Kinetic Investigation of Porphyrin Interaction with Chiral Templates Reveals Unexpected Features of the Induction and Self-Propagation Mechanism of Chiral Memory

Kinetic Investigation of Porphyrin Interaction with Chiral Templates Reveals Unexpected Features of the Induction and Self-Propagation Mechanism of Chiral Memor

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 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

Sequence alignment of human kallikreins (panel A) and three-dimensional structure of PSA (panel B).

<p>Sequence alignment (panel A) is built with those human kallikreins for which the three-dimensional structure is available at the Protein Data Bank. The protein sequences were obtained from the NCBI database (<a href="http://www.ncbi.nlm-nih.gov" target="_blank">http://www.ncbi.nlm-nih.gov</a>). The progressive multiple alignment of PSA (also named kallikrein 3; NCBI entry number: CAD30845.1), kallikrein 1 (also named tissue kallikrein; KLK1; NCBI entry number: AAH05313.1), kallikrein 2 (KLK2; NCBI entry number: AAF08276.1), kallikrein 4 (KLK4; NCBI entry number: AAD38019.1), kallikrein 6 (KLK6; NCBI entry number: AAP35498.1), kallikrein 7 (KLK7; NCBI entry number: NP_644806.1), and human plasma kallikrein (HPK; NCBI entry number: AAF79940.1) was performed by the Clustal-Omega program (<a href="http://www.ebi.ac.uk/Tools/msa/clustalo" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalo</a>). Only the trypsin-like serine protease domain of HPK has been aligned. The “*” symbol means that the residues are identical in all the aligned sequences; the ":" symbol indicate conserved substitutions, and the "." symbol means semi-conserved substitutions. The amino acid sequence of bovine chymotrypsinogen (BCTRP; NCBI entry number: 681083A) has been reported as the template. Three-dimensional structure of PSA (panel B). In both panels, the image was produced using UCSF Chimera molecular graphics package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Pettersen1" target="_blank">[26]</a>. The “kallikrein loop” is in yellow <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Menez1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Tang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Fernndez1" target="_blank">[28]</a>, amino acid residues forming the catalytic triad are in red, and amino acid residues affecting the pH dependence of the catalytic parameters are in cyan.</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

Different parameters at various pH values, as obtained from the analysis of steady-state kinetics according to Eq. (1c) and of pre-steady-state kinetics according to Eq. (1d).

<p>Different parameters at various pH values, as obtained from the analysis of steady-state kinetics according to Eq. (1c) and of pre-steady-state kinetics according to Eq. (1d).</p

Somatostatin modulation on IDE level in BV-2 cells.

<p>(A) IDE mRNA increases after 5 hrs of incubation with somatostatin (white columns); 24 hrs after incubation, the level is similar to the control (grey columns). Basal mRNA levels were measured by real time PCR in individual preparations of BV2. Data were first normalized against GAPDH and then expressed setting the value measured in controls at 1. The results presented are the means ± ES (B) Elisa analysis of conditioned medium indicates that IDE level increases in BV-2 cells after 24 hrs of incubation as a function of somatostatin concentration. The results presented are the means ± SEs of five independent experiments in triplicate. P<0.05, one-way ANOVA, followed by Tukey's test, n = 15.</p

<i>p</i>K_a values from the pH-dependence of various kinetic parameters.

<p><i>p</i>K_a values from the pH-dependence of various kinetic parameters.</p

Time course of the PSA-catalyzed hydrolysis of Mu-HSSKLQ-AMC.

<p>Observation wavelength  = 460 nm, pH = 7.5 and temperature  = 37.0°C. The concentration of PSA was 50 nM. The concentration of Mu-HSSKLQ-AMC was 5 µM.</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