Subcellular localization of GTPase of immunity-associated protein 2 / 北京大学学报(医学版)

OBJECTIVE@#To analyze the subcellular localization of GTPase of immunity-associated protein 2 (GIMAP2) for the further functional study.@*METHODS@#In the study, we first obtained the protein sequences of GTPase of immunity-associated protein 2 (GIMAP2) from National Center for Biotechnology Information (NCBI) database, and then performed a prediction analysis of its transmembrane structure, nuclear localization signal (NLS), nuclear export signal (NES) and subcellular localization through bioinformatics online tools. GIMAP2 gene amplified by PCR was inserted into the expression vector pQCXIP-mCherry-N1 and positive clones were selected by ampicillin resistance. After using methods to extract and purify, the sequenced recombinant plasmid pQCXIP-GIMAP2-mCherry, together with the retroviral packaging plasmids VSVG and Gag/pol, was transferred into HEK293FT cells by liposomes for virus packaging. The virus supernatant was collected 48 h after transfection and directly infected the human breast cancer cell line MDA-MB-436. Immunofluorescence staining was constructed to detect the localization of endogenous and exogenous GIMAP2 in MDA-MB-436 cells. Meanwhile, green fluorescent chemical dyes were used to label mitochondria, endoplasmic reticulum, and lipid droplets in living MDA-MB-436 cells stably expressing the GIMAP2-mCherry fusion protein. Images for the three dye-labeled organelles and GIMAP2-mCherry fusion protein were captured by super-resolution microscope N-SIM.@*RESULTS@#Bioinformatics analysis data showed that GIMAP2 protein composed of 337 amino acids might contain two transmembrane helix (TM) structures at the carboxyl terminus, of which TMs were estimated to contain 40-41 expected amino acids, followed by the residual protein structures toward the cytoplasmic side. NES was located at the 279-281 amino acids of the carboxyl terminus whereas NLS was not found. GIMAP2 might locate in the lumen of the endoplasmic reticulum. Sequencing results indicated that the expression vector pQCXIP-GIMAP2-mCherry was successfully constructed. Fluorescent staining confirmed that GIMAP2-mCherry fusion protein, co-localized well with endogenous GIMAP2, expressed successfully in the endoplasmic reticulum and on the surface of lipid droplets in MDA-MB-436 cells.@*CONCLUSION@#GIMAP2 localizes in the endoplasmic reticulum and on the surface of LDs, suggesting potential involvement of GIMAP2 in lipid metabolism.

Application of liver biopsy in patients after liver transplantation / 国际外科学杂志

Objective To explore the histopathological manifestations of various complications after transplantation,the patients in our hospital after liver transplantation liver puncturebiopsy pathology data were analyzed,and then provide a reliable clinical diagnosis and treatment for the patients scheme.Methods A retrospective analysis of our hospital 198 cases of liver transplantation in 249 cases diagnosis of liver puncture biopsy pathology data,HE staining method to analyze the pathological morphological changes,using rejectionpathological criteria,according to clinical examination and treatment effect of international unified Banff.Results All biopsy materials,acute rejection rate is the highest,a total of 71 cases(28.5％),biliary complications occurred in 39 cases (15.7％),hepatitis B virusinfection and recurrence in 28 cases (11.2％),34 cases of drug-induceddamage (13.7％),reperfusion injury in 35 cases (14.1％),CMV infection of 14cases (5.6％),tumor recurrence in 7 cases (2.8％),chronic rejection in 16cases (6.4％),primary graft non function in 2 cases (0.8％),it is difficult to determine in 3 cases (1.2％).Conclusion Transplantation of liver biopsy can provide correct cause for abnormal liver function,and to guide the clinical treatment of accurate,effective treatment,suggested that thetransplantation center will transhepatic listed for liver transplantationpostoperative routine inspections,periodic biopsies,the survival of the state to better protect the graft.