Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by reduced expression levels of the frataxin gene (FXN) due to expansion of triplet nucleotide GAA repeats in the first intron of FXN. FXN is a mitochondrial protein which plays an important role in the regulation of intracellular iron trafficking, biogenesis of iron-sulfur cluster and heme, and removal of reactive oxygen species. Our previous work showed that tissue-specific expression of FXN in cerebellum and heart generates two novel isoforms. In order to find the isoforms in mouse tissues, we tried to obtain a polyclonal antibody against mouse Fxn with high specificity and sensitivity. Thus, the recombinant plasmid pET24(+)-mFxn was constructed to express his-tagged Fxn in BL21 (DE3) cells. The expressed protein is a mature form with 130 amino acids (aa, 14.38 kDa) without the N-terminal signal peptide (77 aa), purified on Ni-NTA column and further dialyzed with Centrifugal Filtration Device. The polyclonal antibody against Fxn was produced by immunizing rabbits with highly purified protein. The collected antiserums were preliminarily purified by precipitation with (NH4)2SO4. Western blotting analysis and cell immunofluorescence showed that the obtained antibody was able to detect both purified and endogenous Fxn. It also worked well in immunoprecipitation with mouse tissues. This is the first time, to our knowledge, to report that mouse Fxn was used as immunogen to generate antibody with high specificity and sensitivity. This work provides a powerful tool for our further research on mouse Fxn isoforms.
Animals ; Antibodies ; immunology ; metabolism ; Antibody Specificity ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Engineering ; methods ; Immunization ; Iron-Binding Proteins ; genetics ; immunology ; Mice ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology

OBJECTIVETo observe if Porphyromonas gingivalis (Pg) infection could enhance the adhesion of human monocytic cell line (THP-1) to human umbilical vein endothelial cells (HUVEC).

METHODSPgATCC33277 was cultured in anaerobic jar, and THP-1 was infected with various concentrations of PgATCC33277 at multiplicity of infection (MOI) of 1: 100 for 8 and 24 hours, respectively. After removal of the free Pg, THP-1 cells were cocultured with HUVEC for 1 hour to observe the adhesion of THP-1 to HUVEC.HUVEC with adhesive THP-1 cells were co-cultured for additional 23 hours. The medium and cells were separately collected. The expression of related chemotactic cytokine[monocyte chemotactic protein 1(MCP-1) and interleukin 8(IL-8)] and intercellular adhesion molecule-1(ICAM-1) were detected with enzyme-linked immunosorbent assay.

RESULTSThe adhesion of THP-1 to HUVEC was enhanced (13.8%-35.2%, P = 0.006) and the expression of ICAM-1 of HUVEC was increased from (132.5 ± 7.7) to (164.9 ± 9.1) ng/L (P = 0.005) after infection for 24 hours by Pg. Both of the secreted MCP-1 and IL-8 elevated after infection of Pg for 24 hours from (183.2 ± 3.1) to (221.0 ± 4.9) ng/L (P = 0.012) and from (587.2 ± 5.1) to (787.2 ± 10.3) ng/L (P = 0.002), respectively.

CONCLUSIONSPg could enhance the adhesion of monocytes to endothelial cells and stimulate the inflammation, suggesting that Pg infection may be one of the risk factors in promoting the development of atherosclerosis.

Bacteroidaceae Infections ; metabolism ; Cell Line ; Cells, Cultured ; Chemokine CCL2 ; biosynthesis ; Coculture Techniques ; Endothelial Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Intercellular Adhesion Molecule-1 ; biosynthesis ; Interleukin-8 ; biosynthesis ; Monocytes ; Porphyromonas gingivalis ; pathogenicity ; Umbilical Veins

OBJECTIVETo investigate the mechanisms of Porphyromonas gingivalis (Pg) infection-mediated enhancement of adhesion between monocytes THP-1 and human umbilical vein endothelial cells (HUVEC) by detecting the effect of erythropoietin producing hepatomocellular receptor interacting protein B2 (Ephrin B2) and its receptors on the adhesion.

METHODSPgATCC33277 was cultured in an anaerobic jar, and THP-1 cells were infected with various concentrations of Pg at multiplicity of infection (MOI) of 1:100 for 8 and 24 h, respectively. The expression of Ephrin B2 receptor of THP-1 cells was detected. After removal of the free Pg, THP-1 cells were cocultured with HUVEC (overexpress of EphrinB2 or not) for 24 h to detect the expression of Ephrin B2 of HUVEC cells after additional cultivation for 23 h.

RESULTSThe adhesion of THP-1 cells post infection by Pg to HUVEC was enhanced. The mRNA levels of Ephrin B2 receptors, including EphB3 (5.169±0.152, P = 0.005), EphB4 (11.040±1.195, P = 0.001), and EphA4 (4.976± 0.122, P = 0.001) expressed by THP-1, and Ephrin B2 (8.938±0.962, P = 0.008) expressed by HUVEC were significantly elevated 24 h post infection of Pg. Over expression of Ephrin B2 in HUVEC promoted the adhesion of THP-1 to HUVEC.

CONCLUSIONSEphrin B2 and its receptors are involved in Pg infection mediated enhancement of the adhesion of THP-1 to HUVEC cells, suggesting that Ephrin B2 participates in the development of atherosclerosis.

Atherosclerosis ; Cell Adhesion ; Cells, Cultured ; Coculture Techniques ; Endothelium, Vascular ; Ephrin-B2 ; physiology ; Epoetin Alfa ; Erythropoietin ; Human Umbilical Vein Endothelial Cells ; Humans ; Monocytes ; Porphyromonas gingivalis ; pathogenicity ; Recombinant Proteins ; Umbilical Veins ; cytology