1.Transmembrane Segment Analysis of Transient Receptor Potential Channel
Qiuxiang SU ; Liyan ZHANG ; Kun JI ; Gaigai ZHANG ; Lihua ZHANG
Journal of China Medical University 2016;45(7):610-615
Objective To investigate the membrane topology of transient receptor potential(TRP)channel. Methods Glycosylation method was used to investigate the membrane integrations of each hydrophobic segment of canonical TRP(TRPC5). Results In TRPC5 channel,S4?S8 segments were integrated into membrane with Ncyt/Cexo and Nexo/Ccyt orientations sequentially ,and C?terminus was intracellular. S1?S3 segments were integrated into membrane with two possible types. One was that S1 and S3 were integrated into membrane,whereas S2 was left out of the membrane on the cytosolic side;and the other was a mixed type that S1 and S3 were exposed to cytoplasm respectively. Both of them,the N?termi?nus was intracellular. Conclusion S4?S8 segments of TRPC5 are transmembrane segments. The integrations of S1?S3 segments into membrane need to be further investigated.
2.The role of oxygen free radicals in the pathogenesis of acute mononuclear leukemia
Gaigai BAI ; Chenyang LU ; Qiuju SHEN ; Shan MENG ; Lingyun HUI ; Dan SU ; Wanggang ZHANG ; Fuling ZHOU
Journal of Xi'an Jiaotong University(Medical Sciences) 2015;(4):501-504
Objective To study the mechanism of oxidative stress involved in the pathogenesis and relapse of acute monocytic leukemia (M5 ).Methods We detected reactive oxide species (ROS)levels,conducted plasma analysis obtained from 76 M5 patients at diagnosis and at relapse,and observed the ultrastructure of mitochondria of mononuclear cells in peripheral blood by transmission electron microscope.Results Compared with that in the control group,the average fluorescence intensity of intracellular ROS was significantly increased in M5 groups, especially in the relapse patients (P < 0.05 ).Low total antioxidative capacity (T-AOC)and antioxidant enzyme activity were characteristic of M5 at both diagnosis and relapse. However, lactate dehydrogenase (LDH ), malondialdehyde (MDA)and 8-hydroxy-2’-deoxyguanine (8-OHdG)increased significantly at both diagnosis and relapse (P < 0.05 ).Prominent ultrastructural abnormalities (mitochondrial swelling,outer membrane blebs,and aberrant cristae disorder)were present in patients with primary M5,and they were obviously abnormal in relapsing M5 patients.Conclusion Oxidative stress is the initiating factor of M5.Mitochondria are the main intracellular location for ROS generation.To maintain the dynamic balance between ROS and antioxidant defence may be the critical factor for preventing relapse.
3.Development of a cell-based diagnostic system for vitamin K-dependent coagulation factor deficiency 1.
Wenwen GAO ; Hongli LIU ; Gaigai SU ; Yaqi XU ; Yiyi WANG ; Longteng CUI ; Rong HUANG ; Haiping YANG ; Meng GAO ; Shoumin XI ; Guomin SHEN
Chinese Journal of Medical Genetics 2020;37(8):811-814
OBJECTIVE:
To develop a cell-based system for the diagnosis of vitamin K-dependent coagulation factor deficiency 1 (VKCFD1).
METHODS:
In HEK293 cells stably expressing the reporter gene FIX-Gla-PC, the gamma-glutamyl carboxylase (GGCX) gene was knocked out by using CRISPR/Cas9 technology. Enzyme-linked immunosorbent assay (ELISA), DNA sequencing and Western blotting were used to identify the GGCX gene knockout cells. A quickchange point variant method was used to construct the GGCX variant. ELISA was used to assess the influence of GGCX variant on the activity of reporter gene.
RESULTS:
Two monoclonal cell lines with no reporter activity by ELISA was identified. Edition and knockout of the GGCX gene was confirmed by DNA sequencing and Western blotting. The activity of the reporter gene was recovered by transfection of the wild-type GGCX gene. Thereby two monoclonal cells with GGCX knockout were obtained. By comparing the wild-type and pathogenic GGCX variants, the reporter activity was decreased in the pathogenic variants significantly.
CONCLUSION
A cell-based system for the detection of GGCX activity was successfully developed, which can be used for the diagnosis of VKCFD1 caused by GGCX variants.