Antimicrobial resistance, virulence-associated genes, and pulsed-field gel electrophoresis profiles of Salmonella enterica subsp. enterica serovar Typhimurium isolated from piglets with diarrhea in Korea

Salmonella enterica subsp. enterica serovar Typhimurium was isolated from diarrheic piglets in 2 periods, 2000-2001 (n = 25) and 2005-2006 (n = 17). To compare the characteristics of the isolates collected during the 2 periods, all isolates were tested for antimicrobial resistance, the presence of virulence genes, and pulsed-field gel electrophoresis (PFGE) patterns. All 42 isolates were resistant to at least 1 of the 20 antimicrobials tested, and 39 (93%) were resistant to 2 or more antimicrobials. One isolate was resistant to 12 antimicrobials. Profiles of antimicrobial resistance revealed 20 resistance types. Several isolates were also resistant to quinolones and expanded-spectrum cephalosporins. Ten isolates (24%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT); only one isolate had been isolated in 2000-2001, indicating that this type of resistance has rapidly disseminated. Polymerase chain reaction (PCR) assays revealed that all the isolates carried invA. Among the 25 strains isolated in 2000-2001, all carried the sipA, sopA, sopD, sopE2, and ssaR genes, and 96% carried sopB and sifA. Among the 17 strains isolated in 2005-2006, all carried sifA, and approximately 90% carried sipA, sopA, sopB, sopD, sopE2, and ssaR. However, only 6 (14%) of the 42 isolates carried spvC. By PFGE analysis, all 42 strains were classified into 4 major clusters, basically by collection period. The genetic similarity according to PFGE suggests that the strains isolated from diarrheic piglets of this region within the same period may be closely related.     

Molecular epidemiology of rabies virus circulating in South Korea, 1998-2010

We analyzed the nucleotide sequences of the G-L (glycoprotein-large protein) intergenic non-coding region of 33 strains of the rabies virus (RABV) isolated in South Korea in 1998-2010 and compared the sequences with those of previously reported non-Korean strains. The similarities of the nucleotide sequences of the G-L region among all Korean RABV isolates ranged from 97.1 to 100%. Based on the phylogenetic analysis of the G-L region, the Korean RABV isolates were classified into three distinct subgroups with high similarity and were most closely related to the non-Korean NeiMeng1025C isolate, which was isolated from a rabid raccoon dog in eastern China, suggesting that the Korean RABV isolates originate from a rabid raccoon dog in northeastern Asia. Our results indicated that G-L region, as a useful phylogenetic indicator, is equivalent to the nucleoprotein (N) or glycoprotein (G) gene for study of RABV molecular epidemiology and that the Korean RABV isolates showing a few substitutions in the G-L region are continuously circulating in South Korea.     

Mitochondrial dysfunction influences apoptosis and autophagy in porcine parthenotes developing in vitro

Mitochondria are important regulators of both apoptosis and autophagy. One of the triggers for mitochondrial-mediated apoptosis is the production of reactive oxygen species (ROS), which include hydrogen peroxide, superoxide, hydroxyl radical, nitric oxide and peroxynitrite. Recently, several studies have indicated that ROS may also be involved in the induction of autophagy. In the present study, we used H(2)O(2) to induce mitochondrial stress, examined apoptotic- and autophagic-related gene expression and observed LC3 protein (autophagosome presence marker) expression in porcine parthenotes developing in vitro. In porcine four-cell parthenotes cultured for 5 days in NCSU37 medium containing 0.4% BSA, the developmental rate and mitochondrial distribution did not differ from that of the group supplemented with 100 μM H(2)O(2) but was significantly decreased in the group supplemented with 500 μM H(2)O(2) (P<0.05). Transmission electron microscopy (TEM) indicated that whereas normal shaped mitochondria were observed in blastocysts from the control group, abnormal mitochondria (mitophagy) and autophagic vacuoles were observed in blastocysts from the group that received 500 μM H(2)O(2). Furthermore, addition of H(2)O(2) (100 μM and 500 μM) decreased cell numbers (P<0.05) and increased both apoptosis (P<0.05) and LC3 protein expression in the blastocysts. Real-time RT-PCR showed that H(2)O(2) significantly decreased mRNA expression of anti-apoptotic gene Bcl-xL but increased pro-apoptotic genes, Caspase 3 (Casp3) and Bak, and autophagy-related genes, microtubule-associated protein 1 light chain 3 (Map1lc3b) and lysosomal-associated membrane protein 2 (Lamp2). However, the addition of H(2)O(2) had no effect on mRNA expression levels in nuclear DNA-encoded mitochondrial-related genes, cytochrome oxidase (Cox) 5a, Cox5b and Cox6b1, in blastocysts. These results suggest that H(2)O(2) leads to mitochondrial dysfunction that results in apoptosis and autophagy, which is possibly related to porcine early embryo development.     

Immune responses to new vaccine candidates constructed by a live attenuated Salmonella Typhimurium delivery system expressing Escherichia coli F4, F5, F6, F41 and intimin adhesin antigens in a murine model

To construct a novel live oral vaccine candidate for the prevention of pathogenic Escherichia coli infections in neonatal piglets, an expression and secretion plasmid and an attenuated Salmonella delivery system were used. The individual E. coli genes K88ac, K99, FasA, F41 and intimin adhesins were inserted into pBP244 containing asd, lepB, secA and secB, and these plasmids were transformed into a Salmonella Typhimurium ΔcpxR Δlon Δasd. Forty female BALB/c mice were divided into four groups, A to D (ten mice per group). Groups A and B were administered with the mixture containing all constructs and the S. Typhimurium containing pBP244 only as a control, respectively. Groups C and D were primed and boosted with the mixture and the S. Typhimurium harboring pBP244 only, respectively. Each recombinant adhesin secreted from the individual candidates was confirmed by Western blot analysis. The serum IgG and secretory IgA (sIgA) titers to individual adhesins in all immunized groups were higher than those in control. Furthermore, IgG and sIgA levels in group C were higher than those in group A, and the IgG1 titers were increased in Group C but IgG2a titers were similar or decreased in Group C compared to Group A. In addition, the vaccine strains were not detected in fecal samples of any immunized mice. The novel vaccine candidates are not only highly immunogenic, but also safe for vaccinated mice and environment. In addition, the immune responses can be more efficiently induced through the booster-administration.     

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.     

Metabolic dependence of green tea on plucking positions revisited: a metabolomic study

The dependence of global green tea metabolome on plucking positions was investigated through (1)H nuclear magnetic resonance (NMR) analysis coupled with multivariate statistical data set. Pattern recognition methods, such as principal component analysis (PCA) and orthogonal projection on latent structure-discriminant analysis (OPLS-DA), were employed for a finding metabolic discrimination among fresh green tea leaves plucked at different positions from young to old leaves. In addition to clear metabolic discrimination among green tea leaves, elevations in theanine, caffeine, and gallic acid levels but reductions in catechins, such as epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCG), glucose, and sucrose levels were observed, as the green tea plant grows up. On the other hand, the younger the green tea leaf is, the more theanine, caffeine, and gallic acid but the lesser catechins accumlated in the green tea leaf, revealing a reverse assocation between theanine and catechins levels due to incorporaton of theanine into catechins with growing up green tea plant. Moreover, as compared to the tea leaf, the observation of marked high levels of theanine and low levels of catechins in green tea stems exhibited a distinct tea plant metabolism between the tea leaf and the stem. This metabolomic approach highlights taking insight to global metabolic dependence of green tea leaf on plucking position, thereby providing distinct information on green tea production with specific tea quality.     

Simultaneous determination of orysastrobin and its isomers in rice using HPLC-UV and LC-MS/MS

Orysastrobin is a new strobilurin-type fungicide to control leaf and panicle blast and sheath blight in rice. An analytical method was developed to determine the residues of orysastrobin and its two isomers, the main metabolite F001 and the major impurity F033, in hulled rice by the use of high-performance liquid chromatography with ultraviolet photometry (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS/MS). All compounds were extracted with acetone from hulled rice samples. The extract was diluted with saline water, and an extraction step using dichloromethane/n-hexane partition was used to recover analytes from the aqueous phase. An n-hexane/acetonitrile partition and Florisil column chromatography were employed to further remove interfering coextractives prior to instrumental analysis. An octadecylsilyl column was successfully applied to identify orysastrobin and its isomers in sample extracts. Net recovery rates of orysastrobin, F001, and F033 from fortified samples ranged from 80.6 to 114.8% using HPLC-UV and LC-MS/MS. Relative standard deviations for the analytical methods were all <20%, and the quantification limits of the method were in the 0.002-0.02 mg/kg range. The proposed methods were reproducible and sufficiently accurate to evaluate the terminal residue of orysastrobin and its isomers in rice.     

Osthole enhances glucose uptake through activation of AMP-activated protein kinase in skeletal muscle cells

AMP-activated protein kinase (AMPK) is an energy sensor that regulates cellular metabolism. Activation of AMPK in skeletal muscles, the liver, and adipose tissues results in a favorable metabolic milieu for preventing and treating type 2 diabetes, i.e., decreased levels of circulating glucose, plasma lipids, and ectopic fat accumulation and enhanced insulin sensitivity. Osthole was extracted from a Chinese herbal medicine, and we found that it had glucose lowering activity in our previous study. However, the detailed glucose lowering mechanisms of osthole are still unclear. In this study, we used skeletal muscle cells to examine the underlying molecular mechanisms of osthole's glucose lowering activity. A Western blot analysis revealed that osthole significantly induced phosphorylation of AMPK and acetyl-CoA carboxylase (ACC). Next, we found that osthole significantly increased the level of translocation of glucose transporter 4 (GLUT4) to plasma membranes and glucose uptake in a dose-dependent manner. Osthole-induced glucose uptake was reversed by treatment with Compound C, an AMPK inhibitor, suggesting that osthole-induced glucose uptake was mediated in an AMPK-dependent manner. The increase in the AMP:ATP ratio was involved in osthole's activation of AMPK. Finally, we found that osthole counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that the increase in the AMP:ATP ratio by osthole triggered activation of the AMPK signaling pathway and led to increases in plasma membrane GLUT4 content and glucose uptake level. Therefore, osthole might have potential as an antidiabetic agent for treating diabetes.     

Combined genomic-metabolomic approach for the differentiation of geographical origins of natural products: deer antlers as an example

The correct identification of the geographical origin of deer antlers is essential to quality control, as its positive physiological effects correlate with chemical components. In this study, we applied both genomics and metabolomics to the origin-identification of 101 samples from Canada, New Zealand, and Korea. The genomics identified deer species in each country but failed to categorize all the samples, due to the presence of identical species in different countries. For identical species, NMR-based metabolomics gave clean separations, compounds specific to each country were identified, and the validity was confirmed by prediction analysis. As the genomics provided unambiguous read-outs for different species, and the metabolomics cleanly distinguished among identical species from different countries, their combined use could be a robust method for origin-identification even in difficult cases. We believe the method to be generally applicable to many herbal medicinal products for which various species are grown internationally.