15 research outputs found
Table_3_Transmembrane Peptides as Sensors of the Membrane Physical State.DOCX
<p>Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state (MPS) suggested a new approach for the discovery of antimicrobial peptides.</p
Table_2_Transmembrane Peptides as Sensors of the Membrane Physical State.docx
<p>Cell membranes are commonly considered fundamental structures having multiple roles such as confinement, storage of lipids, sustain and control of membrane proteins. In spite of their importance, many aspects remain unclear. The number of lipid types is orders of magnitude larger than the number of amino acids, and this compositional complexity is not clearly embedded in any membrane model. A diffused hypothesis is that the large lipid palette permits to recruit and organize specific proteins controlling the formation of specialized lipid domains and the lateral pressure profile of the bilayer. Unfortunately, a satisfactory knowledge of lipid abundance remains utopian because of the technical difficulties in isolating definite membrane regions. More importantly, a theoretical framework where to fit the lipidomic data is still missing. In this work, we wish to utilize the amino acid sequence and frequency of the membrane proteins as bioinformatics sensors of cell bilayers. The use of an alignment-free method to find a correlation between the sequences of transmembrane portion of membrane proteins with the membrane physical state (MPS) suggested a new approach for the discovery of antimicrobial peptides.</p
On–Off Mechano-responsive Switching of ESIPT Luminescence in Polymorphic <i>N</i>‑Salicylidene-4-amino-2-methylbenzotriazole
We
report the synthesis of a luminescent <i>N</i>-salicylidene
aniline derivative, <i>N</i>-salicylidene-4-amino-2-methylbenzotriazole
(<b>1</b>), and the study of its polymorphism and photophysical
properties. Three phases showing yellow (<b>1-Y</b>), orange
(<b>1-O</b>), and red (<b>1-R</b>) fluorescence have been
isolated and characterized by thermal and single crystal X-ray analysis.
The photoluminescence results from excited-state intramolecular proton
transfer process and the quantum yield is strongly dependent on polymorphism
(Φ<sub>1‑Y</sub> = 0.87, Φ<sub>1‑O</sub> = 0.11, Φ<sub>1‑R</sub> = 0.028). The poorly emitting <b>1-R</b> can be easily prepared, converted to the bright <b>1-Y</b> by grinding, and reverted to <b>1-R</b> through
melting and annealing, giving rise to a luminescence on–off
mechano-responsive cycle. The different photophysical properties are
explained with the variable π-overlap and molecular conformation
changes in the three polymorphs, characterized by a very similar crystal
packing. By DFT calculations, the absorption properties were explained
as dependent on the torsion angle between the two planar portions
of the molecule, which affects the equilibrium between enol and keto
forms in the ground state
2OAA attenuates the increase in iNOS protein levels and NO production induced by LPS in BV2 murine microglial cells.
<p><b>A</b>. Bar diagram showing the dose-dependent effect of 2OAA (50, 120 and 240×10<sup>−6</sup> M; 24 h) on NO production (measured by the Griess assay: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072052#s2" target="_blank">Materials and Methods</a>) by BV2 mouse microglial cells previously challenged with LPS (1 µg/ml; 24 h). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072052#s3" target="_blank">Results</a> are expressed relative to the NO production in untreated cells in the presence of LPS (100%: ** <i>p</i><0.005, *** <i>p</i><0.001; n = 6). <b>B</b>. A representative immunoblot and bar diagram showing the effect of 2OAA (120×10<sup>−6</sup> M; 24 h) on iNOS expression in BV2 mouse microglial cells previously challenged with LPS (1 µg/ml; 24 h). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072052#s3" target="_blank">Results</a> are expressed relative to the NO produced by untreated cells maintained in the presence of LPS (100%: *** <i>p</i><0.001; n = 6).</p
2OAA attenuates the LPS-induced increase in serum TNF-α levels in C57BL6/J mice.
<p><b>A</b>. Bar diagram showing the dose-dependent inhibition of serum TNF-α by 2OAA (50, 200 and 500 mg/kg) in LPS-challenged C57BL6/J mice (100%: *** <i>p</i><0.001 with respect to the LPS-challenged controls). <b>B</b>. Bar diagram showing the inhibitory effects of cortisone (12.5 mg/kg; Co), ibuprofen (7.5 mg/kg; Ib) and 2OAA (500 mg/kg) on serum TNF-α in LPS-challenged C57BL6/J mice (*** <i>p</i><0.001 with respect to controls; †<i>p</i><0.05 with respect to Ib).</p
BV2 cell viability is inhibited by AA but not by 2OAA.
<p>BV2 mouse cells were exposed to 120 and 240×10<sup>−6</sup> M of AA or 2OAA for 24 h. Upper panel. Bar diagram showing the cell viability assayed with <b>A</b>. MTT or <b>B</b> Trypan Blue by AA (120 and 240×10<sup>−6</sup> M) as compared with untreated control cells (100%; n = 6). Lower panel. Bar diagram showing no inhibitory effect on cell viability assayed by <b>C</b> MTT or <b>D</b> Typan Blue by 2OAA (120 and 240×10<sup>−6</sup> M) as compared with untreated control cells (100%: ** p<0.01, *** p<0.001; n = 6).</p
2OAA inhibits COX1 and COX2 activity.
<p>Bar diagram showing the inhibition of <b>A</b>. COX1 or <b>B</b>. COX2 activity determined by measuring PGH<sub>2</sub> production in the presence of 2OAA (250×10<sup>−6</sup> M) and AA (250×10<sup>−6</sup> M), and compared with untreated control cells (100%: ** <i>p</i><0.01, *** <i>p</i><0.001 with respect to controls; †<i>p</i><0.05, †††<i>p</i><0.001 with respect to AA; n = 6).</p
Computational simulations based on molecular docking.
<p><b>A</b>. AA in the COX1 binding site. <b>B</b>. S2OAA in the COX1 binding site. The two carboxyl oxygens (O1 and O2) of AA establish hydrogen bonds with Arg 120 and they have close hydrophobic contacts with Phe 205, Val 344 and Tyr 348. The orientation of S2OAA is very similar to that of AA, with O1 and O2 occupying the same positions in both. The hydroxyl oxygen (O*) of S2OAA is hydrogen bound to Glu 524, although this favorable interaction is counterbalanced by a distortion of the carbon backbone. To facilitate visual inspection of the interactions, the binding site is shaded in grey, the fatty acids are represented by sticks and balls, and only amino acids closer than 3 Ã… are shown. <b>C</b>. AA in the COX2 binding site. <b>D</b>. S2OAA in the COX2 binding site. The carboxylate group of AA is coordinated with Arg 2120 by one hydrogen bond, whereas R2OAA possesses five hydrogen bonds. The O* oxygen occupies the position of O1 of AA and in an analogous manner, O1 of S2OAA occupies the position of the AA O2. Finally, O2 of substituted arachidonic acid is free to hydrogen bond to Tyr 2355. The binding site is shaded in grey, fatty acids are represented by sticks and balls, and only amino acids closer than 3 Ã… are shown. <b>E</b>. The Fukui function f<sup>0</sup>(r) is color-mapped onto the electron density isosurface with equal isovalues.</p
Proteolysis of COX2 by 2OAA.
<p><b>A</b>. Bar diagram showing the upregulation of COX2 mRNA in differentiated U937 cells challenged with LPS (62 ng/ml). 2OAA (120×10<sup>−6</sup> M; 6 h) failed to downregulate COX2 mRNA levels despite its inhibitory effect on COX2 protein. <b>B</b>. Representative immunoblot showing COX2 protein in the presence or absence of the general protein synthesis inhibitor CHX (50×10<sup>−6</sup> M), the chemical blocker of the COX2 active site NS-398 (20×10<sup>−6</sup> M), and the 26S proteasome inhibitor MG132 (20×10<sup>−6</sup> M) plus 2OAA (120×10<sup>−6</sup> M). Constitutive COX1 protein expression is shown as a Western blot loading control.</p