RP-HPLC法制备银杏酸的研究

目的 研究5种银杏酸单体的RP—HPLC制备分离条件。方法 将银杏外种皮的环己烷提取物经过预处理，采用HPLC-MS鉴定，RPHPLC分离、制备银杏酸单体。结果 该制备方法所得的5种银杏酸单体纯度超过98％。结论 通过HPLC—MS检测确定银杏总酚酸不同物质的出峰位置，在制备柱上可以获得多种银杏酸单体，分离产物达到该类产品检测用对照物质的质量要求。     

Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation

Aim:

Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro.

Methods:

HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells.

Results:

Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 μg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells.

Conclusion:

The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.     

Mitochondrial dysfunction resulting from loss of cytochrome c impairs radiation-induced bystander effect

Cytochrome c is a pivotal protein that resides in mitochondria as component of mitochondria respiration and apoptosis initiator. Using murine cells lacking cytochrome c, we showed here that cytochrome c-deficient cells had attenuated reactive oxygen species/nitric oxide and micronuclei induction to radiation-induced bystander signals, indicating cytochrome c is essential for the bystander effect.     

Intracellular chromosome breaks on silicon surface

The genotoxicity of silicon (Si) is investigated by soaking crystalline Si in a complete culture medium for 60 days and conducting micronuclei tests (MNTs) utilizing hamster ovary (CHO) cells and its Ku80 deficient CHO mutant (xrs5) cells (DNA double-strand breaks repair deficiency). The intracellular concentrations of reactive oxygen/nitrogen species (ROS/RNS) on Si are determined to elucidate the relationship between ROS/RNS and Si-induced genotoxicity by using CHO cells. The cells are treated with ROS scavenger (dimethyl sulfoxide) and MNT are performed. The results indicate that the intracellular concentration of ROS and nitrogen oxide (NO) on Si is higher than those on the control group by about 38% and 12%. ROS/RNS include superoxide (O₂^?) anion, NO, and peroxynitrite (ONOO^?) which can injure chromosomes and induce high cellular DNA double-strand breaks (DSBs).     

The oxidative stress response of myclobutanil and cyproconazole on Tetrahymena thermophila

Using Tetrahymena thermophila as experimental models, the oxidative stress of triazole fungicides myclobutanil (MYC) and cyproconazole (CYP) was investigated. Results showed that 24-h EC₅₀ values for MYC and CYP were 16.67 (13.37–19.65) and 20.44 (18.85–21.96) mg/L, respectively; 48-h EC₅₀ values for MYC and CYP were 14.31 (13.13–15.42) and 18.76 (17.09–20.31) mg/L, respectively. Reactive oxygen species was significantly induced and cytotoxicity was caused by MYC and CYP by increasing propidium iodide (PI) fluorescence. Damage of regular wrinkles and appearing of small holes on the cell surface were observed by SEM. Furthermore, MYC and CYP also caused notable changes in enzyme activities and mRNA levels. Overall, the present study points out that MYC and CYP lead to oxidative stress on T. thermophila. The information presented in this study will provide insights into the mechanism of triazoles-induced oxidative stress on T. thermophila.     

Arsenite-induced germline apoptosis through a MAPK-dependent, p53-independent pathway in Caenorhabditis elegans

Many studies have shown that arsenite is a potent inducer of apoptosis both in cells and tissues. However, there is a lack of appropriate in vivo animal models to study the underlying mechanisms of arsenite-induced apoptosis. Caenorhabditis elegans is an excellent model organism for studying many biological processes. We showed previously that C. elegans could be used as an in vivo system to investigate the genotoxic effects of arsenite. In order to elucidate the underlying mechanisms of arsenite-induced apoptosis in vivo, in the present study, we used the mutated alleles of the C. elegans homologue of known mammalian genes that are involved in the regulation of apoptosis. Our results showed that the loss-of-function mutations of p53/ cep-1 and DNA damage response (DDR) genes hus-1, clk-2, and egl-1 exhibited significant increase in germline apoptosis under arsenite exposure, whereas arsenite-induced germline apoptosis was blocked in loss-of-function alleles of extracellular signal-regulated kinase (ERK) (lin-45 (ku51), mek-2 (n1989), and mpk-1 (ku1)), c-Jun N-terminal kinase (JNK) (jkk-1 (km2), mek-1 (ks54), jnk-1 (gk7), mkk-4 (ju91)), and p38 ( nsy-1 (ag3), sek-1 (ag1), and pmk-1 (km25)) MAPK cascades. These results suggest that arsenite-induced apoptosis occurs independently of p53/ cep-1 and the DNA damage response (DDR) genes hus-1, clk-2, and egl-1 and that the C. elegans caspase gene ced-3, Apaf-1 homologue ced-4, and the MAPK signaling pathways are essential for germline apoptosis. Moreover, our study demonstrates that C. elegans could be a mammalian in vivo substitute model to study the mechanisms of apoptosis.     

Low-aspect ratio nanopatterns on bioinert alumina influence the response and morphology of osteoblast-like cells

Topographical features on the nanometer scale are known to influence cellular behavior. The response of specific cell types to various types of surface structures is currently still being investigated. Alumina ceramics play an important role as biomaterials, e.g., in medical and dental applications. In this study, we investigated the influence of nanoscale surface features with low aspect ratio (< 0.1) on the response of osteoblast-like MG-63 cells. To this end, low-energy ion irradiation was employed to produce shallow nanoscale ripple patterns on Al₂O₃(0001) surfaces with lateral periodicities of 24 nm and 179 nm and heights of only 0.7 and 11.5 nm, respectively. The nanopatterning was found to increase the proliferation of MG-63 cells and may lead to pseudopodia alignment along the ripples. Furthermore, focal adhesion behavior and cell morphology were analyzed. We found that MG-63 cells are able to recognize surface nanopatterns with extremely low vertical variations of less than 1 nm. In conclusion, it is shown that surface topography in the sub-nm range significantly influences the response of osteoblast-like cells.     

Escherichia coli mutants induced by multi-ion irradiation

Wild-type Escherichia coli K12 strain W3110 was irradiated by 10 keV nitrogen ions. Specifically, irradiation was performed six times by N⁺ ions, followed by the selection of lac constitutive mutants, and each time a stable S55 mutant was produced. By sequencing the whole genome, the fine map of S55 was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels), and nine structural variations (SVs) were found in the S55 genome. Among the 18 SNPs, 11 are transversional from A, T or C to G, accounting for 55.6% of point mutations. GCCA insertion occurs in the target gene lacI. Four SNPs, including three in rlpB and one in ygbN, are connected with cell envelope and transport. All nine structural variations of S55 are deletions and contain insertion sequence (IS) elements. Six deleted SVs contain disrupted ISs, nonfunctional pseudogenes, and one more 23 252 bp SV in the Rac prophage region. Overall, our results show that deletion bias observed in E. coli K12 genome evolution is generally related to the deletion of some nonfunctional regions. Furthermore, since ISs are unstable factors in a genome, the multi-ion irradiations that caused these deleted fragments in S55 turn out to be beneficial to genome stability, generating a wider mutational spectrum. Thus, it is possible that the mutation of these genes increases the ability of the E. coli genome to resist etch and damage caused by ion irradiation.