Varied patterns of DNA methylation change between different satellite regions in bovine preimplantation development

Global reduction of DNA methylation, a part of genome reprogramming processes, occurs in a gradual manner until before implantation and is recognized as a conserved process in mammals. Here, we reported that in bovine, satellite regions exhibited varied patterns of methylation changes when one-cell egg advanced to the blastocyst; a maintenance methylation was observed in satellite I sequences, a decrease in alpha satellites, and an increase in satellite II regions. Cloned embryos exhibited similar changes for DNA methylation in the satellite I and alpha. We also observed that the satellite I and alpha sequences were methylated more in inner cell mass region of the blastocyst whereas the satellite II showed selective demethylation in this region. Together, these findings point that individual satellite sequences carry their own methylation patterns under the pressure of global demethylation, suggesting that local methylation control system acts on the satellite regions in early bovine embryos.     

Lactobacillus acidophilus expressing recombinant K99 adhesive fimbriae has an inhibitory effect on adhesion of enterotoxigenic Escherichia coli

The most common enteric colibacillosis in neonatal and newborns is caused by enterotoxigenic Escherichia coli(ETEC). Colonization of ETEC in the small intestine is associated with adhesions using fimbriae, which is known as a specific adhesion factor and provides highly specific means for anchoring and prerequisite for an infectious agent. In the present study we have engineered Lactobacillus acidophilus to produce recombinant K99 fimbriae, which is used for the colonization to the intestine of pigs. The expression of K99 fimbrial protein was confirmed using SDS-PAGE, immunoblot and agglutination analyses. To evaluate a function of the K99 fimbrial protein, inhibition and competition tests were performed on pre-screened intestinal brush border from pigs. The tests showed that recombinant L. acidophilus, not control L. acidophilus, had a significant inhibitory effect to and competition against K99+ E. coli in a dose dependent manner. In conclusion, we demonstrated that recombinant K99 fimbriae producing L. acidophilus was able to prevent E. coli binding to intestinal brush border.     

ERK/ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expression through co-activation of CHOP and Elk1

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers apoptosis upon binding to its ligand or when overexpressed. Its expression is induced by certain small molecule drugs, including celecoxib, through mechanisms that have not been fully elucidated. The current study has revealed a novel ERK/ribosomal S6 kinase (RSK)-dependent mechanism that regulates DR5 expression primarily using celecoxib as a DR5 inducer. Both C/EBP homologous protein (CHOP) and Elk1 are required for celecoxib-induced DR5 expression based on promoter deletion and mutation analysis and siRNA-mediated gene silencing results. Co-expression of both CHOP and Elk1 exhibited enhanced effects on increasing DR5 promoter activity and DR5 expression, indicating that CHOP and Elk1 co-operatively regulate DR5 expression. Because Elk1 is an ERK-regulated protein, we accordingly found that celecoxib increased the levels of phosphorylated ERK1/2, RSK2, and Elk1. Inhibition of either ERK signaling with a MEK inhibitor or ERK1/2 siRNA, or RSK2 signaling with an RSK2 inhibitor or RSK2 siRNA abrogated DR5 up-regulation by celecoxib as well as other agents. Moreover, these inhibitions suppressed celecoxib-induced CHOP up-regulation. Thus, ERK/RSK-dependent, CHOP and Elk1-mediated mechanisms are critical for DR5 induction. Additionally, celecoxib increased CHOP promoter activity in an ATF4-dependent manner, and siRNA-mediated blockade of ATF4 abrogated both CHOP induction and DR5 up-regulation, indicating that ATF4 is involved in celecoxib-induced CHOP and DR5 expression. Collectively, we conclude that small molecules such as celecoxib induce DR5 expression through activating ERK/RSK signaling and subsequent Elk1 activation and ATF4-dependent CHOP induction.     

四种中草药对大鼠半乳糖性白内障氧化还原物质及糖类含量的影响

对正常和半乳糖性白内障及给中草药的大鼠晶状体中某些吡啶核苷酸成分、糖类、非蛋白质巯基的含量进行了比较。结果表明,在白内障晶状体中,NADPH及非蛋白质巯基的含量明显低于正常晶状体的,而NADP、半乳糖及半乳糖醇的含量明显高于正常晶状体的;当注射半乳糖的同时分别用黄岑、石斛、菟丝子及玉蝴蝶四种中草药水煎剂灌胃,上述变化为基本恢复至正常晶状体的水平。表明四种中草药对晶状体中的异常生化变化具有阻止及纠正作用。     

Enzymatic activity of glucose oxidase covalently wired via viologen to electrically conductive polypyrrole films

The surface functionalization of an electrically conductive polypyrrole film (PPY) with a viologen, (N-(2-carboxyl-ethyl)-N'-(4-vinyl-benzyl)-4,4'-bipyridinium dichloride, or CVV) for the covalent immobilization of glucose oxidase (GOD) has been carried out. The viologen was first synthesized and graft polymerized on PPY film. It then served as an anchor via its carboxyl groups for the covalent immobilization of GOD. The surface composition of the as-functionalized substrates was characterized by X-ray photoelectron spectroscopy (XPS). The effects of the CVV monomer concentration on the CVV-graft polymer concentration and the amount of GOD immobilized on the surface were investigated. The activity of the immobilized GOD was compared with that of free GOD and the kinetic effects were also obtained. The cyclic voltammetric (CV) response of the GOD-functionalized PPY substrates was studied in a phosphate buffer solution under an argon atmosphere. The CV results support the mechanism in which CVV acts as a mediator to transfer electron between the electrode and enzyme, and hence regenerating the enzyme in the enzymatic reaction with glucose. High sensitivity and linear response of the enzyme electrode was observed with glucose concentration ranging from 0 to 20 mM.     

Non–Laboratory-Based Self-Assessment Screening Score for Non-Alcoholic Fatty Liver Disease: Development,Validation and Comparison with Other Scores

Background

Non-alcoholic fatty liver disease (NAFLD) is a prevalent and rapidly increasing disease worldwide; however, no widely accepted screening models to assess the risk of NAFLD are available. Therefore, we aimed to develop and validate a self-assessment score for NAFLD in the general population using two independent cohorts.

Methods

The development cohort comprised 15676 subjects (8313 males and 7363 females) who visited the National Health Insurance Service Ilsan Hospital in Korea in 2008–2010. Anthropometric, clinical, and laboratory data were examined during regular health check-ups and fatty liver diagnosed by abdominal ultrasound. Logistic regression analysis was conducted to determine predictors of prevalent NAFLD and to derive risk scores/models. We validated our models and compared them with other existing methods using an external cohort (N = 66868).

Results

The simple self-assessment score consists of age, sex, waist circumference, body mass index, history of diabetes and dyslipidemia, alcohol intake, physical activity and menopause status, which are independently associated with NAFLD, and has a value of 0–15. A cut-off point of ≥8 defined 58% of males and 36% of females as being at high-risk of NAFLD, and yielded a sensitivity of 80% in men (77% in women), a specificity of 67% (81%), a positive predictive value of 72% (63%), a negative predictive value of 76% (89%) and an AUC of 0.82 (0.88). Comparable results were obtained using the validation dataset. The comprehensive NAFLD score, which includes additional laboratory parameters, has enhanced discrimination ability, with an AUC of 0.86 for males and 0.91 for females. Both simple and comprehensive NAFLD scores were significantly increased in subjects with higher fatty liver grades or severity of liver conditions (e.g., simple steatosis, steatohepatitis).

Conclusions

The new non–laboratory-based self-assessment score may be useful for identifying individuals at high-risk of NAFLD. Further studies are warranted to evaluate the utility and feasibility of the scores in various settings.     

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.     

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.