Diseño del canal de venta a través de Internet para la tienda de calzado MD

Muchos estudios recientes indican un incremento sustancial en el comercio electrónico en América Latina y el Caribe, y muy especialmente en el espacio B2C (Business-to-Customers, relaciones entre negocios y clientes). En el año 2008, las transacciones representaron cerca de USD$16.025 millones, y su crecimiento anual fue casi del 35%. En la región centroamericana, no existen tiendas en la Web que se especialicen en calzado para dama, por lo que una incursión a corto plazo permitiría obtener el liderazgo en el mercado. El aumento en la penetración de tarjetas de crédito internacionales en el sector femenino en la región, crea un mercado potencial de más de dos millones de personas. De estas, cerca del 60% estaría dispuesta a comprar zapatos a través de Internet. MD es la actual marca de tiendas orientadas a calzado de moda para mujeres entre 18 y 40 años, propiedad de Distribuciones Diversas, S.A. de C.V. El fundador, actual socio mayoritario y presidente es el Ingeniero Carlos Cabrera. Su hija, Amanda Cabrera, diseñó la imagen y estrategia actual de MD que la ha dado una muy buena posición en el mercado de calzado femenino, especialmente en países como Guatemala y El Salvador. MD.biz será una división de MD, que comercializará calzado a través de Internet, y estará dirigida hacia el mismo mercado objetivo. Operará a nivel de la región centroamericana, y estará establecida en las oficinas centrales de Distribuciones Diversas, S.A. de C.V., en El Salvador. Su misión será la de comercializar calzado y accesorios relacionados en Internet con un alto componente de moda, que cumplan con estándares de calidad, comodidad y diseño, para satisfacer las necesidades del mercado regional y ser reconocida como una empresa comprometida con sus clientes, empleados y accionistas, en los países que opera. MD.biz enfocará sus productos y estrategias de marketing a mujeres entre 23 y 40 años de edad, que tengan acceso a tarjetas de crédito, familiarizadas con el uso de Internet para hacer compras en línea y que residan en cualquier ciudad donde llegue el servicio de courier oficial en países de América Central (excepto Belice y Panamá)

Co-Evolution of Mitochondrial tRNA Import and Codon Usage Determines Translational Efficiency in the Green Alga Chlamydomonas

Mitochondria from diverse phyla, including protozoa, fungi, higher plants, and humans, import tRNAs from the cytosol in order to ensure proper mitochondrial translation. Despite the broad occurrence of this process, our understanding of tRNA import mechanisms is fragmentary, and crucial questions about their regulation remain unanswered. In the unicellular green alga Chlamydomonas, a precise correlation was found between the mitochondrial codon usage and the nature and amount of imported tRNAs. This led to the hypothesis that tRNA import might be a dynamic process able to adapt to the mitochondrial genome content. By manipulating the Chlamydomonas mitochondrial genome, we introduced point mutations in order to modify its codon usage. We find that the codon usage modification results in reduced levels of mitochondrial translation as well as in subsequent decreased levels and activities of respiratory complexes. These effects are linked to the consequential limitations of the pool of tRNAs in mitochondria. This indicates that tRNA mitochondrial import cannot be rapidly regulated in response to a novel genetic context and thus does not appear to be a dynamic process. It rather suggests that the steady-state levels of imported tRNAs in mitochondria result from a co-evolutive adaptation between the tRNA import mechanism and the requirements of the mitochondrial translation machinery

Correction: Co-Evolution of Mitochondrial tRNA Import and Codon Usage Determines Translational Efficiency in the Green Alga Chlamydomonas.

[This corrects the article DOI: 10.1371/journal.pgen.1002946.]

Respiratory enzyme activities of T11-10, T11-10/2, and T22-11 transformants.

<p>Respiratory activities were measured on membrane fractions of T11-10, T11-10/2 and T22-11 mutants. NADH:Duroquinone corresponds to the rotenone-sensitive NADH:duroquinone oxidoreductase activity (nmol of NADH oxidized min⁻¹ mg of proteins⁻¹); NADH:Ferricyanide corresponds to the NADH:Fe(CN)₆³⁻ oxidoreductase activity (nmol of K3Fe(CN)₆³⁻ reduced min⁻¹ mg of proteins⁻¹); CII+III corresponds to the succinate:cytochrome <i>c</i> oxidoreductase activity (nmol cytochrome <i>c</i> reduced min⁻¹ mg of proteins⁻¹); CIV corresponds to the cytochrome <i>c</i> oxidase activity (nmol of cytochrome <i>c</i> oxidized min⁻¹ mg of proteins⁻¹). Asterisks indicate statistically significantly differences using Student <i>t</i> test with a significance threshold of 0.05. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002946#s2" target="_blank">Results</a> are means of 3 to 6 independent experiments.</p

Confocal microscopy of mitochondria from T22-WT, T22-11, and <i>dum22</i> stained with MitoTracker dyes.

<p>Visualization in T22-WT, T22-11 and <i>dum22</i> strains (A) of mitochondria by MitoTracker dye, (B) of chloroplast by chlorophyll autofluorescence and (C) of the overlapping images in <i>Chlamydomonas</i> cells. The white line corresponds to 5 µm.</p

Mitochondrial <i>in organello</i> protein synthesis of T22-11 transformant.

<p>(A) Mitochondrial proteins of T22-WT and T22-11 (25 µg) were loaded on SDS-PAGE after mitochondrial <i>in organello</i> protein synthesis and stained with Coomassie Blue. (B) Mitochondrial protein samples (10 µg) coming from the same experiment as described in (A) were separated by SDS-PAGE, blotted and probed with antisera against the VdacI <i>Chlamydomonas</i> protein. (C) Mitochondrial translated proteins from experiment (A) were visualized by autoradiography. Expected migration of the eight mitochondrial proteins are indicated. Major bands obtained in the <i>in organello</i> protein synthesis are indicated from b1 to b9. (D) Major bands (b1 to b9) were quantified. The histogram corresponds to the percentage of variation in the T22-11 transformant as compared to the T22-WT strain. The experiment was repeated two times and showed the same decrease of the annotated bands.</p

Total respiration and doubling time in T11-10, T11-10/2, and T22-11 transformants.

<p>Dark whole-cell respiratory rates are expressed in nmol of O₂ min⁻¹ 10⁻⁷ cells ± SD (mean of 3 independent experiments). Doubling times were measured in heterotrophic conditions (D) and mixotrophic conditions (L) and are expressed in hours ± SD (mean of 3 independent experiments). Asterisks indicate statistically significantly differences using Student <i>t</i> test with a significance threshold of 0.05.</p

Analysis of the import status of mitochondrial tRNAs in T22-11 transformant.

<p>(A) Northern blot analysis of mitochondrial tRNAs extracted from the T22-WT strain and T22-11 transformant. Hybridizations were performed with radiolabeled oligonucleotides specific for cytosolic tRNA^Gly(GCC) (G1), tRNA^Gly(UCC) (G2), tRNA^Gly(CCC) (G3), tRNA^Val(AAC) (V) and tRNA^Leu(AAG) (L); for mitochondrial tRNA^Met (M mt), tRNA^Gln (Q mt) and for the mitochondrial L3a rRNA (L3a mt). (B) Signals were quantified and normalized with the L3a mt signal. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002946#s2" target="_blank">Results</a> are the means of 3 to 5 independent experiments and correspond to the percentage of variation of tRNA steady-state levels in the T22-11 transformant as compared to the T22-WT strain. Asterisks indicate statistically significant differences using Student <i>t</i> test with a significance threshold of 0.05.</p

Molecular characterization of the transformants.

<p>(A) Schematic map of the <i>Chlamydomonas reinhardtii</i> mitochondrial genome. Boxes represent protein-coding genes: (<i>cob</i>) apocytochrome <i>b</i> of complex III; (<i>nd1</i>, <i>2</i>, <i>4</i>, <i>5</i> and <i>6</i>) subunits of complex I; (<i>cox1</i>) subunit 1 of complex IV; (<i>rtl</i>) reverse transcriptase-like protein. W, Q, and M represent tRNAs for Trp, Glu, and Met, respectively. The bidirectional origin of transcription between <i>nd5</i> and <i>cox1</i> genes is represented by a dashed vertical line and two horizontal arrows. Terminal inverted repeats are shown by short arrows and <i>Sac</i>I digestion site at position 5.5 kb (GenBank u03843 numbering) is indicated. Region where modifications on the <i>nd4</i> gene were found on T11-10, T11-10/2 and T22-11 transformants is indicated in grey. Position and name of primers are indicated above the map. Primers with a star are specific for the modified gene version (for primer sequence see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002946#pgen.1002946.s004" target="_blank">Table S1</a>). Positions of the <i>dum11</i> and <i>dum22</i> deletions are shown. Mitochondrial DNA fragments contained in pND4-LP, pCucob and pCumut are schematized. Grey boxes represent the modified genes where GGC/GGT codons were changed in GGG codons. (B) Detection of the <i>cob</i> gene in transformants obtained after biolistic transformation with pND4-LP (T-ND4-LP) and pCucob (T-cucob) constructs. PCR analyses were performed with cobF/cobR (1) and telF/cobR (2) pair primers. (C) Detection of the mutated and the wild-type <i>nd4</i> genes on T11-10, T11-10/2 and T22-11 transformants. PCR analyses were performed with 4F2*/4R2 and 4F2/4R2 pair primers for the modified <i>nd4</i> gene (<i>nd4*</i>) and for wild-type <i>nd4</i> gene (<i>nd4</i>) respectively. (D–E) Reconstitution of complete mitochondrial genome in T11-10, T11-10/2 and T22-11 transformants. Southern blot analyses were performed (D) on total DNA with the <i>nd6</i> PCR probe and (E) on <i>Sac</i>I digested DNA with <i>nd4</i> and <i>nd6</i> PCR probes. (F) Transcript levels of <i>nd4</i> and <i>nd6</i> genes in T11-10, T11-10/2 and T22-11 transformants. Northern blot analyses were performed on total RNA with <i>nd4</i> and <i>nd6</i> PCR probes. Loadings of rRNAs are shown.</p