Ferrichrome-iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA. To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of beta-sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N-terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.

The reversible, enzymatically driven removal and readdition of its carboxy-terminal tyrosine are major posttranslational modifications of alpha-tubulin. To study these processes isoform-specific antibodies were produced and subsequently used to characterize tyrosinated and detyrosinated tubulin in the brine shrimp, Artemia. Tyrosinated tubulin existed in relatively constant amounts on western blots of cell-free protein extracts from Artemia at all developmental stages examined, whereas detyrosinated tubulin was present after 20-24 h of postgastrula growth. In agreement with the blots, the detyrosinated isoform was observed in immunofluorescently stained larvae after 24 h of incubation, appearing first in structures of a transient nature, namely spindles and midbodies. The elongated muscle cells encircling the gut and the epithelium bordering the gut lumen were stained extensively with antibody to detyrosinated tubulin. Detyrosination was accompanied by the appearance of a tubulin-reactive carboxypeptidase, which used both nonpolymerized and polymerized tubulin as substrate. The enzyme bound to microtubules very poorly, if at all, under conditions used in this work. Several inhibitors of carboxypeptidase A had no effect on the carboxypeptidase from Artemia and revealed similarities between this enzyme and others thought to be tubulin specific. The use of inhibitors also indicated that the carboxypeptidase from Artemia recognized aspects of tubulin structure in addition to the carboxy-terminal tyrosine. Our results support the idea that detyrosinated tubulin appears in microtubules of varying stability, and they demonstrate that Artemia possess a carboxypeptidase with the potential to detyrosinate tubulin during growth of larvae.

This study aims to investigate the mechanisms involved in the action of lutein (LU) alleviating arsenic-induced reproductive toxicity using mice model. Forty male Kunming mice were received following treatments by gavage: normal saline solution (control), arsenic trioxide (ATO; 5 mg/kg/day), LU (40 mg/kg/day), and ATO + LU (5 mg/kg/day + 40 mg/kg/day). At the end, the mice were killed by cervical dislocation and weighed. Pathological examination was done on the testis. The biomedical parameters including superoxide dismutase (SOD), glutathione (GSH), total antioxidative capability, malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), and reproductive indexes were analyzed. The messenger RNA (mRNA) and protein expression of Nrf2, heme oxygenase 1 (HO-1), glutathione S-transferase (GST), nicotinamide adenine dinucleotide phosphate dehydrogenase, quinone 1 (NQO1) in testis were detected by real-time polymerase chain reaction and Western blot. We found that there was a decrease in sperm count; testis somatic index; the activities of SOD, GSH, total antioxidative capacity ( p < 0.01, respectively) in ATO-treated mice, while there was an increase in the levels of sperm abnormalities, MDA, and 8-OHdG than control ( p < 0.01, respectively). The groups treated with ATO + LU showed recovery of the measured parameters between those of ATO or saline-treated group. The antagonized interaction between ATO and LU was statistically significant ( p < 0.01). Mice treated with ATO + LU also showed greater mRNA expression of Nrf2, HO-1, NQO1, and GST than ATO or saline-treated groups. These findings suggest that LU alleviates reproductive toxicity induced by arsenic in male mice via Nrf2 signaling, which implicates a possible mechanism of LU in preventing the reproductive injury, and elucidates that consuming the rich plant sources of LU will alleviate the reproductive toxicity induced by chemicals.

No abstract

Epstein-Barr virus (EBV) infection in tumor cells is generally restricted to the latent forms of viral infection. Switching the latent form of viral infection into the lytic form may induce tumor cell death. An important nuclear factor, nuclear factor (NF)-kappaB, is thought to play an essential role in EBV lytic infection; high levels of NF-kappaB can inhibit EBV lytic replication. In this study, we tested the effect of inducing EBV lytic replication using two NF-kappaB inhibitors: Bay11-7082 and Z-LLF-CHO, to reveal the possibility of targeting EBV-positive cancer therapy with these two NF-kappaB inhibitors. Our results showed that Bay11-7082 and Z-LLF-CHO reactivated EBV in a dose-dependent manner, thus resulting in EBV-positive 5-8F cell death. In contrast, there was no significant effect on EBV-negative HNE3 cells. When ganciclovir was used in combination with either Bay11-7082 or Z-LLF-CHO to treat 5-8F cells, the cytotoxic effect of the NF-kappaB inhibitor was amplified. The finding indicates that inhibiting the NF-kappaB activity of EBV-positive cells can induce lytic replication of EBV and cause lytic cytotoxicity against these cells.