit D*.</p>a, b<p>significant differences (p = 0.0002, Mann-Whitney U test).</p><p>IVIM parameters of SCCs and lymphomas.</p