Size-Dependent Cytotoxicity and Reactive Oxygen Species of Cerium Oxide Nanoparticles in Human Retinal Pigment Epithelia Cells.

PURPOSE: The use of cerium oxide nanoparticles (CeO2 NPs), a lanthanide element oxide and bivalent compound, has been growing continuously in industry and biomedicine. Due to their wide application, the potential human health problems of CeO2 NPs have attracted attention, but studies on the toxicity of this compound to human eyes are lacking. This study investigated the cytotoxicity and reactive oxygen species (ROS) of CeO2 NPs in human retinal pigment epithelial cells (ARPE-19 cells). METHODS: Using the transmission electron microscope (TEM), the size distribution and shape of CeO2 NPs were characterized. To explore the effect of CeO2 NP size on ophthalmic toxicity in vitro, three sizes (15, 30 and 45 nm) of CeO2 NPs were investigated using ATP content measurement, LDH release measurement and cell proliferation assay in ARPE-19 cells. ROS values and mitochondrial membrane potential depolarization were evaluated by H2DCF-DA staining and JC-1 staining. Morphology changes were detected using a phase-contrast microscope. RESULTS: The cytotoxicity of 15 nm CeO2 NPs was found to be the highest and hence was further explored. Treatment with 15 nm CeO2 NPs caused the morphology of ARPE-19 cells to change in a dose- and time-dependent manner. Moreover, the treatment induced excessive ROS generation and mitochondrial membrane potential depolarization. In addition, cytotoxicity was attenuated by the application of a ROS scavenger N-acetyl-L- cysteine (NAC). CONCLUSION: CeO2 NPs induced cytotoxicity in ARPE-19 cells and excessive production of ROS and decreasing mitochondrial membrane potential. The Overproduction of ROS partially contributes to CeO2 NP-induced cytotoxicity.

Effect of graphene oxide with different morphological characteristics on properties of immobilized enzyme in the covalent method.

Although graphene oxide (GO) has great potential in the field of immobilized enzyme catalysts, the detailed effects of GO with different morphological structures on immobilized enzyme are not well understood. GOs were prepared from 8000 mesh and nanoscale graphite at different reaction temperatures, and used as carriers to immobilize alpha-amylase by cross-linking method. The properties of GOs were characterized through Atomic force microscope, Fourier-transformed infrared, X-ray photoelectron spectroscopy, Raman and UV-Vis. Furthermore, the dosage of cross-linking agent, cross-linking time, optimum temperature/pH, thermal/pH/storage stability, reusability and kinetic parameters of immobilized enzymes were investigated. The results showed that the loading of alpha-amylase on GOs was 162.3-274.2 mg g-1. The reusability experiments revealed high activity maintenance of immobilized alpha-amylase even after seven reaction cycles. Moreover, the storage stability of immobilized enzyme improved via immobilization in comparison with free one and it maintained over 70% of their initial activity after 20 days storage at 4 °C.

Ultraviolet-Visible (UV-Vis) and Fluorescence Spectroscopic Investigation of the Interactions of Ionic Liquids and Catalase.

The inhibitory effects of nine ionic liquids (ILs) on the catalase activity were investigated using fluorescence, absorption ultraviolet-visible spectroscopy. The interactions of ILs and catalase on the molecular level were studied. The experimental results indicated that ILs could inhibit the catalase activity and their inhibitory abilities depended on their chemical structures. Fluorescence experiments showed that hydrogen bonding played an important role in the interaction process. The inhibitory abilities of ILs on catalase activity could be simply described by their hydrophobicity and hydrogen bonding abilities. Unexpected less inhibitory effects of trifluoromethanesulfonate (TfO-) might be ascribed to its larger size, which makes it difficult to go through the substrate channel of catalase to the active site.

Spectroscopic studies on the inhibitory effects of ionic liquids on lipase activity.

The effects of ionic liquids (ILs) on the lipase activity were studied by UV-Vis spectroscopy and the IL-lipase interaction mechanism at the molecular level was investigated by fluorescence technique. Experimental results indicated that the lipase activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. The inhibitory ability of the Cl(-)- and Br(-)-based ILs increased with increasing the alkyl chain length in the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of lipase with the addition of ILs. Both ΔH and ΔS were positive suggesting hydrophobicity was the major driven force for the Cl(-)- and Br(-)-based ILs. For the BF4(-)-, CF3SO3(-)-, ClO4(-)- and N(CN)2(-)-based ILs, hydrogen bonding was the main driven force. For a more comprehensive understanding of the effects of ILs on lipase activity, the roles of hydrophobicity and hydrogen bonding must be considered simultaneously. A regression-based equation was developed to describe the relationship of the inhibitory ability of ILs and their hydrophobicity and hydrogen bonding ability.

Evaluation of the toxicity of ionic liquids on trypsin: A mechanism study.

The toxicity of ionic liquids (ILs) was evaluated by using trypsin as biomarker. Experimental results indicated that the trypsin activity was inhibited by ILs and the degree of inhibition highly depended on the chemical structures of ILs. Primary analysis illustrated that hydrophobicity of ILs was one of the driven forces ruling the ILs-trypsin interaction. Thermodynamic parameters, Gibbs free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of trypsin in the presence of ILs. Both negative ΔH and ΔS suggested hydrogen bonding was the major driven force underlying the IL-trypsin interaction. To assess the toxicity of ILs, it should be considered the combination of the hydrogen bonding ability and hydrophobicity of ILs. A regression based model was established to correlate the relationship of the inhibitory ability, hydrophobicity and hydrogen bonding ability of ILs.

Inhibitory effects of ionic liquids on the lactic dehydrogenase activity.

Ionic liquids (ILs) were widely used in scientific and industrial application and have been reported to possess potential toxicity to the environment and human health. The effects of six typical N-methylimidazolium-based ILs ([Cnmim]X, n=4, 6, 8; X=Br(-), Cl(-), BF4(-), CF3SO3(-)) on the lactic dehydrogenase (LDH) activity and the molecular interaction mechanism of ILs and the LDH were investigated with the aid of spectroscopic techniques. Experimental results showed that the LDH activity was inhibited in the presence of ILs. For the ILs with the same anion but different cations, their inhibitory ability on the LDH activity increased with increasing the alkyl chain length on the IL cation. Thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) were obtained by analyzing the fluorescence behavior of LDH with the addition of ILs. Both positive ΔH and ΔS suggested that hydrophobicity was the major driven force in the interaction process as expected.

Optimization of ultrasound-assisted extraction parameters of chlorophyll from Chlorella vulgaris residue after lipid separation using response surface methodology.

An investigation into ultrasound-assisted extraction (UAE) was conducted for the extraction of chlorophyll from Chlorella vulgaris residue after lipid separation. The best possible combination of extraction parameters was obtained with the response surface methodology (RSM), at a three-variable, three-level experiment Box-Behnken design (BBD). The optimum extraction parameters were as follows: extraction temperature, 61.4 °C, extraction time, 78.7 min, ethanol volume, 79.4 %, at a fixed ultrasonic power of 200 W. Under the modified optimal conditions, the model predicted a total chlorophyll content of 30.1 mg/g. Verification of the optimization showed that chlorophyll extraction of 31.1 ± 1.56 mg/g was observed under the optimal conditions, which well matches with the predicted value. Under these conditions, two stage extraction could sufficiently reach the maximal chlorophyll yield (35.2 mg/g), and the extraction rate reached up to 88.9 %. The present paper provides a feasible technology route for comprehensive utilization of bioactive substances from Chlorella and microalgal biomass biorefinery.

[Molecular analysis of soluble methane monooxygenase and 16S rDNA from a type II methanotroph].

UNLABELLED: The soluble methane monooxygenase (sMMO) from Methylosinus trichosporium IMV 3011 catalyzes the conversion of methane to methanol. OBJECTIVE: To identify the novel species Methylosinus trichosporium IMV 3011 and to describe its evolution status. METHODS: With the aid of the information from GenBank, we designed several sets of primers for PCR amplification and sequencing, the 16S rDNA and complete of genes sequence for soluble methane monooxygenases were gene sequenced and analyzed with biology software. RESULTS: We obtained a 5319 bp of full-length DNA of soluble methane monooxygenases and a 1290 bp of 16S rDNA. Software analysis for six open reading frames and the deduced amino acid sequences of soluble methane monooxygenases has shown that 99.0% to 82.7% identity to the counterpart of Methylosinus trichosporium OB3b, 99.4% to 81.8% identity and 99.8% to 89.2% similarity to the predicted amino acid of mmoX genes in compared five strains. The multiple alignments of MMOX amino acid residues reveal that there is high conservation in MMOX, especially in two Fe binding regions. CONCLUSION: These results indicated that strain IMV 3011 should be a true member of Methylosinus trichosporium, and it is closer to the species Methylosinus trichosporium OB3b.

[Sequence analysis of 16S rDNA and genes of soluble methane monooxygenase from Methylomonas sp. GYJ3].

Soluble methane monooxygenase (MMO) from methanotrophs is a member of binuclear iron-containing multicomponent oxygenases, which can catalyze bioconversion of methane to methanol at ambient temperature and regulate methane recycle in nature. The research focused mainly on the sequence analysis of 16S rDNA and sMMO genes from Methylomonas sp. GYJ3. With the aid of the information from GenBank, the PCR primers and the sequence primers were designed, obtained a 5690bp of sMMO fragment and a 1280bp of 16S rDNA. Sequence comparison for MMOX with counterpart of other five strains showed that from 78% to 99% identity in protein level and from 71 % to 97% identity in gene level, in the separate comparison of six components, only orfY component had a lower identical. The multiple alignment of MMOX amino acid sequence with other four strains showed that there is a high conservation, especially in two Fe binding regions. 16S rDNA phylogenetic analysis demonstrated that Methylomonas sp. GYJ3 is relative with gamma proteobacteria. Phylogenetic analysis of MMOX amino acid sequence showed that Methylomonas sp. GYJ3 is closer to Methylomonas sp. KSW III of type I methanotrophs. It was concluded that Methylomonas sp. GYJ3 is belong to the genus of type I methanotroph Methylomonas, and the result was a direct evidence for the sMMO can be expressed in type I methanotrophs. The theoretical pI of hydroxylase was 6.28 and the theoretical MW of hydroxylase was 248874.41Da.

[Methane monooxygenases hydroxylase from a type II methanotroph: purification and physical-chemical properties].

Methanotrophs can catalyze hydroxylate of methane and some hydrocarbon. Which play an important role in mitigating global warming and have also potential significance for industrial applications or bioremediation. A high activity of hydroxylase, a crucial component in sMMO, from Methylosinus trichosporium IMV 3011 has been purified to homologues by using chromatographic techniques. The molecular weight of the hydroxylase determined by gel filtration is 201.3 kD, and SDS-PAGE showed that hydroxylase consists of three subunits(alpha beta gamma) with molecular weights of 58kD, 36kD and 23kD respectively, drawing a comparison both methods indicated that the hydroxylase is a homodimer with an (alpha beta gamma)2 configuration. Purified hydroxylase has a pI at 5.2 judged by thin layer isoelectric focusing. The purified hydroxylase contains 3.02 mol of iron per mol of protein. The stability pH for the hydroxylase in solution is 5.8-8.0 and the stability temperature is below 35 degrees C. The cells form show a long, bent, and rod-shaped with even surface observed by scanning electron microscopy.