Endoplasmic reticulum stress is involved in podocyte apoptosis induced by saturated fatty acid palmitate / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Podocyte apoptosis is recently indicated as an early phenomenon of diabetic nephropathy. Pancreatic β-cells exposed to saturated free fatty acid palmitate undergo irreversible endoplasmic reticulum (ER) stress and consequent apoptosis, contributing to the onset of diabetes. We hypothesized that palmitate could induce podocyte apoptosis via ER stress, which initiates or aggravates proteinuria in diabetic nephropathy.</p><p><b>METHODS</b>Podocyte apoptosis was detected by 4',6-diamidio-2-phenylindole (DAPI) stained apoptotic cell count and Annexin V-PI stain. The expressions of ER molecule chaperone glucose-regulated protein 78 (GRP78), indicators of ER-associated apoptosis C/EBP homologous protein (CHOP), and Bcl-2 were assayed by Western blotting and real-time PCR. GRP78 and synaptopodin were co-localized by immunofluorescence stain.</p><p><b>RESULTS</b>Palmitate significantly increased the percentage of cultured apoptotic murine podocytes time-dependently when loading 0.75 mmol/L (10 hours, 13 hours, and 15 hours compared with 0 hour, P < 0.001) and dose-dependently when loading palmitate ranging from 0.25 to 1.00 mmol/L for 15 hours (compared to control, P < 0.001). Palmitate time-dependently and dose-dependently increased the protein expression of GRP78 and CHOP, and decreased that of Bcl-2. Palmitate loading ranging from 0.5 to 1.0 mmol/L for 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to control (P < 0.001), with the maximum concentration being 0.75 mmol/L. Palmitate 0.5 mmol/L loading for 3 hours, 8 hours, and 12 hours significantly increased mRNA of GRP78 and CHOP, and decreased that of Bcl-2 compared to 0 hour (P < 0.001), with the maximum effect at 3 hours. Confocal microscopy demonstrated that GRP78 expression was significantly increased when exposed to 0.5 mmol/L of palmitate for 8 hours compared to control.</p><p><b>CONCLUSION</b>Palmitate could induce podocyte apoptosis via ER stress, suggesting podocyte apoptosis and consequent proteinuria caused by lipotoxic free fatty acid could be ameliorated by relief of ER stress.</p>

Prokaryotic expression and purification of mitochondrial transcription complex proteins / 中国医学科学院学报

<p><b>OBJECTIVE</b>To obtain human mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M), and mitochondrial transcription factor B2 (TFB2M) that were expressed efficiently in E. coli BE21 and to purify the target proteins.</p><p><b>METHODS</b>TFAM, TFB1M, and TFB2M segments were designed and synthesized. After having been sequenced, the reconstructed expression vectors were constructed by enzyme digestion and by cloning into an expression vector pET42a. Then the reconstructed vectors were transformed into E. coli BL21. Recombinant glutathione S transferase (GST) fusion proteins were expressed via the induction of IsoPropyl beta-D-ThioGalactoside (IPTG) and purified by glutathione Sepharose 4B.</p><p><b>RESULTS</b>The expression plasmids of pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB1M were successfully constructed. The sequences of the cloned gene segments were identical with GenBank reported. The protein bands with relative molecular masses of 56 000, 67 000, and 69 000 appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after the expressed GST-TFAM, GST-TFB1M, and GST-TFB2M fusion proteins were separated by SDS-PAGE. The expressed fusion proteins were purified to high purity.</p><p><b>CONCLUSION</b>The recombinant plasmids pET42a-TFAM, pET42a-TFB1M, and pET42a-TFB2M were successfully constructed, and the GST-fused target proteins were prepared.</p>