Role of the CPC sequence in the antioxidant activity of GcGAST protein in E.coli.

Gibberellic acid stimulated transcriptional protein from Gymnadenia conopsea (GcGAST) is a novel member of GA-induced cysteine-rich protein family, which shared 12 highly conserved cysteine residues with other members in C-terminal domain. In the present paper, the recombinant plasmid, as well as two mutants Serine-Proline-Cysteine (SPC) and Cysteine-Proline-Serine (CPS), were constructed to investigate for the first time the effects of the cysteines in Cysteine-Proline-Cysteine (CPC) sequence on the antioxidant activity of GcGAST protein. It was found that E.coli expressing wt GcGAST exhibited significant resistance against exogenous H(2)O(2). Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to H(2)O(2). Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate H(2)O(2), whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of H(2)O(2)-eliminating ability. Fourier transform Infrared spectroscopy analysis indicated that the IB of CPS mutant contained more ß-sheet secondary structure than wt and SPC mutant. Non-reducing SDS-PAGE combined western-blotting analysis revealed that the disulfide bonds were important for the formation of IBs of wt GcGAST and SPC mutant, whereas non-reducing SDS-PAGE of resolubilized IBs showed that hydrophobic interaction favored the aggregation of IBs in CPS mutant. Taken together, these results suggested that GcGAST possessed antioxidant activity in the level of IB, which made some contribution to cellular resistance to H(2)O(2). More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.

[Application of polarization fluorescence to the study on the effects of oxidative stress on wheat chloroplast].

In the present paper wheat flag leaves were collected during the tasseling period, and then 1 mmol x L(-1) hydrogen peroxide was added to induce oxidative stress on leaves. In comparison, the detached leaves were also kept under drought or darkness condition for 24 h for the same purpose. Following the preparation of chloroplasts, polarization fluorescence spectroscopic method was utilized to measure fluorescence emission spectra and fluorescence excitation spectra of chloroplasts in the case of VV, VH, HV and HH, where V and H is representative of vertical polarization and horizontal polarization, respectively. Gaussian deconvolution was done on emission spectra, and the fitting data revealed that no matter whether Chla or Chlb molecules were excited upon excitation at 436 nm or 475 nm, the ratio of fluorescence peak area at 684 nm and 720 nm, i. e. A684/ A720, tends to increase slightly after oxidative stress. In addition, some useful information was available from polarization excitation spectra, where it was observed that the treatment of oxidative stress gave rise to higher ratio of excitation peak intensity between 436 nm and 475 nm (E436/E475), indicating that Chla made more contribution to PSII fluorescence emission than Chlb did. Simultaneously, the ratio of 475 nm and 600 nm (E475/E600), representing the energy transfer efficiency from Car to Chlb, was also found to be higher after the detached leaves were treated. In addition, both fluorescence polarization and viscosity were calculated in this paper, and the data showed that oxidative stress should be responsible for higher fluorescence polarization at 680 nm and higher viscosity in microenviroment. The above-mentioned phenomenon is consistent with the lipid peroxidation of unsaturated fatty acids. It also provides a simple and feasible method to study oxidative stress.

Diaqua-bis(5-carb-oxy-2-methyl-1H-imidazole-4-carboxyl-ato-κN,O)manganese(II).

The title complex, [Mn(C(6)H(5)N(2)O(4))(2)(H(2)O)(2)], was obtained by hydro-thermal synthesis. The Mn(II) atom, which lies on an inversion centre, displays a slightly distorted octa-hedral geometry. In the crystal packing, complex mol-ecules are linked by inter-molecular O-H⋯O and N-H⋯O hydrogen bonds to form a three-dimensional supramolecular structure. The title complex is isostructural with the corresponding cadmium(II) complex [Nie, Wen, Wu, Liu & Liu (2007 ▶). Acta Cryst. E63, m753-m755].