Sa Mitjalluna: Minería Prehistórica de cobre en Illa den Colom (Mahón, Menorca)

Se presentan los resultados preliminares de la excavación arqueológica realizada en abril de 2012 en los restos mineros prehistóricos, identificados previamente por prospección superficial, del depósito mineral de Sa Mitjalluna. Este depósito está localizado en la parte centro oriental de Illa den Colom, una pequeña isla situada en el término municipal de Mahón (Menorca, Islas Baleares), frente a la población de Es Grau. La intervención, planteada a través de seis sondeos de diagnóstico, ha permitido la documentación de un frente de trabajo minero que explotaba una veta de cuarzo con mineralización de calcopirita y especies secundarias de cobre (malaquita y azurita fundamentalmente), y de una de las escombreras asociadas a la actividad minera, con una secuencia estratigráfica prehistórica compleja, conteniendo numerosas mazas líticas mineras prehistóricas, fabricadas con cantos rodados de origen erosivo marino local. Estas herramientas, junto con los fragmentos de cerámica recuperados, han permitido datar la explotación minera en torno a mediados del II milenio a.C

Binding energy to COX isoforms.

<p>Binding energy to COX isoforms.</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 Å 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 Å are shown. <b>E</b>. The Fukui function f⁰(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 (120×10⁻⁶ 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 (50×10⁻⁶ M), the chemical blocker of the COX2 active site NS-398 (20×10⁻⁶ M), and the 26S proteasome inhibitor MG132 (20×10⁻⁶ M) plus 2OAA (120×10⁻⁶ M). Constitutive COX1 protein expression is shown as a Western blot loading control.</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 PGH₂ production in the presence of 2OAA (250×10⁻⁶ M) and AA (250×10⁻⁶ 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

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 240×10⁻⁶ 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 (120×10⁻⁶ 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 240×10⁻⁶ 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 240×10⁻⁶ 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 240×10⁻⁶ M) as compared with untreated control cells (100%: ** p<0.01, *** p<0.001; n = 6).</p