Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Expression of KA1 kainate receptor subunit in the substantia gelatinosa of the trigeminal subnucleus caudalis in mice

The KA1 kainate receptor (KAR) subunit in the substantia gelatinosa (SG) of the trigeminal subnucleus caudalis (Vc) has been implicated in the processing of nociceptive information from the orofacial region. This study compared the expression of the KA1 KAR subunit in the SG of the Vc in juvenile, prepubescent and adult mice. RT-PCR, Western blot and immunohistochemistry analyses were used to examine the expression level in SG area. The expression levels of the KA1 KAR subunit mRNA and protein were higher in juvenile mice than in prepubescent or adult mice. Quantitative data revealed that the KA1 KAR subunit mRNA and protein were expressed at levels approximately two and three times higher, respectively, in juvenile mice than in adult mice. A similar expression pattern of the KA1 KAR subunit was observed in an immunohistochemical study that showed higher expression in the juvenile (59%) than those of adult (35%) mice. These results show that the KA1 KAR subunits are expressed in the SG of the Vc in mice and that the expression level of the KA1 KAR subunit decreases gradually with postnatal development. These findings suggest that age-dependent KA1 KAR subunit expression can be a potential mechanism of age-dependent pain perception.     

An Outbreak of Highly Pathogenic Avian Influenza at a Public Animal Exhibit in Seoul,Korea, During 2008

This study describes the first recorded outbreak of HPAI in the city of Seoul, in captive birds held in an exhibition for public viewing at a local district office. The index cases were two pheasants, which had been introduced into the exhibit on 24 April, 4 days prior to death, from a store in a local market in Gyeonggi‐do. Ducks and chickens from an HPAI outbreak farm, subsequently confirmed on 4 May, had also been held in this store. This outbreak highlights the potential role of local markets in AIV transmission. This outbreak led to considerable public health concern in Korea, however, no human cases were reported. The non‐commercial poultry sector needs to be considered in national plans for preparedness and response.     

Induced synthesis of caffeoylserotonin in pepper fruits upon infection by the anthracnose fungus, Colletotrichum gloeosporioides

Caffeoylserotonin (CaS) is a serotonin derivative that belongs to the family of phenylpropanoid amides, but has not previously been identified in plants. In this study, pepper fruits challenged with the anthracnose fungus, Colletotrichum gloeosporioides, were found to synthesize CaS, which accumulated at concentrations of up to 2.3 μg/g fresh weight. The induction of CaS was accompanied by the upregulation of both 4-coumarate-CoA ligase (4CL) and serotonin N-hydroxycinnamoyl transferase (SHT) in unripe pepper fruits. However, no induction of SHT and 4CL was observed in ripe pepper fruits consistent with the lower level of CaS production.     

Comparison of the glucosinolate-myrosinase systems among daikon (Raphanus sativus, Japanese white radish) varieties

Myrosinase is a cytosolic plant enzyme present in daikon ( Raphanus sativus, Japanese white radish) roots that hydrolyzes 4-methylthio-3-butenyl glucosinolate (MTBGLS) into the natural pungent agent 4-methylthio-3-butenyl isothiocyanate (MTBITC), which possesses antimicrobial, antimutagenic, and anticarcinogenic properties. The concentration of MTBGLS, myrosinase activity, and production of MTBITC in seven daikon varieties (one conventional and six heirlooms) were determined to rank the activity of the glucosinolate-myrosinase system and identify critical factors influencing the production of MTBITC. The six heirloom varieties produced 2.0-11.5 times higher levels of MTBITC as compared to the conventional variety, Aokubi, which is consumed by the present Japanese population. The myrosinase was located exclusively in the outer epidermal layer in Aokubi, and MTBGLS was widely distributed throughout the root tissue. Although the skin is a potentially rich source of myrosinase in Aokubi, the skin is usually peeled off in the current practice of preparing daikon for cooking. New practices are therefore proposed for the preparation of daikon tubers that eliminate the peeling of the skin to avoid removing the enzyme needed to convert MTBGLS to the health-beneficial MTBITC. It is also concluded that the consumption of heirloom daikon varieties in addition to changes in food preparation will optimize the health benefits of daikon.     

Inhibition of gap junctional intercellular communication by the green tea polyphenol (-)-epigallocatechin gallate in normal rat liver epithelial cells

(-)-Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, is a promising chemopreventive agent against cancer due to its strong antiproliferative effects on cancer cells; however, its possible toxicity and carcinogenicity must be investigated before EGCG can be used as a dietary supplement for chemoprevention. The inhibition of gap junctional intercellular communication (GJIC) is strongly associated with carcinogenesis, particularly the tumor promotion process; thus, we investigated the effects of EGCG on GJIC in WB-F344 normal rat liver epithelial (RLE) cells. EGCG, but not (-)-epicatechin (EC), another polyphenol found in green tea, inhibited GJIC in a dose-dependent and reversible manner in RLE cells. EGCG also induced the phosphorylation of connexin 43 (Cx43), a major regulator of GJIC. The phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) was also observed in EGCG-treated RLE cells. The inhibition of GJIC and phosphorylation of Cx43 and ERK1/2 by EGCG were completely blocked by U0126, a pharmacological inhibitor of mitogen-activated protein kinase/ERK kinase. EGCG generated a larger amount of hydrogen peroxide than EC in a dose-dependent manner. Furthermore, catalase partially inhibited the EGCG-induced inhibition of GJIC and the phosphorylation of Cx43 and ERK1/2. These results indicated that EGCG inhibited GJIC mainly due to its prooxidant activity.     

Sesquiterpenoids isolated from the flower buds of Tussilago farfara L. inhibit diacylglycerol acyltransferase

Inhibition of acyl CoA:diacylglycerol acyltransferase (DGAT), which is a key enzyme in triglyceride synthesis in eukaryotic organisms, has been proposed as one of the drug targets for treating obesity, type II diabetes mellitus, and metabolic syndrome. Bioassay-guided fractionation of EtOH extract of the flower buds of Tussilago farfara , using an in vitro DGAT enzyme assay, resulted in the isolation of four known sesquiterpenoids, tussilagonone (1), tussilagone (2), 7beta-(3-ethyl-cis-crotonoyloxy)-1alpha-(2-methylbutyryloxy)-3,14-dehydro-Z-notonipetranone (3), and 8-angeloylxy-3,4-epoxy-bisabola-7(14),10-dien-2-one (4). DGAT1 inhibitory activity was studied by in vitro DGAT assay using rat liver microsomes and HepG2 cell microsomes. They showed DGAT1 inhibition with IC(50) values of 99.2 (1), 18.8 (2), 47.0 (3), and 211.1 (4) microM (for rat liver microsomes) and >1 mM (1), 49.1 (2), 160.7 (3), and 294.4 (4) microM (for HepG2 cell microsomes), respectively. Compound 2 showed the most potent inhibition against microsomal DGAT1 derived from rat liver and human hepatocellular carcinoma HepG2 cells and also significantly inhibited triglyceride synthesis by suppressing incorporation of [(14)C]acetate or [(14)C]glycerol into triglycerides in HepG2 cells. These findings suggest that tussilagone is a potential lead compound in the treatment of obesity and type 2 diabetes.     

红壤稻田施用控释肥与氮素转化的关系

在湘南红壤稻田连续两年(2002-2003)采用田间试验与室内分析相结合的研究方法,对施用控释肥和尿素的稻田肥料氮转化与去向进行研究.结果表明,稻田氨挥发是氮素损失的主要原因.施用尿素,其氨挥发损失量达到施氮素总量的39.28%,施用控释肥(LCU70、LCU50),其氨挥发损失量分别占其施氮总量的19.99%和10.91%,比尿素的氨挥发氮素损失量降低19.29%～28.37%.田间表面水NH4-N浓度高峰期出现在施肥后的第1天,10d后下降到对照水平.田间氨挥发高峰期出现在施肥后的第3天,7d后下降到对照水平.施用控释肥(LCU70、LCUS0),水稻对氮素的吸收利用率分别为80.5%、64.7%,比施尿素高50.5%、34.7%,差异极显著.