Examination of hypoxic ischemia injury in predevelopmental mouse brain.

<p><i>A</i>: Cerebral blood flow immediately after carotid artery ligation in a P2–P3 mouse. <i>B</i>: Experimental paradigm. Hypoxic injury was followed by 24 h of normothermia or hypothermia, and mice were sacrificed at the indicated time points (P4, 5 weeks). Note that behavioral tests were conducted at 4 weeks of age. <i>C, D</i>: Representative examples of hematoxylin staining for normothermia (C) or hypothermia (D) treated mice. In the normothermia group, the boundary was obscure between the superficial layers and the deep layers (white asterisk). Scale, 500 µm. High magnification images of the contralateral (<i>left</i>) and ipsilateral (<i>right</i>) hemisphere. Scale, 200 µm.</p

Apoptosis (TUNEL staining) in the cortex 24 h after hypoxic ischemia injury.

<p><i>A</i>: Tissue sections are shown from brain hemispheres subjected to mild ischemic injury (<i>top</i>) or severe ischemic injury in mice from the normothermia (<i>middle</i>) and hypothermia (<i>bottom</i>) groups. <i>Arrow</i> indicates TUNEL-positive cells. Scale, 500 µm. <i>B</i>: <i>(left</i>) Average number of TUNEL-positive cells (mean±SE) in the superficial and deep cortical layers in the normothermia (n = 9) and hypothermia (n = 8) groups. *<i>P</i><0.05 (Wilcoxon test). <i>(Right</i>) Distribution of the number of TUNEL-positive cells in the superficial layers and deep cortical layers.</p

Disruptions in laminar structure of mouse brain cortex after hypoxic ischemia injury.

<p><i>A</i>: (<i>left</i>) Histological tissue sections show the laminar structure of the cortex in the normothermia group. Scale bar, 500 µm. The designated areas are enlarged in the bottom panels for clarity. Scale bar, 200 µm; <i>(right</i>) average areas of superficial and deep cortical layers measured on mouse brain sections (n = 6, mean±SE). *<i>P</i><0.05 (Wilcoxon test). <i>B:</i> Laminar structure in the hypothermia group (n = 6, mean±SE). Other notations defined in (<i>A</i>).</p

Neuronal cell density reduced in adult mouse brains after hypoxic ischemia injury.

<p><i>A</i>: Immunohistochemistry shows NeuN-positive cells in the cortex (<i>Top</i>) and striatum (<i>Bottom</i>) of mice subjected to hypoxic ischemia injury, followed by normothermia. Scale, 500 µm. Average density of NeuN cells (n = 11 mouse brains, mean±SE) was assessed in the contralateral (black) and ischemic ipsilateral (white) hemispheres. <i>B</i>: High magnification images of the cortices of mice in normothermia and hypothermia groups. Scale, 200 µm. *<i>P<</i>0.05 (Wilcoxon test). Other notations are defined in (<i>A</i>).</p

Fatty Acid-Binding Protein 5 Facilitates the Blood–Brain Barrier Transport of Docosahexaenoic Acid

The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood–brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA <i>in vitro</i> and <i>in vivo</i>, respectively. hFAPB5 was recombinantly expressed and purified from <i>Escherichia coli</i> C41­(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of ¹⁴C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An <i>in situ</i> transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (<i>K</i>_in) of ¹⁴C-DHA between FABP5-deficient (FABP5^–/–) and wild-type (FABP5^+/+) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in ¹⁴C-DHA cellular uptake. By using optimized conditions for the <i>in situ</i> transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of ¹⁴C-DHA (1 min)), the <i>K</i>_in of ¹⁴C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5^+/+ mice, the <i>K</i>_in of ¹⁴C-DHA decreased 36.7 ± 12.4% in FABP5^–/– mice. This study demonstrates that FABP5 binds to DHA and is involved in the brain endothelial cell uptake and subsequent BBB transport of DHA, confirming the importance of this cytoplasmic carrier protein in the CNS exposure of this PUFA essential for neuronal function

Inhibition of Fatty Acid Synthase Decreases Expression of Stemness Markers in Glioma Stem Cells

<div><p>Cellular metabolic changes, especially to lipid metabolism, have recently been recognized as a hallmark of various cancer cells. However, little is known about the significance of cellular lipid metabolism in the regulation of biological activity of glioma stem cells (GSCs). In this study, we examined the expression and role of fatty acid synthase (FASN), a key lipogenic enzyme, in GSCs. In the <i>de novo</i> lipid synthesis assay, GSCs exhibited higher lipogenesis than differentiated non-GSCs. Western blot and immunocytochemical analyses revealed that FASN is strongly expressed in multiple lines of patient-derived GSCs (G144 and Y10), but its expression was markedly reduced upon differentiation. When GSCs were treated with 20 μM cerulenin, a pharmacological inhibitor of FASN, their proliferation and migration were significantly suppressed and <i>de novo</i> lipogenesis decreased. Furthermore, following cerulenin treatment, expression of the GSC markers nestin, Sox2 and fatty acid binding protein (FABP7), markers of GCSs, decreased while that of glial fibrillary acidic protein (GFAP) expression increased. Taken together, our results indicate that FASN plays a pivotal role in the maintenance of GSC stemness, and FASN-mediated <i>de novo</i> lipid biosynthesis is closely associated with tumor growth and invasion in glioblastoma.</p></div

<i>De novo</i> lipogenesis using glucose and acetate as carbon source in GSCs and differentiated non-GSCs.

<p>G144, Y10 and G179 cells were incubated with [¹⁴C]-glucose (A) or [¹⁴C]-acetate (B) for 24 or 8 h to measure glucose or acetate incorporation into total lipid, respectively. Data are represented as means ± SEM for three independent experiments for each cell line. * P < 0.01.</p

Expression of FASN in human glioblastoma cells and GSC lines.

<p>(A) Human glioblastoma cells show the strong expression of FASN (blue) and the neural stem cell marker, Sox2 (red). Sox2 expression is confined to the nuclei, whereas FASN is expressed in the cytosol. The white arrows show FASN⁺Sox2⁺ cells. (B) Western blotting showing the expression of FASN, CD133, Sox2, and FABP7 in G144, Y10, G179, Y02, Y04 and Y14 GSC lines. Upon differentiation in the presence of FBS, FASN expression, similar to that of CD133, Sox2, and FABP7, was down-regulated. Expression of β-actin was used as an internal control. (C) Expression of FASN in GSC lines before and after differentiation. GSCs show strong expression of FASN and other neural stem cell markers, Sox2, nestin and CD133. Upon differentiation in the presence of FBS, FASN expression is down-regulated, similar to that of Sox2, nestin, and CD133. Immunofluorescence micrographs showing the co-expression of FASN with Sox2 (a, e, a’) and nestin (b, f, b’) in G144 and Y10 GSC lines. Phase contrast micrographs showing the morphology of GSC lines (G144 and Y10) in the presence of EGF and FGF (d, h) or after differentiation in the presence of FBS (d’). Bars in a-c, e-g, a’-c’ = 50 μm, Bars in d, h, d’ = 20 μm.</p

Effects of cerulenin on stemness and differentiation status.

<p>(A) qPCR results showing the expression of nestin, CD133, Sox2 and FABP7 in G144 GSC lines before and after administration of cerulenin. * P < 0.05, ^# P < 0.01 (B) The protein level of nestin, CD133, Sox2 and FABP7 was down-regulated by cerulenin. * P < 0.05, ^# P < 0.01 C. Expression of GFAP and NeuN in GSC lines before and after administration of cerulenin. * P < 0.01.</p

Additional file 1: of Modulation of anti-cancer drug sensitivity through the regulation of mitochondrial activity by adenylate kinase 4

Supplemenal Fig. 1 Evaluation of adenylate kinase 4 (AK4) in A549 cells. (A) Western blotting evaluation of AK4 (25 kDa) and α-tubulin (55 kDa) expression at the indicated time-points under hypoxic conditions (1 % O2). (B) Western blotting analysis of AK4 expression after deferoxamine (DFO) treatment. (C) Western immunoblotting showing AK4 knockdown in cells by siRNA. AK4, 25 kDa; phosphorylated 5΄ AMP-activated protein kinase (p-AMPK), 64 kDa; β-actin, 42 kDa. (D) Cell numbers were counted 2 days after seeding under either normoxic or hypoxic (1 % O2) conditions. *P < 0.05, **P < 0.01. (E) Evaluation of drug sensitivity after AK4 knockdown showing the half maximal inhibitory concentration (IC50) for cis-diamminedichloro-platinum(II) (CDDP). (Upper) IC50 curves of CDDP at 24 h, (Lower) IC50 of CDDP at 24 h. **P < 0.01 (PDF 246 kb