BDE-47 induced apoptosis in zebrafish embryos through mitochondrial ROS-mediated JNK signaling.

2,2,4,4-tetrabromodiphenyl ether (BDE-47) has received considerable attention because of its high detection level in biological samples and potential developmental toxicity. Here, using zebrafish (Danio rerio) as the experimental animal, we investigated developmental effects of BDE-47 and explored the potential mechanism. Zebrafish embryos at 4 h post-fertilization (hpf) were exposed to 0.312, 0.625 and 1.25 mg/L BDE-47 to 74-120 hpf. We found that BDE-47 instigated a dose-related developmental toxicity, evidenced by reduced embryonic survival and hatching rate, shortened body length and increased aberration rate. Meanwhile, higher doses of BDE-47 reduced mitochondrial membrane potential and ATP production but increased apoptosis in zebrafish embryos. Expression of genes involved in mitochondrial oxidative phosphorylation (OXPHOS) (ndufb8, sdha, uqcrc1, cox5ab and atp5fal) were negatively related to BDE-47 doses in zebrafish embryos. Moreover, exposure to BDE-47 at 0.625 or 1.25 mg/L impaired mitochondrial biogenesis and mitochondrial dynamics. Our data further showed that BDE- 47 exposure induced excessive reactive oxygen species (ROS) and oxidative stress, which was accompanied by the activation of c-Jun N-terminal Kinase (JNK). Antioxidant NAC and JNK inhibition could mitigate apoptosis in embryos and improve embryonic development in BDE-47-treated zebrafish, suggesting the involvement of ROS/JNK pathway in embryonic developmental changes induced by BDE-47. Altogether, our data suggest here that developmental toxicity of BDE-47 may be associated with mitochondrial ROS-mediated JNK signaling in zebrafish embryo.

A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes.

Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub(F45W)-Xaa) and di-ubiquitin chains (Ub(F45W)-diUb). After removal of the intact substrate by Ni(2+)-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub(F45W) product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.

The mechanism of the molecular interaction between cerium (III) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).

The mechanism of the molecular interaction between Ce3+, a member of rare earth elements, and Rubisco in vitro is investigated. The carboxylase activity of Rubisco greatly increased under low concentrations of Ce3+ and decreased under high concentrations of Ce3+. The ultraviolet absorption spectra show that the various concentrations of Ce3+ treatment do not shift the characteristic peaks of Rubisco while the characteristic peak intensity of Rubisco increases with increasing Ce3+ concentration. The Rubisco-Ce3+ interactions also do not cause any noticeable change in the λmax of Rubisco fluorescence spectra. However, the fluorescence intensity of Rubisco is found quenched by the addition of Ce3+, which strongly suggests that Ce3+ could directly bind to the Rubisco protein. and the binding sites is estimated to 1.52 per protein. The binding between Ce3+ and Rubisco is also proved by extended X-ray absorption fine-structure essay; Ce3+ coordinated with eight oxygen atoms of Rubisco in first shells and six oxygen atoms in second shells. The results implied that Ce3+ might improve the microenvironment of Rubisco and, in turn, affected the carboxylase capacity of Rubisco greatly.

Effect of Nd3+ ion on carboxylation activity of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach.

Neodymium (Nd), as a member of rare earth elements, proved to enhance the photosynthesis rate and organic substance accumulation of spinach through the increase in carboxylation activity of Rubisco. Although the oxygenase activity of spinach Rubisco was slightly changed with the Nd(3+) treatment, the specific factor of Rubisco was greatly increased. It was partially due to the promotion of Rubisco activase (R-A) activity but mainly to the formation of Rubisco-Rubisco activase super-complex, a heavier molecular mass protein (about 1200kD) comprising both Rubisco and Rubisco activase. This super-complex was found during the extraction procedure of Rubisco by the gel electrophoresis and Western-blot studies. The formation of Rubisco-R-A super-complex suggested that the secondary structure of the protein purified from the Nd(3+)-treated spinach was different from that of the control. Extended X-ray absorption fine structure study of the 'Rubisco' purified from the Nd(3+)-treated spinach revealed that Nd was bound with four oxygen atoms and two sulfur atoms of amino acid residues at the Nd-O and Nd-S bond lengths of 2.46 and 2.89A, respectively.

[Effect of Pb2+ on spectral characteristics of DNA from fish intestines].

The interaction of Pb2+ and DNA of fish intestines was investigated using absorption spectrum, fluorescence emission spectrum, and electrophoresis. Ultraviolet absorption spectra indicated that with the addition of Pb2+, the DNA generated obvious hypochromic effect, and the absorption peak at 207 nm of DNA was blue-shifted. The fluorescence emission spectra showed that with the addition of Pb2+ , the emission peak of DNA at about 419 nm did not have obvious changes, but the intensity of fluorescence reduced gradually, showing that Pb2+ has quenching effect on the DNA endogenous fluorescence. Pb2+ has 0.8 binding sites to DNA, and the fluorescence quenching of DNA caused by Pb2+ belongs to static quenching. The binding constants of binding sites are 6.08 x 10(4) L x mol(-1) and 2.82 x 10(4) L x mol(-1) from the Lineweaver-Burk plot. The experiments of ultraviolet and fluorescence spectra showed that exogenous Pb2+ could cause some changes to the conformation of DNA, but analysis of electrophoresis proved that exogenous Pb2+ at different concentration did not result in DNA breakage.

[Study of the effect of Pb2+ on alpha-amylase activity by spectroscopy].

The activity of alpha-amylase from porcine pancreas was enhanced under the treatment by Pb2+ at low concentration (0.5-4 mumol.L-1), but was inhibited by Pb2+ at high concentration (above 4 mumol.L-1). Pb2+ at high concentration could competitively displace Ca2+ from alpha-amylase. The EXAFS demonstrated that Pb2+ was bound to the active site of alpha-amylase, the coordination atom was oxygen, the coordination number was 2, and the Pb-O bond length was 0.234 nm. Circular dichroism spectra showed that the secondary structure of trypsin was greatly changed by Pb2+ at high concentration, as alpha-helix, beta-turn and random coil contents decreased, while beta-sheet, aromatic and disulfide bond contents increased. It was suggested that Pb2+ was bound to result in an alpha-amylase conformational change, and the enzyme activity decreased.

[Mechanism of heavy metal ions on RNase activity from bovine pancreas].

This paper studied mechanism of Ce3+, Cd2+, Pb2+ on RNase activity from bovine pancreas. The results showed that the activity of RNase was enhanced under the treatment by Ce3+, Cd2+, Pb2+ at lower concentration (10-60 or 10-30 mumol.L-1), but was inhibited by Ce3+, Cd2+, Pb2+ at higher concentration (40 or 70 mumol.L-1 above), and the inhibition was in the order as Pb2+ > Cd2+ > Ce3+. The equilibrium dialysis demonstrates that RNase may have one Ca(2+)-binding site. The fluorescence titration showed that one molecule of RNase has one binding site for Ce3+, the association constant k for its low-affinity Ce(3+)-binding site is 1.22 x 10(8) L.mol-1. However, it can bind three Cd2+ or Pb2+ and the association causing constant k for its low-affinity Cd2+ or Pb(2+)-binding site is 1.8 x 10(8) L.mol-1, 2.01 x 10(8) L.mol-1, respectively, and caused the conformational changes of RNase.