Mutagenicity of 7-ketocholesterol in CHO cells: The role of lipid peroxidation.

As an important cholesterol oxide, 7-ketocholesterol plays a deleterious role in the occurrence of cancer. Although the fact had been proved that 7-ketocholesterol could induce several biological phenomena, including apoptosis, DNA damage, et al., this issue whether 7-ketocholesterol led to mutagenesis in mammalian cells remains largely unexplored. Here, we investigated the major role of lipid peroxidation in the genotoxic response to 7-ketocholesterol in chinese hamster ovary (CHO) cells. The results showed that 7-ketocholesterol induced gene mutation and DNA double-strand breaks (DSBs) in concentration- and time-dependent manner. After CHO cells were treated with 25 µM 7-ketocholesterol for 48 h, the mutation frequency at hprt gene loci and the level of γ-H2AX protein were both significantly increased. Exposure to 7-ketocholesterol resulted in a concentration-dependent increase in the apoptotic rate and the protein expression of cleaved caspase-3 and -7 in CHO cells. Moreover, a significant increase of superoxide dismutase (SOD) activity and content of malondialdehyde (MDA) was also observed. Using a inhibitor of lipid peroxidation (butylated hydroxytoluene), it was found to remarkably inhibit the genotoxicity and MDA levels caused by 7-ketocholesterol. These findings indicated that lipid peroxidation was involved in the mutagenic process of 7-ketocholesterol in CHO cells.

Genotoxicity of microcystin-LR in mammalian cells: Implication from peroxynitrite produced by mitochondria.

Microcystin-LR (MC-LR) is a widely known hepatotoxin which could induce the occurrence and metastasis of hepatocellular carcinoma. In recent years, with the frequent outbreak of cyanobacteria, the harm of MC-LR has gradually attracted more attention. Hence, this study focused on the effect of MC-LR on DNA damage in HepG2 cells, identifying the types and sources of free radicals that make an important function on this issue. Our data suggested that MC-LR induced concentration- and time-dependent increasement of DNA double-strand breaks (DSBs). After exposure to 1 µM MC-LR for 3 days, the protein expression and immunofluorescence staining of γ-H2AX was significantly increased. Using a scavenger of mitochondrial O2.- (4-hydroxy-tempo), a inhibitor of mitochondrial NOS (7-nitroindazole), and a scavenger of ONOO- (uric acid), it was revealed that ONOO- originated from mitochondria made a significant contribution to the genotoxicity of MC-LR. Moreover, a significant decreasement of mitochondrial membrane potential (MMP) was observed. These findings suggested that peroxynitrite targeting mitochondria plays a vital role in the MC-LR-induced genotoxic response in mammalian cells.

[The design and use of a mobile intensive care unit ambulance].

OBJECTIVE: To improve the range of use and efficiency of first-aid in a disaster, a mobile intensive care unit (ICU) ambulance was designed and evaluated. METHODS: The data of disasters, accidental injury in astronauts, injuries occurred in conflicts in foreign nations were reviewed and analyzed, together with our clinical experience of first-aid, a mobile ICU in an ambulance was designed. RESULTS: Provided with automatic cardiopulmonary resuscitation apparatus and instruments for surgical life-saving operation, the mobile ICU ambulance could allow the occupants to perform effective rescue means during rush hour of urban traffic. The new model of ambulance was proved to be able to afford an increased rescue rate in disaster scenario. CONCLUSION: The disability rate and fatality of accidental injuries and local conflicts could be reduced significantly by the use of our mobile ICU ambulance.

[Investigation of fucoidanase by FTIR spectra].

Fucoidanase was isolated and purified from marine fungus LD8 by solid state fermentation, extraction with citric acid buffer, acetone precipitatation and column chromatography on Sephadex G-100. A single band on PAGE shows that pretty pure fucoidanase has been obtained. FT-IR spectra and its derivation, self-deconvolution and curve-fitting methods were used to analyze the secondary structure of the fucoidanase. Composite bands of the amide I and amide III were studied by using Fourier self-deconvolution (FSD) with an enhancement factor of K = 2.2 and a half width of 20.2 cm(-1). The relative average fractions of alpha-helix, beta-sheet, random coil, beta-turn are 11.5%, 58.6%, 14.5% and 15.9%, respectively, according to amide I region, while the content of alpha-helix is 12%, beta-sheet 57.3%, random coil 14.5%, and beta-turn 16.3% on amide III region. In other words, both the conclusions were exactly consistent. All the above results show that beta-sheet was the dominant component, which is about 58%, and that beta-turn is about 15%, random coil 15%, and alpha-helix 12% at room temperature.

The studies of FT-IR and CD spectroscopy on catechol oxidase I from tobacco.

A novel copper-containing enzyme named COI (catechol oxidase I) has been isolated and purified from tobacco by extracting acetone-emerged powder with phosphate buffer, centrifugation at low temperature, ammonium sulfate fractional precipitation, and column chromatography on DEAE-sephadex (A-50), sephadex (G-75), and DEAE-celluse (DE-52). PAGE, SDS-PAGE were used to detect the enzyme purity, and to determine its molecular weight. Then the secondary structures of COI at different pH, different temperatures and different concentrations of guanidine hydrochloride (GdnHCl) were studied by the FT-IR, Fourier self-deconvolution spectra, and circular dichroism (CD). At pH 2.0, the contents of both alpha-helix and anti-parallel beta-sheet decrease, and that of random coil increases, while beta-turn is unchanged compared with the neutral condition (pH 7.0). At pH 11.0, the results indicate that the contents of alpha-helix, anti-parallel beta-sheet and beta-turn decrease, while random coil structure increases. According to the CD measurements, the relative average fractions of alpha-helix, anti-parallel beta-sheet, beta-turn/parallel beta-sheet, aromatic residues and disulfide bond, and random coil/gamma-turn are 41.7%, 16.7%, 23.5%, 11.3%, and 6.8% at pH 7.0, respectively, while 7.2%, 7.7%, 15.2%, 10.7%, 59.2% at pH 2.0, and 20.6%, 9.5%, 15.2%, 10.5%, 44.2% at pH 11.0. Both alpha-helix and random coil decrease with temperature increasing, and anti-parallel beta-sheet increases at the same time. After incubated in 6 mol/L guanidine hydrochloride for 30 min, the fraction of alpha-helix almost disappears (only 1.1% left), while random coil/gamma-turn increases to 81.8%, which coincides well with the results obtained through enzymatic activity experiment.

[Study on the interaction of cystine and polyphenol oxidase from nicotian tobaccum].

The interaction of cystine and polyphenol oxidase (PPO) from nicotian tobaccum has been studied. The results show that cystine has an inhibitory effect on the enzymatic activity, the mechanism of which might be that the thiolether in cystine coordinates with Cu2+ in the active site of PPO, and the inactivation constant value of PPO by cystine is 0.633 min(-1), while the substrates or the products inhibit their combination. Cystine can also be combined with the products of enzymatic reaction to form a colourless compound. Cystine can inhibit the enzymatic activity completely when the molar ratio of cystine to PPO approaches 16,000:1. The effect of cystine on the fluorescence changes with the molar ratio of cystine to the PPO. However, when the molar ratio gets to 75:1, cystine has no longer the effect on either the fluorescence spectra or the synchronous spectra. Microenvironment of trp residue in PPO is more hydrophobic than that of free trp in water.

[Fluorescence study on the interaction of salicylic acid and bovine serum albumin].

The interaction between salicylic acid and bovine serum albumin has been studied by fluorescence spectroscopy. The results show that the quenching mechanism of the combination of bovine serum albumin with salicylic acid is a static quenching procedure, the quenching constant K(sv) is 1.097 x 10(4) (mol x L(-1))(-1), and the equilibrium constant is 7.377 x 10(4). The number of binding sites is 1 and it is a strong one. When the ratio of molar concentration of salicylic acid to bovine serum albumin is lower than 1:1, it binds to Trp residue first but it doesn't result in any microenvironment changes of Trp residue. The binding distance between salicylic acid and bovine serum albumin and the energy transfer efficiency were obtained based on the theory of Förester spectroscopy energy transfer.