OBJECTIVETo investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.

METHODSROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.

RESULTS20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.

CONCLUSIONSSodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; biosynthesis ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

PURPOSETo investigate the effects of salvianolic acid B (SAB) on tumor necrosis factor a (TNF-α) induced alterations of cerebral microcirculation with a bone-abrading model.

METHODSThe influences of craniotomy model and bone-abrading model on cerebral microcirculation were compared. The bone-abrading method was used to detect the effects of intracerebroventricular application of 40 μg/kg·bw TNF-α on cerebral venular leakage of fluorescein isothiocyanate (FITC)- albulmin and the rolling and adhesion of leukocytes on venules with fluorescence tracer rhodamine 6G. The therapeutical effects of SAB on TNF-α induced microcirculatory alteration were observed, with continuous intravenous injection of 5 mg/kg·h SAB starting at 20 min before or 20 min after TNF-α administration, respectively. The expressions of CD11b/CD18 and CD62L in leukocytes were measured with flow cytometry. Immunohistochemical staining was also used to detect E-selectin and ICAM-1 expression in endothelial cells.

RESULTSCompared with craniotomy method, the bone-abrading method preserved a higher erythrocyte velocity in cerebral venules and more opening capillaries. TNF-α intervention only caused responses of vascular hyperpermeability and leukocyte rolling on venular walls, without leukocyte adhesion and other hemodynamic changes. Pre- or post-SAB treatment attenuated those responses and suppressed the enhanced expressions of CD11b/CD18 and CD62L in leukocytes and E-selectin and ICAM-1 in endothelial cells induced by TNF-α.

CONCLUSIONSThe pre- and post-applications of SAB during TNF-α stimulation could suppress adhesive molecular expression and subsequently attenuate the increase of cerebral vascular permeability and leukocyte rolling.

Animals ; Benzofurans ; pharmacology ; Blood Flow Velocity ; Cerebrovascular Circulation ; drug effects ; Craniotomy ; Disease Models, Animal ; E-Selectin ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Mice ; Mice, Inbred C57BL ; Microcirculation ; drug effects ; Random Allocation ; Reference Values ; Tumor Necrosis Factor-alpha ; administration & dosage

To study the biological function of the N-glycosylation modification of prion proteins (PrP), various eukaryotic expression vectors for the mutants with N-glycosylation modification of human PrP had been constructed and expressed. With site-direct mutation technique, human PRNP gene was mutated and the obtained mutants were subcloned into eukaryotic expressing plasmid pcDNA3.1 and transiently expressed in Hela cervical adenocarcinoma cell. The expression products of the mutated PrP were identified with Western blotting assay and the PNGase digestion assay. Several mutants with specific glycosylation modification were identified from the expressed products by Western blot, including two mutants with one glycosylation site mutated and one without any mutation at glycosylation sites. The expressed products were digested with PNGase F. The wild type proteins and those with one of glycosylation sites mutated were digested, resulting in their molecular weights reduced, while the molecular weights of products with mutations at both glycosylation sites were not changed. The mutant of wild type human PRNP gene at N-glycosylation modification sites and six modified mutants with mono- or non-N-glycosylation had been obtained successfully in the study. Moreover, the modified PrP with mono- and non-N-glycosylation were able to be expressed transitantly in Hela cells, which could be a useful means for studying prions.
Escherichia coli ; genetics ; metabolism ; Glycation End Products, Advanced ; biosynthesis ; genetics ; Glycosylation ; HeLa Cells ; Humans ; Mutagenesis, Site-Directed ; Mutant Proteins ; biosynthesis ; genetics ; Prions ; biosynthesis ; genetics ; Transfection

ObjectiveTranscription factor NFE2 was observed abnormal expression in myeloproliferative neoplasm (MPN) patients. However, how NFE2 is transcriptionally regulated remains ambiguous. This study aims to explore the elements and molecular mechanisms involved in the transcriptional regulation of NFE2. MethodsActive enhancers were predicted by public NGS data and conformed experimentally via dual luciferase reporter assay. After that, PRO-seq and GRO-seq data was used to detect enhancer RNAs transcribed from these enhancers. RACE was utilized to clone the full length enhancer RNA (eRNA) transcripts, and RT-qPCR was used to measure their expression in different leukemia cell lines as well as the transcript levels during induced differentiation. Finally, to investigate the molecular function of the eRNA, overexpression and knockdown of the eRNA via lentivirus system was performed in K562 cells. ResultsWe identified three enhancers regulating NFE2 transcription, which located at -3.6k, -6.2k and +6.3k from NFE2 transcription start site (TSS) respectively. At the -3.6k enhancer, we cloned an eRNA transcript and characterized that as a lncRNA which was expressed and located in the nucleus in three types of leukemia cell lines. When this lncRNA was overexpressed, expression of NFE2 was upregulated and decreases of K562 cell proliferation and migration ability were observed. While knocking down of this lncRNA, the level of NFE2 decreases correspondingly and the proliferation ability of K562 cells increases accordingly. ConclusionWe identified an enhancer lncRNA that regulates NFE2 transcription positively and suppresses K562 cell proliferation.