Fluorescence assay for the detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity in human blood samples / 北京大学学报(医学版)

OBJECTIVE@#To develop a simple, sensitive and robust method for rapid detection of human apurinic/apyrimidinic endonuclease 1 (APE1) in various biological samples.@*METHODS@#An abasic site-containing DNA probe with a sequence of 5'-T*T*C*C*T*C*T(ROX)AGAGXCGTT (BHQ2)C*A*C*T*G*T*AGTTTATA*C*A*G*T*GAATCTCTCTAG*T*C*T-3' ["X" represents AP site; The phosphorothioated nucleotides (at 3' side) are indicated with an asterisk after the nucleotides; ROX is 6-carboxy-X-rhodamine and BHQ2 is Black Hole quencher 2] was synthesized and used for the detection. In the presence of APE1, the DNA probe could be specifically hydrolyzed by the enzyme and release the fluorophore, resulting in strong fluorescence emission. The activity of APE1 was determined according to the rate of increase in fluorescence intensity. In this work, we modified the reaction buffer and significantly improved the performance of the method. Moreover, the method was further extended to measure the contents of APE1 in the protein extraction from peripheral blood mononuclear cells (PBMCs) extracted from human whole blood samples by density gradient centrifugation. The assay was also applied to measure the activity of APE1 in human serum samples.@*RESULTS@#With a new reaction buffer composed of 0.04% (V/V) Triton X-100, 50 mmol/L KAc, 20 mmol/L Tris-Ac, 10 mmol/L Mg(Ac)2 and 1 mmol/L dithiothreitol (DTT), the method achieved a detection limit of 0.005 U/mL (3 pg/mL) and a linear response ranging from 6 pg/mL to 1.2 ng/mL. The contents of APE1 in the protein extraction from PBMCs of eight blood samples were measured to be in the range from 0.061 to 0.40 ng/μg protein, with an average of 0.16 ng/μg protein. The recovery was 98%±5% (n=3). The levels of APE1 in the sera from 102 normal individuals (51 male and 51 female, age range: 59-75 years) were observed to be from 0.13 to 0.34 ng/mL, with a recovery of 96%±15% (n=3).@*CONCLUSION@#The new fluorescence assay was simple, rapid and sensitive, providing a practical tool to measure the activity of APE1 in serum samples and cell extracts. It also holds great potential in measurement of APE1 in many other biological samples for clinical test and laboratory research.

The role of endoplasmic reticulum stress in pulmonary hypertension in rat induced by chronic hypoxia and hypercapnia / 中国应用生理学杂志

OBJECTIVE@#To observe the pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia, and to explore the role of endoplasmic reticulum stress in pulmonary hypertension.@*METHODS@#Forty SD rats were random-ly divided into four groups:normoxic control group (N), hypoxia hypercapnia group (HH), ERS inhibitor 4-phenylbutyric acid group (4-PBA), endoplasmic reticulum stress (ERS) pathway agonist tunicamycin group (TM), ten rats in each group.The mean pulmona-ry artery pressure (mPAP), mean carotid artery pressure (mCAP) and right ventricular hypertrophy index of rats in each group were measured.Pulmonary artery smooth muscle cells were identified by immunofluorescence α-smooth muscle actin (α-SMA).Morphologi-cal changes of lung tissue and pulmonary artery were observed by electron microscope.The apoptotic index of pulmonary artery smooth muscle cells in each group was detected by TUNEL.Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), c-Jun N-terminal kinase (JNK) and cysteinyl aspartate specific proteinase-12 (caspase-12) mRNA and protein in each group.@*RESULTS@#①Compared with the N group, the mPAP, the ratio of right ventricle weight to left ventricle plus ventricular septum weight[RV/(LV+S)]and the ratio of pulmonary artery wall area to total tube area (WA/TA) were increased (<0.01), and the ratio of pulmonary artery luminal area to total tube area (LA/TA) were decreased (<0.01), pulmonary artery smooth muscle cell apoptosis index were decreased (<0.05 or <0.01) in HH group, 4-PBA group and TM group.ERS related protein and mRNA expressions were increased, the differences were statistically significant.②Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of 4-PBA group were decreased ( <0.01), LA/TA and pulmonary artery smooth muscle cell apoptosis index were increased (<0.01, <0.05).The expressions of ERS related protein and mRNA were all decreased (<0.05 or <0.01).③Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of TM group were increased (<0.05 or <0.01), pulmonary artery middle layer thickened, LA/TA and pulmonary artery smooth muscle cell apoptotic index were decreased (<0.01).ERS related protein and mRNA expressions were increased with statistical significance except GRP78 protein.@*CONCLUSIONS@#Pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia may be related to the excessive proliferation of pulmonary artery smooth muscle cells and too little apopto-sis;ERS related factors (JNK, caspase-12 and CHOP) are involved in the regulation of pulmonary hypertension induced by hypoxia hypercapnia.

Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia / 生理学报

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca]in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca]were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].

Effect of ERK1/2 on rat pulmonary artery smooth muscle cells Kv1.5 channel in the process of hypoxia / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To explore the effect of ERK1/2 MAPK pathway on the expression of Kv1.5 channel, a voltage-gated potassium ion channel, in rat pulmonary artery smooth muscle cells (PASMCs) and its mechanisms during the process of hypoxia.</p><p><b>METHODS</b>The PASMCs derived from SD rats were cultivated primarily. The third to sixth generation of PASMCs were divided into 5 groups randomly: (1) Normal group (N); (2) Hypoxic group (H); (3) Demethy sulfoxide(DMSO) group (HD); (4) U0126 group (HU): 10 micromol/L U0126; (5) Anisomycin group (HA): 10 micromol/L anisomycin. There were three dishes of cells in each group. The cells in normal group were cultured in normoxic incubator (5% CO2, 37 degrees C), the cells in other groups were added to 0.05% DMSO in the hypoxic incubator (5% CO2, 2% O2, 37 degrees C), all cells were cultured for 60 h. RT-PCR and Western blot were used to detected the espressions of Kv1.5 mRNA and protein in PASMCs.</p><p><b>RESULTS</b>Compared with N group, the expressions of Kv1.5 mRNA and protein in H, HD and HA groups were reduced significantly (P < 0.05); Compared with H group and HD groups, Kv1.5 mRNA and protein expressions in HU group were increased sharply (P < 0.05). Compared with the HU group, Kv1.5 mRNA and protein expressions in HA groups were significantly lower (P < 0.05).</p><p><b>CONCLUSION</b>Low oxygen reduced Kv1.5 mRNA and protein expressions, U0126 could resistant the Kv1.5 channel lower expression caused by hypoxia. Anisomycin had no significant effect on Kv1.5 channel expression under hypoxia, but the expression of Kv1.5 was still significantly lower than the normal oxygen group. These data suggest that hypoxia may cause hypoxic pulmonary hypertension by interfering ERK1/2 signaling pathway to inhibit Kv1.5</p>

Production and characterization of monoclonal antibody against recombinant human erythropoietin / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To produce specific monoclonal antibody (mAb) against recombinant human erythropoietin (rHuEPO) for development of highly efficient methods for erythropoietin detection in biological fluids.</p><p><b>METHODS</b>rHuEPO was covalently coupled with bovine serum albumin (BSA) and the conjugate was used to immunize mice to produce specific mAb against rHuEPO based on hybridoma technology. The obtained F3-mAb was characterized by enzyme-linked immunosorbent assay (ELISA), SDS-PAGE and Western blot.</p><p><b>RESULTS</b>The isotype of F3-mAb was found to be IgM with an affinity constant of 2.1 x 10(8) L/mol. The competitive ELISA using the obtained IgM showed a broader linear range and lower detection limit compared with previous work.</p><p><b>CONCLUSIONS</b>The modification of rHuEPO was proved to be successful in generating required specific mAb with high avidity to rHuEPO.</p>