NtMEK2, which is the tobacco MAPK kinase that is upstream of SIPK and WIPK, was identified using the dexamethasone (DEX)-inducible gain-of-function transgenic system. Expression of $NtNEK2^{DD}$, a constitutively active mutant of NtNEK2, leads to HR-like cell death, which indicates that the NtMEK2-SIPK/WIPK cascade controls defense responses in tobacco. However, little is known about the downstream target substrates or defense-related genes that are regulated by the NtMEK2-SIPK/ WIPK cascade. In this study, ACP-based differential display RT-PCR was used to isolate the downstream effectors mediated by the NtMEK2-SIPK/WIPK cascade in $NtNEK2^{DD}$ transgenic plants. The results identified 6 novel differentially expressed genes (DEGs). These included pathogen induced protein 2-4 (pI2-4), monoterpene synthase 2 (MTS2), seven in absentia protein (SINA), cell death marker protein 1 (CDM1), hydroxyproline-rich glycoprotein (HRGP) and unknown genes (DEG45). The induction of these genes was confirmed by RT-PCR of samples obtained from $NtNEK2^{DD}$ plants. Additionally, when compared with other isolated DEGs, the pI2-4, CDM1 and HRGP genes were significantly up-regulated in response to treatment with salicylic acid and tobacco mosaic virus. Taken together, these results suggest that three novel DEGs were regulated by the NtMEK2-SIPK/WIPK cascade involved in disease resistance in tobacco.
Salicylic acid(SA) is a phytohormone that is related to plant defense mechanism. The SA accumulation is triggered by abiotic and biotic stresses. SA acts as a signal molecular compound mediating systemic acquired resistance and hypersensitive response in plant. Although the role of SA has been studied extensively, an understanding of the SA regulatory mechanism is still lacking in plants. In order to comprehend SA regulatory mechanism, we have been transformed with a SID2 promoter:GUS::LUC fusion construct into siz1-2 mutant and wild plant(Col-0). SIZ1 encodes SUMO E3 ligase and negatively regulates SA accumulation in plants. SID2(SALICYLIC ACID INDUCTION DEFICIENT2) is a crucial enzyme of SA biosynthesis. The Arabidopsis SID2 gene encodes isochorismate synthase(ICS) that controls SA level by conversion of chorismate to isochorismate. We compared the regulation of SID2 in wild-type and siz1-2 transgenic plants that express SID2 promoter:GUS::LUC constructs respectively. The expressions of $\beta$-GLUCURONIDASE and LUCIFERASE were higher in siz 1-2 transgenic plant without any stress treatment. SID2 promoter:GUS::LUC/siz1-2 transgenic plant will be used as a starting material for isolation of siz1-2 suppressor mutants and genes involved in SA-mediated stress signaling pathway.
Wood of Robinia pseudoacacia and bark of Populus alba$\times$P. glandulosa, Fraxinus rhynchophylla and Ulmus davidiana var. japonica were collected and extracted with acetone-water(7:3, v/v) in glass jar to examine whether its bioactive compounds exist. The concentrated extracts were fractionated with hexane, chloroform, ethylacetate and water, and then freeze-dried for column chromatography and bioactive tests. The isolated compounds were sakuranetin-5-O-$\beta$-D-glucopyranoside from Populus alba $\times$Pl glandulosa, 4--ethyoxy-(+)-leucorobinetinidin frm R. pseudoacacia and fraxetion from F. rhynchophylla and were characterized by $^1H$ and$^{13}C $ NMR and positive FAB-MS. Decay-resistant activity was expressed by weight loss ratio and hyphae growth inhibition in the wood dust agar medium inoculated wood rot fungi. R. pseudoacacia showed best anti-decaying property in both test and its methanol untreated samples, indicating higher activity than methanol treated samples in hyphae grwoth test. In antioxidative test, $\alpha$-tocopherol, one of natural antioxidants, and BHT, one of synthetic antioxidants, were used as references to cmpare with the antioxidant activities of the extacted fractions. Ethylacetate fraction of F. rhynchophylla bark indicated the hightest activity in this test and all fractions of R. pseudiacacia extractives also indicated higher activities compared with the other fractions. In the isolated compounds, aesculetin isolated from F. rhynchophylla bark showed best activity and followed by robonetinidin from R. pseudoacaica.
This study has been carried out in order to investigate the bacterial quality of fish meat paste products and the characteristics of isolated thermodurics from the products. Twenty samples of crab-flavored fish stick (Kematsal), 23 samples of plate fish meat paste (Panomuk, Kamaboko), 5 samples of fried fish meat paste (Tigimomuk), 2 samples of roasted fish meat paste (Puduromuk, Chikuwa), 20 samples of fish sausage were collected from processing plants and supermarkets in Pusan, Korea during the period from May to October in 1984. The results obtained are as follows. Amont the samples collected from supermarkets, roasted fish meat paste and fried fish meat paste marked hish counts in coliforms and fungi while very low in the samples of crab-flavored fish stick and plate fish meat paste. Salmonella was not detected in all the samples examined and Staphylococcus aureus was detected only in fried fish meat paste, Thermoduric bacteria were detected less than 10$^2$/g in the samples of crab-flavored fish stick and plate fish meat paste, which might come from subsidiary materials such as starch and seasonings. Among the isolated bacteria, distribution of the proteolytics were more than 87% and the lipolytics were less than 20%. Gram positive bacteria was more than 70% in crab-flavored fish stick and plate fish meat paste, 47.3% in fried fish meat paste. And rod in shape was almost more than 90% in all the samples. The most heat resistant bacterium isolated from the samples was identified as a Bacillus licheniformis(named B. licheniformis CR-11). The strain showed strong proteolytic activity and also grew well at above 2$0^{\circ}C$. The growth rate and generation time of CR-11 strain were 0.31 hr$^{-1}$ , 2.24 hr at 2$0^{\circ}C$, 0.64 hr$^{-1}$ , 1.09 hr at 3$0^{\circ}C$ and 0.78 hr$^{-1}$ , 0.89 hr at 35$^{\circ}C$. Heat resistance value of the spores of CR-11 strain suspended in phosphate buffer solution was D$_{85}$$^{\circ}C$=41.9 min, D$_{90}$$^{\circ}C$=27.9 min, D$_{95}$$^{\circ}C$=10.2 min, D$_{100}$$^{\circ}C$=4.3 min (Z=13.8$^{\circ}C$)
Proceedings of the Korean Society for Food Science of Animal Resources Conference
/
2004.05a
/
pp.89-119
/
2004
This study was conducted to isolate lactobacilli having probiotic characteristics to be used as health adjuncts with fermented milk products. Acid tolerant strains were selected in Lactobacilli MRS broth adjusted to pH 4.0 from 80 healthy persons (infants, children and adults). And bile tolerant strains were examined in Lactobacilli MRS broth in which 1.0% bile salt was added. By estimation above characteristics, the strains No. 27, which was isolated from adult feces, was selected and identified as Lactobacillus salivarius subsp. salivarius based on carbohydrate fermentation and 16S rDNA sequencing. It was used as a probiotic strain in fermented milk products. The pH of fermented milk decreased from pH 6.7 to 5.0 and titratable acidity increased from 0.3% to 1.0% by L. salivarius subsp. salivarius (isolation strain 20, 35, and 37), when incubated for 36 h at $37^{\circ}C$. The number of viable cell counts of fermented milk was maximized at this incubation condition. The SDS-PAGE evidenced no significant change of casein but distinct changes of whey protein were observed by isolated L. salivarius subsp. salivarius for titratable acidity being incubated by $0.9{\sim}1.0%$ at $37^{\circ}C$. All of the strains produced 83.43 to 131.96 mM of lactic acid and 5.39 to 26.85 mM of isobutyric acid in fermented products. The in vitro culture experiment was performed to evaluate ability to reduce cholesterol levels and antimicrobial activity in the growth medium. The selected L. salivarius subsp. salivarius reduced $23{\sim}38%$ of cholesterol content in lactobacilli MRS broth during bacterial growth for 24 hours at $37^{\circ}C$. All of the isolated L. salivarius subsp. salivarius had an excellent antibacterial activity with $15{\sim}25$ mm of inhibition zone to E. coli KCTC1039, S. enteritidis KCCM3313, S. typhimurium M-15, and S. typhimurium KCCM40253 when its pH had not been adjusted. Also, all of the isolated L. salivarius subsp. salivarius had partial inhibition zone to E. coli KCTC1039, E. coli KCTC0115 and S. enteritidis KCCM3313 when it had been adjusted to pH 5.7. The selected strains were determined to have resistances of twelve antibiotic. Strains 27 and 35 among the L. salivarius subsp. salivarius showed the highest resistance to the antibiotics. Purified ${\alpha}$-galactosidase was obtained by DEAE-Sephadex A-50 ion exchange chromatography, Mono-Q ion exchange chromatography and HPLC column chromatography from L. salivarius subsp. salivarius 27. The specific activity of the purified enzyme was 8,994 units/mg protein, representing an 17.09 folds purification of the original cell crude extract. The molecular weight of enzyme was identified about 53,000 dalton by 12% SDS-PAGE. Optimal temperature and pH for activity of this enzyme were $40^{\circ}C$ and 7.0 respectively. The enzyme was found to be stable between 25 and $50^{\circ}C$. ${\alpha}$-galactosidase activity was lost rapidly below pH 5.0 and above pH 9.0. This enzyme was liberated galactose from melibiose, raffinose, and stachyose, and also the hydrolysis rate of substrate was compound by HPLC. These results indicated that some of the L. salivarius subsp. salivarius (strain 27 and 35) are considered as effective probiotic strains with a potential for industrial applications, but the further study is needed to establish their use as probiotics in vivo.
The incidence of sexually transmitted disease, especially gonorrhoea has risen despite the progress in its diagnosis and treatment. For the effective control programs of sexually transmitted disease, it should be required socio-medical approaches. A study on gonorrhoea and penicillinase-producing Neisseria gonorrhoeae(PPNG) was conducted in Jeonju and Kunsan area from March, 1982 through August, 1982. The 221 entertrainers were studied in order to determine the prevalences of gonorhoea and PPNG. Socio-demographic informations of the entertainers were obtained by interviewing them. Gonococci were cultured on Thayer-Martin enrichment media for isolation, and PPNG was confirmed using beta-lactamase reagent ($PADAC^{tm}$ Beta-Lactamase Test Strips, Galbiochem-Behring). The results of the study are summarized as follows; 1. The average age of the entertainers studied was $26.1{\pm}4.7$ years. 2. The average years of working in entertaining business was $2.4{\pm}1.4$ years, and the average income per month was $239,592{\pm}90,480$ won. On the education level, 70.6% of the entertainers were middle or high school graduates 3. 47.5% of the entertainers were using contraceptives. 90.5% have experienced artificial abortion. 4. 37(16.7%) out of 221 entertainers were revealed to gonorrhoea, and 13 (35.1%) of gonorrhoea patients were by PPNG. 5. The prevalence rates of gonorrhoea and the proportion of PPNG by age were not significant statistically. Meanwhile, the colelations between the rates of gonorrhoea and education, frequency of love-making with customers and type of sexual partner were highly significant statistically. 6. 37 strains of gonococci isolated were almostly resistant to several antimicrobial agents, especially amikacin, clindamycin and chloramphenicol. Furthermore PPNG strains were completely resistant to not only above drugs but also penicillin.
The present study investigated whether leukemia-maintaining cells reside in a differentiation-resistant fraction using a megakaryocytic differentiation model of K562 cells. Treatment with phorbol-12-myristate-13-acetate (PMA) significantly inhibited the colony-forming efficiency of the K562 cells. At a PMA concentration of 1 nM or higher, colony was not formed, but approximately 40% of K562 cells still survived in soft agar. Approximately 70% of colony-forming cells that were isolated following the removal of PMA after exposure to the agent were differentiated after treatment with 10 nM PMA for 3 days. The differentiation rate of the colony-forming cells was gradually increased and reached about 90% 6 weeks after colony isolation, which was comparable to the level of a PMA-treated K562 control. Meanwhile, imatinib-resistant variants from the K562 cells, including K562/R1, K562/R2, and K562/R3 cells, did not show any colony-forming activity, and most imatinib-resistant variants were CD44 positive. After 4 months of culture in drug-free medium, the surface level of CD44 was decreased in comparison with primary imatinib-resistant variants, and a few colonies were formed from K562/R3 cells. In these cells, Bcr-Abl, which was lost in the imatinib-resistant variants, was re-expressed, and the original phenotypes of the K562 cells were partially recovered. These results suggest that leukemia-maintaining cells might reside in a differentiation-resistant population. Differentiation therapy to eliminate leukemia-maintaining cells could be a successful treatment for leukemia if the leukemia-maintaining cells were exposed to a differentiation inducer for a long time and at a high dose.
Lactic acid bacteria obtained from traditional Kimchi were selected on the basis of their caseinolytic activity and lactose usability and examined for availability as a starter in probiotic activity. Thirty-two strains were selected as lactic acid producing bacteria in BCP agar, and two strains (KC23 and KF26) with more than 90% resistance for both acid and bile salts were selected. The two strains were identified as L. plantarum (KC23) and L. paracasei (KF26) by API 50 CHL system and 16S rRNA sequence analysis. L. plantarum (KC23) was finally selected based on its biochemical characteristics for lactose and raffinose usability. Free tyrosine content increased rapidly in 10% skimmed milk medium, from $24.1{\mu}g/mL$ after 8 h to $43.9{\mu}g/mL$ after 16 h. Additionally, the caseinolytic clear zone of 12 mm of L. plantarum (KC23) was greater than the 9 mm zone of commercial L. acidophilus CSLA. The bacterium exhibited mesophilic growth and yielded $8.9{\times}10^8CFU/mL$ when incubated at $37^{\circ}C$ for 12 h at pH 4.25. Moreover, L. plantarum KC23 exhibited antibacterial activity as it formed a clear zone of 8-13 mm for the 5 pathogens. Adherent activity was 2.23 fold higher than that of LGG. The acidity of 10% skimmed milk fermented for 12 h was 0.74%.
This study aimed to isolate wild yeasts obtained from flowers and soil of the Wolpyung park, Daejeon city and Gykpo beach, Buan, Jeollabuk-do in Korea, and to further characterize previously unrecorded wild yeast strains. In total, 88 strains of 62 different species of wild yeasts were isolated from 75 samples obtained from the Wolpyung park. Among these, six strains of Trichosporon moniliiforme and four strains each of Papiliotrema flavescens and Candida melibiosica were isolated. Additionally, 39 strains of 30 different species of wild yeasts were isolated from 35 samples collected from the Gykpo beach. Among the 127 isolated wild yeast strains, 10 strains, including Apiotrichum porosum ASCM32-1, were previously unrecorded. All the 10 previously unrecorded yeasts were oval or global in shape, and three strains, including Candida athensensis WP4-90-3, formed spores. Three strains, including Vishniacozyma taibaiensis WP13-2, were halophilic yeasts which grew in 15% NaCl-containing YPD(yeast extract-peptone-dextrose) medium. Five strains, including C. athensensis WP4-90-3, showed 15% ethanol resistance. Cell-free extracts from Candida oleophila WP5-19-1 and Wickerhamomyces anomalus HO9-2 showed the highest β-glucuronidase inhibitory activity (49.0%) and neutrophil elastase inhibitory activity (38.4%), respectively.
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