Engineering of β-carotene biosynthetic pathway.

<p>(A) A schematic representation of the β-carotene biosynthetic pathway in <i>Y</i>. <i>lipolytica</i>. Integrated genes include geranyl diphosphate synthase (<i>crtE</i>), phytoene synthase (<i>crtYB</i>) and carotene desaturase (<i>crtI</i>). (B) Scheme for the construction of the β-carotene producing strain and the efficiency of targeted gene integration. The <i>crtI</i>, <i>crtYB</i> and <i>crtE</i> genes, driven by their individual 4UAS1B promoters, were integrated into the <i>pox1</i>, <i>pox2</i> and <i>pox3</i> sites in the <i>ku70</i>-disrupted <i>Y</i>. <i>lipolytica</i> strain, respectively. Cells treated with HU were transformed with the gene replacement cassette with 50 bp of homology arm to the target gene. The gene targeting rate (%) is shown, and the numbers in parentheses represent the correct integrants/total transformants screened. The experiments were performed in duplicate. HMG-CoA, 3-hydroxy-3-methylgluratyl-coenzyme A; MVA, mevalonic acid; DMPAA, dimethylallyl pyrophosphate; IPP, isopentyl pyrophosphate; PP, pyrophosphate.</p

ESI-mass spectrum and representative expansion of β-carotene produced by the resulting β-carotene producing strain, crtI/YB/E.

<p>(A) ESI-mass spectrum of the wild-type <i>Y</i>. <i>lipolytica</i> strain. (B) ESI-mass spectrum of the resulting β–carotene producing strain (crtI/YB/E). All strains was cultivated for 6 days at 30°C in 50 mL of YPD medium containing 20 g/L glucose. The experiments were performed in duplicate, and the representative results are shown.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Efficiency of gene deletion.

<p>Cells treated or untreated with HU were transformed with a <i>pex10</i>-deletion cassette with 50 bp of homology arm to the <i>pex10</i> gene. The <i>pex10</i> deletion rates (%) are shown and the number of total transformants screened is included in parentheses. WT indicates the wild-type <i>Y</i>. <i>lipolytica</i> Po1f strain. The experiments were performed in duplicate.</p

Efficiency of URA3 marker deletion by HR.

<p>For URA3 selection marker reuse, the strains integrated with deletion or integration cassette containing URA3 marker were grown overnight in the YPD liquid medium and then plated on the 5-FOA selection medium. The percentage of URA3 marker deletion is shown and the number of total colonies screened is included in parentheses. The experiments were performed in duplicate.</p

Time-course effects of 1-methoxyoctadecan-1-ol on calpain activation, STEP cleavage, and p38 MAPK phosphorylation after glutamate exposure.

<p>Cortical neurons were treated with glutamate (300 µM, 15 min) (A and C) and pretreatment with 1-methoxyoctadecan-1-ol (0.1 µg/ml, 24 h) followed by exposure to glutamate (B and D) and both were maintained in the original medium for the specified time. Equal amounts of proteins and each sample were subjected to Western blot assays using the indicated antibodies. Equal protein loading was confirmed by actin expression.</p

Effects of 1-methoxyoctadecan-1-ol on calpain activation, STEP cleavage and p38 MAPK phosphorylation.

<p>The significant calpain1 activation (A), STEP cleavage (B) and p38 MAPK phosphorylation (C) were shown in the ipsilateral (Ipsil) cerebral hemisphere of photothrombotic ischemic mice compared with the contralateral (Con). **<i>P</i><0.01, ***<i>P</i><0.001, <i>vs</i>. vehicle group, Data are expressed as mean±SEM of three separate experiments.</p

Effects of 1-methoxyoctadecan-1-ol on infarct volume, neurological evaluation, and wire-grip test in a photothrombotic ischemic model.

<p>Mice received intraperitoneal administration of DMSO or 1, 3/kg of 1-methoxyoctadecan-1-ol at 30 min before the ischemic insult. Representative photographs of coronal brain sections stained with TTC in vehicle (Veh)- and 1-methoxyoctadecan-1-ol-treated mice (A). White indicates the infarct area. Quantification of the infarct volume, neurological score, and wire-grip test (B). *<i>P</i><0.05, **<i>P</i><0.01 <i>vs</i>. vehicle group. Data are expressed as mean±SEM of three separate experiments.</p

Flow cytometry analysis for neuronal death.

<p>Cortical neurons were pretreated with 1-methoxyoctadecan-1-ol (0.01 and 0.1 µg/ml) for 24 h, followed by exposure to 200 µM glutamate for 6 h. Cells were harvested and stained with Annexin V-FITC/PI, as described under methods and analyzed using flow cytometry. Annexin V⁺PI⁻ cells indicate early apoptotic cells, whereas Annexin V⁺PI⁺ cells are late apoptotic cells. The estimates (%) of gated cells in different compartments are given for each dot blot. Representative flow cytometry analysis scatter-grams of Annexin V/PI staining (A) and quantitative analysis of the histograms (B and C).^#<i>P</i><0.05 and ^##<i>P</i><0.01 <i>vs</i>. control; *<i>P</i><0.05 <i>vs</i>. treatment with glutamate alone. Data are represented as the mean±SEM of three independent experiments.</p

¹H (500 MHz in CDCl₃), ¹³C NMR (125 MHz in CDCl₃) data and key HMBC correlations of 1-methoxyoctadecan-1-ol.

<p>¹H (500 MHz in CDCl₃), ¹³C NMR (125 MHz in CDCl₃) data and key HMBC correlations of 1-methoxyoctadecan-1-ol.</p