Infarct volume 24 h after middle cerebral artery occlusion and reperfusion in each group.

<p>Infarction volume was determined as a percentage of the contralateral hemisphere. Values were expressed as mean ± SEM (n=6-8 for each group). *<i>P</i><0.05 vs. Non-DB-Con or Non-DB-5HD+Sev; † <i>P</i><0.05, Ins-DB-Con or Ins-DB-Sev vs. DB-Con, §<i>P</i><0.05, Ins-DB-Con vs. Ins-DB-Sev.</p

Neurological deficit scores (A) and motor coordination (B) 24 h after middle cerebral artery occlusion and reperfusion in each group.

<p>Values were expressed as mean ± SEM (n=6-8 for each group). *<i>P</i><0.05 vs. Non-DB-Con or Non-DB-5HD+Sev; † <i>P</i><0.05, Ins-DB-Con or Ins-DB-Sev vs. DB-Con, §<i>P</i><0.05, Ins-DB-Con vs. Ins-DB-Sev.</p

Representative Western blots (A) and quantitative comparison of protein levels for Kir6.2 (B) and SUR1 (C) from non-diabetic rats and diabetic rats treated with or without insulin.

<p>Values are expressed as means ± SEM (n=4 for each group). *<i>P</i><0.05 vs. Non-DB; † <i>P</i><0.05 vs. DB.</p

Representative laser confocal images (A) and Quantitative comparison of Kir6.2 (B) and SUR1 (C) fluorescence in the brain cortex from non-diabetic rats and diabetic rats treated with or without insulin.

<p>Scale bar, 200 µm. Values are expressed as means ± SEM (n=3 for each group). *<i>P</i><0.05 vs. Non-DB; † <i>P</i><0.05 vs. DB.</p

Representative RT-PCR product bands (A) and quantitative comparison of mRNA expression for brain mitoK_ATP channel subunits Kir6.2 (B) and SUR1 (C) from non-diabetic rats and diabetic rats treated with or without insulin.

<p>Values are expressed as means ± SEM (n=6 for each group). *<i>P</i><0.05 vs. Non-DB; † <i>P</i><0.05 vs. DB.</p

Expression of AmCDase in the yeast double knockout strain Δypc1Δydc1.

<p>a) Determination of whole-cell lysates (20 μg protein per lane) by Western blot with anti-His antibody at different time points. b) D-erythro-C12-NBD-ceramide content in different groups after the reaction. Values are the means ± SD of three replicates from each independent experiment.</p

Conserved domain of neutral ceramidases.

<p>DNAMAN alignment of the neutral ceramidases from a variety of species showing the highly conserved hexapeptide sequence GDVSPN within the broader conserved amidase domain NXGDVSPNXXGXXC that is crucial for ceramidase activity.</p

Biochemical characterization of the purified recombinant AmCDase.

<p>Hydrolysis of D-erythro-C12-NBD-ceramide was measured by the method described in the “Materials and Methods”. The purified recombinant AmCDase was used for enzyme activity detection, and the reaction continued for 1 h. Values are the means ± SD of three replicates from each independent experiment. (a) Michaelis–Menten represented AmCDase activity by increasing concentrations of D-erythro-C12-NBD-ceramide. (b) Lineweaver-Burk plots for AmCDase. (c) Effects of different cations on AmCDase activity. (d) pH optimum of AmCDase. The buffers were used as described in the experimental procedures.</p

Phylogenetic tree of neutral ceramidases from different species.

<p>The MrBayes analyses were performed with a mixed amino acid model. Species names are (<i>Amorphophallus muelleri; Jatropha curcas; Prunus persica; Sesamum indicum; Arabidopsis thaliana; Anthurium amnicola; Oryza sativa Japonica; Hordeum vulgare; Triticum aestivum; Dicchanthelium oligosanthes; Human; Fukomys damarensis; Microcebus murinus; Castor Canadensis</i>).</p

Primers for amplifying cDNA of AmCDase.

<p>Primers for amplifying cDNA of AmCDase.</p