Effect of somatostatin on MMP-9 expression.

<p>(A) Zymographyc analysis of cell culture supernatants harvested after 24 hrs of incubation indicate that somatostain administration does not modulate gelatinolytic activity of MMP-9 in each of the experimental conditions. (B) In Western blotting analysis of cell lysates a polyclonal anti-MMP-9 antibody recognizes a single band corresponding to the pro-MMP-9 (92 kDa) which is not modulated over each experimental condition. Analysis of β-tubulin represents the internal control. The results presented are the means ± ES of three indipendent experiments in triplicate, n = 9.</p

Somatostatin induces an increase of IDE expression in microglia cells.

<p>Western blot analysis of normalized lysis samples from rat primary microglia (A) and BV-2 (B) indicates that IDE level increases after 24 hrs of somatostatin incubation, while the internal control ß-tubulin is constant. The additional incubation with sst after 6 hrs from first round strengthens the effect on IDE expression (left panel). Densitometric analysis of IDE WB signals, average ± ES of 5 independent experiments in triplicate (right panel). *P<0.05, one-way ANOVA, followed by Tukey's test, n = 15.</p

Normalized averaged time courses of the NO₂^–-mediated nitrosylation of Ma-Pgb*-Fe(II), in the absence and presence of CO, at pH 7.4 and 20°C, λ = 430 nm.

<p>The NO₂^– concentration was 2.5×10^–3 M (traces a and b) and 8.0×10^–3 M (trace c). The CO concentration was 1.0×10^–4 M (trace a). CO inhibits the NO₂^–-mediated nitrosylation of Ma-Pgb*-Fe(II) (trace a). The time course analysis according to Eqn. 1 allowed the determination of the following parameters: trace b, α₁ = 0.58±0.05, <i>k</i>_obs1 = (2.3±0.2) ×10^–2 s^–1, α₂ = 0.42±0.04, and <i>k</i>_obs2 = (3.2±0.3)×10^–3 s^–1; trace c, α₁ = 0.56±0.05, <i>k</i>_obs1 = (7.9±0.8)×10^–2 s^–1, α₂ = 0.44±0.04, and <i>k</i>_obs2 = (9.0±0.1)×10^–3 s^–1. For details, see text.</p

Difference absorbance spectra of Ma-Pgb*-Fe(II) <i>minus</i> Ma-Pgb*-Fe(II)-NO, at pH 7.4 and 20°C.

<p>The overall difference spectrum, the difference spectrum of the fast phase, and the difference spectrum of the slow phase are represented by diamonds, squares, and circles, respectively. For details, see text.</p

Dependence of <i>h</i> on the Ma-Pgb*-Fe(III) and Ma-Pgb*-Fe(III)-azide concentration (open and filled triangles, respectively) for the peroxynitrite isomerization, at pH 7.4 and 20°C.

<p>The continuous line was calculated according to Eq. 5 with <i>h</i>_app = 3.8×10⁴ M^–1 s⁻¹ and <i>k</i>₀ = 2.8×10^–1 s⁻¹. The average value of <i>h</i>₀ obtained in the presence of Ma-Pgb*-Fe(III)-azide (filled triangles) is 2.7×10^–1 s⁻¹. The peroxynitrite concentration was 2.5×10^–4 M. The azide concentration was 1.0×10^–1 M. Where not shown, the standard deviation is smaller than the symbol. For details, see text.</p

Values of the second-order rate constant for peroxynitrite isomerization by ferric heme-proteins.

a<p>pH 7.4 and 20°C. Present study.</p>b<p>pH 7.0 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi7" target="_blank">[32]</a>.</p>c<p>pH 7.0 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Coppola1" target="_blank">[31]</a>.</p>d<p>pH 7.0 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Herold2" target="_blank">[22]</a>.</p>e<p>pH 7.5 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Herold4" target="_blank">[24]</a>.</p>f<p>pH 7.5 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Herold2" target="_blank">[22]</a>.</p>g<p>pH 7.2 and 22°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi4" target="_blank">[28]</a>.</p>h<p>pH 7.0 and 20°C. Cardiolipin was 1.6×10^–4 M. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi5" target="_blank">[29]</a>.</p

Values of the second-order rate constant for the nitrite-reductase activity of ferrous heme-proteins.

a<p>pH 7.0; unknown temperature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Sturms1" target="_blank">[42]</a>.</p>b<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Tiso2" target="_blank">[45]</a>.</p>c<p>CysE20Ser mutant. pH 7.4 and 20°C. Present study.</p>d<p>pH 7.6 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Helbo1" target="_blank">[49]</a>.</p>e<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Tiso1" target="_blank">[43]</a>.</p>f<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Huang1" target="_blank">[37]</a>.</p>g<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Tiso1" target="_blank">[43]</a>.</p>h<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Petersen1" target="_blank">[40]</a>.</p>i<p>pH 7.0 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Li1" target="_blank">[44]</a>.</p>j<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Tiso1" target="_blank">[43]</a>. In “Human neuroglobin CysCD4-CysD5”, the CysCD4 and CysD5 residues form an intramolecular disulphide bond.</p>k<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Tiso1" target="_blank">[43]</a>. In “Human neuroglobin CysCD4/CysD5”, the CysCD4 and CysD5 residues do not form the intramolecular disulphide bond.</p>l<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Tiso1" target="_blank">[43]</a>.</p>m<p>pH 7.4 and 20°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Ascenzi8" target="_blank">[46]</a>.</p>n<p>pH 7.4 and 25°C. From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Li1" target="_blank">[44]</a>.</p

Somatostatin regulates IDE activity enhancing IDE-dependent Aβ degradation.

<p>(A) BV-2 cells transfected with a specific IDE siRNA pool show reduced levels of IDE protein, compared to cells transfected with a nonspecific control siRNA pool (right panel). (B) Aβ(1–40) quantification through sandwich ELISA reveals that the levels of Aβ are drastically reduced in the presence of IDE steady level (grey columns) compared to IDE-silencing samples (white columns). The results presented are the means ± ES of three independent experiments in triplicate. P<0.05, one-way ANOVA, followed by Tukey's test, n = 9. *Significantly different from internal control. ⁺Significantly different from silenced sample in the absence of somatostatin. ^×Significantly different from not-silenced sample in the absence of somatostatin.</p

Ma-Pgb* fold.

<p>(Panel A) The secondary structure elements are labeled A–H’. The 20 <i>N</i>-terminal residues and the extended CE and FG loops that seal the heme pocket and prevent the access of small ligands to the heme distal cavity are in orange. The pre-A Z-helix is in green. The heme (red) is displayed edge on. The proximal HisF8 residue is shown on the left hand side of the heme. The picture includes the mutated SerE20 residue, located at the <i>C</i>-terminus of the E-helix. The HisF8 and SerE20 side chains and residues building up the heme distal pocket are drawn as skeletal models (C atoms yellow, N atoms blue, and O atoms red) and labeled. (Panel B) Mono views of “tunnel 1” (top) and “tunnel 2” (bottom) access sites in Ma-Pgb*. Helices flanking the tunnel entries are labelled. The heme group (seen through the tunnel apertures) is shown in red. The protein is correctly oriented in both images, to bring each tunnel in the direction of sight. The images are rotated by 90°. The pictures have been drawn by UCSF - Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Pettersen1" target="_blank">[55]</a>. For details, see ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095391#pone.0095391-Nardini1" target="_blank">[11]</a>.</p

Somatostatin analogue octreotide increases IDE expression and secretion.

<p>(A) WB (left panel) and densitometric analysis of IDE (right panel) after 24 hrs incubation with the indicated concentrations of octreotide. (B) ELISA analysis on BV-2 medium after octreotide incubation reveals that the sst analogue induces IDE secretion. In every case, the results presented are the means ± ES of four independent experiments in triplicate. * P<0.05, oneway ANOVA, followed by Tukey's test, n = 12.</p