Evaluation of recombinant 28 kDa outer membrane protein of Brucella abortus for the clinical diagnosis of bovine brucellosis in Korea

Brucella spp. are Gram-negative, facultative, intracellular coccobacilli that are pathogenic to a variety of mammals, including ruminants and humans. The conventional serological test for diagnosing brucellosis in cattle in Korea is the standard tube agglutination test. However, agglutination tests sometimes give false-positive results due to cross-reactions with other pathogens. The outer membrane proteins of Brucella species have been extensively studied for their immunogenicity and serodiagnostic applications. However, an application of B. abortus OMPs for serodiagnosis has not been successfully established. In this study, cloning and expression of B. abortus Omp28, a group 3 antigen, were accomplished by PCR amplification cloning into a pMAL expression system, and purification of a recombinant Omp28 (rOmp28). The immunogenicity of rOmp28 was confirmed by Western blot analysis with Brucella-positive bovine serum. To determine whether rOmp2 has a potential benefit for use in the serodiagnosis of bovine brucellosis, rOmp28-based ELISA and latex bead agglutination test were performed. B. abortus positive (n=122) or negative (n=88) from TAT were positive (118/122, 96.7%) or negative (84/88, 95.4%) in ELISA and were positive (94/122, 77%) or negative (71/88, 81.7%) in that the latex bead agglutination test, respectively. The sensitivity, specificity and accuracy were 96.7, 95.4, 96.2% in ELISA and 77, 80.6, 78.5% in latex bead agglutination test, respectively. These findings suggest that the rOmp28 of B. abortus might be a good candidate for developing serological diagnostic tools for bovine brucellosis.     

Phenotypic and genetic characterization of Erwinia rhapontici isolated from diseased Asian pear fruit trees

In 1995, a serious necrotic disease appeared in Asian pear trees in the orchards of Chuncheon, South Korea. Large numbers of bacterial samples were collected, and the causative microorganism was identified as a novel pathogen, Erwinia pyrifoliae. Among the samples, four bacterial isolates with atypical characteristics were further analyzed through biochemical tests and 16S rRNA gene sequence analysis. By phenotypic and genetic analysis these isolates were identified as E. rhapontici. Phylogenetic analysis using 16S rRNA sequence data revealed that E. rhapontici forms a discrete clade with high bootstrap value close to the E. amylovora group. Pathogenicity tests on leaves of tobacco plants (Nicotiana tabacum) elicited hypersensitivity responses, but artificial inoculations on immature fruits and shoots of Asian pear (Pyrus pyrifolia) did not show any necrotic symptoms. The developed primers showed no cross reactivity when tested against other phytopathogens and were able to detect E. rhapontici from mixed culture and in planta.     

Antarctic Marine Algae Extracts as a Potential Natural Resource to Protect Epithelial Barrier Integrity

The intestine and skin provide crucial protection against the external environment. Strengthening the epithelial barrier function of these organs is critical for maintaining homeostasis against inflammatory stimuli. Recent studies suggest that polar marine algae are a promising bioactive resource because of their adaptation to extreme environments. To investigate the bioactive properties of polar marine algae on epithelial cells of the intestine and skin, we created extracts of the Antarctic macroalgae Himantothallus grandifolius, Plocamium cartilagineum, Phaeurus antarcticus, and Kallymenia antarctica, analyzed the compound profiles of the extracts using gas chromatography-mass spectrometry, and tested the protective activities of the extracts on human intestinal and keratinocyte cell lines by measuring cell viability and reactive oxygen species scavenging. In addition, we assessed immune responses modulated by the extracts by real-time polymerase chain reaction, and we monitored the barrier-protective activities of the extracts on intestinal and keratinocyte cell lines by measuring transepithelial electrical resistance and fluorescence-labeled dextran flux, respectively. We identified bioactive compounds, including several fatty acids and lipid compounds, in the extracts, and found that the extracts perform antioxidant activities that remove intracellular reactive oxygen species and scavenge specific radicals. Furthermore, the Antarctic marine algae extracts increased cell viability, protected cells against inflammatory stimulation, and increased the barrier integrity of cells damaged by lipopolysaccharide or ultraviolet radiation. These results suggest that Antarctic marine algae have optimized their composition for polar environments, and furthermore, that the bioactive properties of compounds produced by Antarctic marine algae can potentially be used to develop therapeutics to promote the protective barrier function of the intestine and skin.     

Porphyra tenera Protects against PM2.5-Induced Cognitive Dysfunction with the Regulation of Gut Function

To evaluate the biological effects of Porphyra tenera (P. tenera), we tried to confirm the possibility that the intake of P. tenera could modulate cognitive and intestinal functions in PM_2.5-induced cognitive decline mice. P. tenera attenuated PM_2.5-induced learning and memory impairment through antioxidant and anti-inflammatory effects by regulating the mitochondrial function and TLR-initiated NF-κB signaling. In addition, P. tenera effectively alleviated Aβ production/tau phosphorylation by inhibiting the JNK phosphorylation. Also, the bioactive constituents of P. tenera determined the sulfated galactan, mycosporine-like amino acids (MAAs), and chlorophyll derivatives. Moreover, the bioactive compounds of P. tenera by gut fermentation protected against gut dysbiosis and intestinal tight junction damage with a decrease in inflammatory response and short-chain fatty acid production. Based on these results, our findings suggest that P. tenera with sulfated galactan and MAAs is a potential material for cognitive function improvement.     

Taeanamides A and B,Nonribosomal Lipo-Decapeptides Isolated from an Intertidal-Mudflat-Derived Streptomyces sp.

Two new lipo-decapeptides, namely taeanamides A and B (1 and 2), were discovered from the Gram-positive bacterium Streptomyces sp. AMD43, which was isolated from a mudflat sample from Anmyeondo, Korea. The exact molecular masses of 1 and 2 were revealed by high-resolution mass spectrometry, and the planar structures of 1 and 2 were elucidated using NMR spectroscopy. The absolute configurations of 1 and 2 were determined using a combined analysis of ¹H-¹H coupling constants and ROESY correlations, the advanced Marfey’s method, and bioinformatics. The putative nonribosomal peptide synthetase pathway for the taeanamides was identified by analyzing the full genome sequence data of Streptomyces sp. AMD43. We also found that taeanamide A exhibited mild anti-tuberculosis bioactivity, whereas taeanamide B showed significant bioactivity against several cancer cell lines.     

Prevalence of Lawsonia intracellularis, Salmonella spp. and Eimeria spp. in healthy and diarrheic pet rabbits

A total of 170 fresh fecal samples (healthy; n=137, diarrheic; n=33) were collected from pet rabbits. By using PCR and formol-ether concentration method, a total 13/137 healthy rabbit feces were positive for L. intracellularis, 6/137 for Salmonella, and 13/137 for Eimeria. On the other hand, a total 17/33 diarrheic rabbit fecal samples were positive for L. intracellularis, 10/33 for Salmonella, and 21/33 for Eimeria. From these results, more than 20% of clinically normal and 97% of diarrheic rabbits were positive for single or concurrent infection of three pathogens. To the best of our knowledge, this is the first report to describe the prevalence of the microorganisms L. intracellularis, Salmonella and Eimeria in pet rabbits.     

Pseudoalteromone A,a Ubiquinone Derivative from Marine Pseudoalteromonas spp., Suppresses Melanogenesis

An ubiquinone derivative, pseudoalteromone A (1), has been isolated from two marine-derived Pseudoalteromonas spp., APmarine002 and ROA-050, and its anti-melanogenesis activity was investigated. The anti-melanogenic capacity of pseudoalteromone A was demonstrated by assessing the intracellular and extracellular melanin content and cellular tyrosinase activity in the B16 cell line, Melan-a mouse melanocyte cell line, and MNT-1 human malignant melanoma cell line. Treatment with pseudoalteromone A (40 μg/mL) for 72 h reduced α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin production by up to 44.68% in B16 cells and 38.24% in MNT-1 cells. Notably, pseudoalteromone A induced a concentration-dependent reduction in cellular tyrosinase activity in B16 cell, and Western blot analyses showed that this inhibitory activity was associated with a significant decrease in protein levels of tyrosinase and tyrosinase-related protein 1 (Tyrp-1), suggesting that pseudoalteromone A exerts its anti-melanogenesis activity through effects on melanogenic genes. We further evaluated the skin-whitening effect of pseudoalteromone A in the three-dimensional (3D) pigmented-epidermis model, MelanoDerm, and visualized the 3D distribution of melanin by two-photon excited fluorescence imaging in this human skin equivalent. Collectively, our findings suggest that pseudoalteromone A inhibits tyrosinase activity and expression and that this accounts for its anti-melanogenic effects in melanocytes.     

In situ and in vitro antioxidant activity of sweetpotato anthocyanins

Anthocyanins from a variety of fruits and vegetables have been shown to possess potent antioxidant activity in vitro, but scavenging of free radicals by anthocyanins has only been demonstrated in situ in the leaves of certain plants. We report on a new sweetpotato that exhibits mottled purple flesh attributable to high concentrations of anthocyanins. By perfusing transverse sweetpotato sections with the reactive oxygen species H(2)O(2), followed by the H(2)O(2) sensitive fluorochrome scopletin, we show that anthocyanins act as antioxidants in situ within the sweetpotato storage roots. We also demonstrate in vitro antioxidant activity by sweetpotato anthocyanins, where an additive effect with hydroxycinnamic acids is observed. Anthocyanic foods have been shown to offer protection against a variety of degenerative disease processes. Given that sweetpotato can be eaten several hundred grams at a time and as a staple, these data are consistent with the possibility of superior health protection by anthocyanic varieties of sweetpotato in comparison to most common fruits and vegetables.     

A new method for assessing foliar uptake of fungicides using Congo Red as a tracer

In order to develop a new method for measuring foliar uptake of fungicides, Congo Red was selected as a tracer, and optimum procedures were established for washing, extracting and analyzing it and fungicides from leaf surfaces. Congo Red, a water‐soluble dye, was not absorbed into cucumber or rice leaf, even in the presence of various surfactants, and was completely washable from leaf surfaces by aqueous acetonitrile solutions. Congo Red and fungicides in washings were quantified to calculate the amount of foliar uptake of the latter, by comparing the ratio to Congo Red. The optimum concentration of Congo Red in a formulation should be established in order to minimize its influence on fungicide uptake. Although Congo Red has proved to be useful with a conventional droplet application method, it will give more realistic and practical results with the spraying method used in the present study. © 2001 Society of Chemical Industry     

Recruit of porcine oocytes excluded from nuclear transfer program for the production of embryos following parthenogenetic activation

To evaluate whether oocytes excluded from somatic cell nuclear transfer (SCNT) could be utilized for embryo production by parthenogenetic activation (PA), porcine oocytes with poor morphology after maturation culture were excluded from SCNT and subsequently used for PA with different stimuli. In the first set of experiment, either electric pulse of different strengths (1.75, 2.0 or 2.25 kV/cm for 30 microsec each) or chemicals with different treatment durations [7% ethanol for 5 min followed by exposure to 6-dimethylaminopurine (6-DMAP) for 0, 2, 3 or 4 hr] was employed. Development to the 8-cell and morula stages was significantly (P<0.05) improved by electric stimulation of 2.0 kV/cm, while blastocyst formation was enhanced by chemical treatment of ethanol and 6-DMAP for 4 hr. Subsequently, oocytes were parthenogenetically activated by one of four stimuli; 1) optimal electric (2.0 kV/cm for 30 microsec), 2) optimal chemical (ethanol followed by 6-DMAP for 4 hr), 3) electric then chemical and 4) vice versa. On the other hand, oocytes with normal morphology were subjected to the same experimental treatments for the control. Regardless of oocyte type, a combination of electric and chemical stimulations did not further stimulate preimplantation development, compared with electric activation only. However, combinational treatment greatly increased the cell number of blastocysts in SCNT-excluded oocytes (21.9 to 22.9 vs. 16.9 cells/blastocyst), while such effect was not found in normal oocytes (22.2 to 23.3 cells/blastocyst). In conclusion, porcine oocytes excluded from SCNT still have a potential to develop blastocysts after PA and this might contribute to increasing the efficiency of SCNT for various purposes. A combined activation by electricity and chemical yielded the best rate of preimplantation development with increasing the quality of blastocyst.