A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.     

Effects of combined administration of calcium,iron, zinc,chrysanthemum flavonoids,and DMSA on the treatment of lead intoxication in mice

The effect of combined administration of calcium (Ca), iron (Fe), zinc (Zn), chrysanthemum flavonoids, and meso‐2,3‐dimercaptosuccinic acid (DMSA) on the treatment of lead (Pb) intoxication in mice was studied. One hundred ninety female mice (SPF level, aged 18‐22 days) were randomly divided into two groups as experimental animals. Mice in group I (10 mice) served as normal control animals, and were administered deionized water containing 12.5 μL/L acetate acid for 6 weeks, whereas mice in group II (180 mice) were exposed to 0.1% (wt/vol) of lead acetate in deionized water for 6 weeks and served as experimental animals. After 6 weeks of successful modeling, 180 mice from group II (lead‐exposed) were divided into 18 groups of 10 mice each, 16 of which were treated by the combined administration of Ca, Fe, Zn, chrysanthemum flavonoids, and DMSA by L₁₆ (2¹⁵) orthogonal design. The remaining two groups were given treatment with low and high doses of DMSA, respectively. After three weeks of intervention (ig), the optimal treatment group was identified according to its blood lead level, as well as some antioxidant indices in the blood, liver, and hippocampus. The results indicated that the combined administration of Fe, Zn, chrysanthemum flavonoids, and DMSA with low dosage had the most significant effect on increasing the activities of blood delta‐aminolevulinic acid dehydratase and superoxide dismutase (SOD), hepatic SOD and hippocampus nitric oxide synthase while decreasing the blood lead level, the content of hepatic malondialdehyde and hippocampus nitric oxide; this was considered the optimal treatment group. There was no difference in the level of blood hemoglobin between the optimal treatment group and the model control group (the first group of the orthogonal experiment). The activities of blood glutathione (GSH), hepatic GSH and glutathione peroxidase of the optimal treatment group were the same as other groups’, and the recovery of the related indexes in the optimal effect group closely resembled the high dosage DMSA group. It can be concluded that the coadministration of Fe, Zn, and chrysanthemum flavonoids along with a low‐dose DMSA effectively reduces Pb poisoning and lead‐induced oxidative damage in lead‐exposed mice; the result may provide a theoretical reference for the treatment of Pb poisoning.     

Direct Modulation of Protein Level in Arabidopsis

Dear Editor, The general way to probe functions of a protein in vivo is to perturb its level and then observe subsequent phe- notypic changes. In plants, modulation of protein level is mainly carried out at DNA or RNA level, which is indirect and thus affected by stability of the target protein. Thus, experimental approaches to perturb protein level directly are needed, but still limited in plants. In mammalian cells, a technique to modulate protein level directly has been developed. Engineered destabilizing domain （DD） of the human FKBP12 protein can confer instability to other pro- teins when fused to it. A small synthetic molecule ligand Shield 1 （Shldl） can bind DD and shield it from degrada- tion. The level of DD fused protein can be controlled by adjusting Shldl concentration （Banaszynski et al., 2006）.     

Subnuclear distribution of SSX regulates its function

SSX, a family of genes clustered on the X chromosome, has been identified as a cancer–testis antigen and also forms a part of the SYT–SSX fusion gene found in synovial sarcoma, implying that it has an important role in tumorigenesis. However, knowledge of the molecular regulation of SSX is still limited. In this study, we demonstrate that SSX or its SYT fusion protein is distributed as nuclear speckles, in which it is co-localized with B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi1), which is a core factor of polycomb repressor complex 1. The C-terminal residues of SSX are indispensable for the nuclear speckle distribution, while the N-terminal domain is necessary for the recruitment of Bmi1, indicating that intact SSX must be needed for interaction with Bmi1 both spatially and functionally. In addition, the N-terminus of SSX also proved to contain an intrinsic nucleolar localization signal, which mediates the nucleolar translocation of SSX in particular kinds of cell stress such as the oxidation of hydrogen peroxide or heat shock. This stress-induced translocation is reversible and accompanied by HSP 70 or p14ARF traffic, suggesting that SSX is a stress response gene. It is of note that nucleolar translocation of SSX can result in disassociation of SSX from Bmi1, with consequent down-regulation of Bmi1 activity. These novel findings regarding distinct domains of SSX and its interaction with Bmi1 may shed light on the mechanism by which synovial sarcoma develops and on the up-regulation of SSX in cancer cells.     

Performance of heterogeneous ZrO2 supported metaloxide catalysts for brown grease esterification and sulfur removal

In order to achieve a viable biodiesel industry, new catalyst technology is needed which can process a variety of less expensive waste oils, such as yellow grease and brown grease. However, for these catalysts to be effective for biodiesel production using these feedstocks, they must be able to tolerate higher concentrations of free fatty acids (FFA), water, and sulfur. We have developed a class of zirconia supported metaloxide catalysts that achieve high FAME yields through esterification of FFAs while simultaneously performing desulfurization and de-metallization functions. In fact, methanolysis, with the zirconia supported catalysts, was more effective for desulfurization than an acid washing process. In addition, using zirconia supported catalysts to convert waste grease, high in sulfur content, resulted in a FAME product that could meet the in-use ASTM diesel fuel sulfur specification (<500 ppm). Possible mechanisms of desulfurization and de-metallization by methanolysis were proposed to explain this activity.     

Chronic Stress Suppresses the Expression of Cutaneous Hypothalamic–Pituitary–Adrenocortical Axis Elements and Melanogenesis

Chronic stress can affect skin function, and some skin diseases might be triggered or aggravated by stress. Stress can activate the central hypothalamic–pituitary–adrenocortical (HPA) axis, which causes glucocorticoid levels to increase. The skin has HPA axis elements that react to environmental stressors to regulate skin functions, such as melanogenesis. This study explores the mechanism whereby chronic stress affects skin pigmentation, focusing on the HPA axis, and investigates the role of glucocorticoids in this pathway. We exposed C57BL/6 male mice to two types of chronic stress, chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS). Mice subjected to either stress condition showed reduced melanogenesis. Interestingly, CRS and CUMS triggered reductions in the mRNA expression levels of key factors involved in the HPA axis in the skin. In mice administered corticosterone, decreased melanin synthesis and reduced expression of HPA axis elements were observed. The reduced expression of HPA axis elements and melanogenesis in the skin of stressed mice were reversed by RU486 (a glucocorticoid receptor antagonist) treatment. Glucocorticoids had no significant inhibitory effect on melanogenesis in vitro. These results suggest that, high levels of serum corticosterone induced by chronic stress can reduce the expression of elements of the skin HPA axis by glucocorticoid-dependent negative feedback. These activities can eventually result in decreased skin pigmentation. Our findings raise the possibility that chronic stress could be a risk factor for depigmentation by disrupting the cutaneous HPA axis and should prompt dermatologists to exercise more caution when using glucocorticoids for treatment.     

NFKB1 insertion/deletion promoter polymorphism increases the risk of advanced ovarian cancer in a Chinese population

Ovarian cancer is the leading cause of death among all gynecological cancers. This is mainly attributed to its frequent presentation at an advanced stage (International Federation of Gynecology and Obstetrics stage III-IV). Nuclear factor-kappaB (NF-κB) is critically involved in the carcinogenesis and development of ovarian cancer. A functional insertion/deletion polymorphism (-94 ins/del ATTG) in the promoter region of the NFKB1 gene, which encodes the p50 subunit of the NF-κB protein, has been recently identified and shown to increase the susceptibility to many diseases. The purpose of this study was to explore the association between this polymorphism and the risk of advanced ovarian cancer in a Chinese population. A total of 179 advanced ovarian cancer patients and 223 healthy controls were recruited into this study. Genotypes were determined using polymerase chain reaction-capillary electrophoresis method. The insertion increased the risk of advanced ovarian cancer (odds ratio?=?2.111, 95% confidence intervals?=?1.125-3.961, p?=?0.019 for heterozygote insertion, and odds ratio?=?2.656, 95% confidence intervals?=?1.397-5.051, p?=?0.002 for homozygote insertion) compared with homozygote deletion. Similar results were seen in age-adjusted analyses (p?相似文献   

Identification and characterization of paramyosin from cyst wall of metacercariae implicated protective efficacy against Clonorchis sinensis infection

Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.