• Journal of Plant Biotechnology
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• v.40 no.1
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• pp.18-26
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• 2013

Genetically modified (GM) rice Agb0101, which expresses the insecticidal toxin modified cry1Ac (mcry1Ac1) gene, was developed by the Rural Development Administration in Korea. To monitor the probable release of Agb0101 in the future, it is necessary to develop a reliable detection method. Here, we developed the PCR detection method for monitoring and tracing of GM rice. The primer pair (RBEgh-1/-2) from a starch branching enzyme (RBE4) gene was designed as an endogenous reference, giving rise to an expected PCR amplicon of 101 bp. For the qualitative PCR detection, construct- and event-specific primers were designed on the basis of integration sequence of T-DNA. Event-specific PCRs amplified specifically 5'- or 3'-junction region spanning the native genome DNA and the integrated gene construct, while none of amplified product was shown on crops, rice varieties, and other insect-resistant transgenic rice lines. The event-specific real-time PCR method was performed using TaqMan probe and plasmid pRBECrR containing both rice endogenous gene RBE4 sequence and 5'-junction sequence as the reference molecule. The absolute limit of quantification (LOQ) of real-time PCR was established with around 10 copies for one plasmid molecule pRBECrR. Thereafter, the different amounts of transgenic rice (1, 3, 5, and 10%, respectively) were quantified by using the established real-time PCR method, with a range below 19.55% of the accuracy expressed as bias, 0.06-0.40 of standard deviation (SD) and 3.80-7.01% of relative standard deviations (RSD), respectively. These results indicate that the qualitative and quantitative PCR methods could be used effectively to detect the event Agb0101 in monitoring and traceability.

• Journal of the Korean Society of Food Science and Nutrition
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• v.34 no.6
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• pp.915-923
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• 2005

We monitored paralytic shellfish poison (PSP) by HPLC method in shellfishes including a sea squirt, and its products produced in domestic and imported, total 35 species, 850 samples, collected at 9 cities such as Seoul, Busan, Daegu, Ulsan, Pohang, Masan, Tongyeong, Geoje and Sacheon, 10 times by month (2 times in April and May) from March to October, 2004. PSPs were detected in a few raw samples on March, and it showed highest toxic ratio $(72.9\%)$ on third week of April (51 samples toxic in total 70 samples) and decreased suddenly to $2.4\%$ on third week of May. After then, PSPs were detected in a few samples, sporadically, and disappeared after August. Most raw shellfishes in the market were safe from PSP, showing the toxicity below 1 MU/g (quarantine level 4.0 MU/g), except oyster collected on April at Masan (1.9 MU/g), blue mussel on April (1.8 MU/g) and purplish washington clam on May at Sacheon (2.1 MU/g). However, it was strongly suggested to survey and under control for the imported scallops and ark shells which showed highly toxic in the quarantine level on May, July, even September. PSP were not detectable in the all shellfish products collected on May, July and September, except 2 boiled and dried mussels contained trace amount (0.01 MU/g), and all those products were safe as below quarantine toxin level (4.0 MU/g).

• Development and Reproduction
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• v.11 no.1
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• pp.49-54
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• 2007

Ethane 1,2-dimethane sulfonate(EDS), a toxin which specifically kills Leydig cells(LC), has been widely used to prepare the reversible testosterone(T) depletion rat model. In the present study, we monitored the gene expression profiles of pituitary gonadotropins, LH and FSH, up to 7 weeks after EDS injection. Adult male Sprague-Dawley rats($300{\sim}350\;g$ B.W.) were injected with a single dose of EDS(75 mg/kg i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. Total RNAs were purified from each pituitary, and the message levels of common alpha subunit($C{\alpha}$) of pituitary glycoprotein hormones, LH beta subunit($LH{\beta}$), FSH beta subunit($FSH{\beta}$) and GnRH receptor(GnRH-R) were evaluated by semi-quantitative RT-PCRs. The message levels of $C{\alpha}$ increased sharply during weeks 1-4, then return to the control level on week 5. The mRNA levels of $LH{\beta}$ were elevated after week 2, reached the peak at week 4, then declined to the control level after week 5. The message levels of $FSH{\beta}$ were elevated after week 2, reached the peak at week 3, then declined to the nadir at week 5. Similarly, the mRNA levels of GnRH-R were elevated after week 2, reached the peak at week 3, then gradually declined to the control level after week 5. The present study indicated that EDS treatment could induce reversible alterations in the transcriptional activities of gonadotropin subunits and GnRH-R in the anterior pituitary from male rats. EDS injection model might be useful to understand the mechanism of hormonal regulation of hypothalamus- pituitary neuroendocrine axis in male rats.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.2
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• pp.107-113
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• 1994

Among the currently recognized pathogenic vibrios, V. vulnificus and V. cholerae non O1 are the most serious bacteria from the point of view of sea food hygiene in Korea. In this paper, the authors compared the hemolytic activities of the crude hemolysin produced by V. vulnificus and V. cholerae non O1 isolated from shellfish collected in Chungmoo, Korea. The authors also attempted to improve the purification method of V. vulnificus hemolysin(VVH) and tried to make antiserum with the purified hemolysin. VVH was produced in abundance in heart infusion broth containing $2\%$ NaCl in a shaking cultivation process(140rpm) at $37^{\circ}C$ for 15 hours. While hemolysin production patterns of V. cholerae non O1 were quite different by the strain during the culture times compared with the V. vulnificus. Hemolytic activity of the VVH on sheep erythrocytes was stronger than those of rabbit, but hemolytic activities of the hemolysin produced by V. cholerae non O1 on rabbit erythrocytes were as much as twice as strong as on those of sheep and horse. VVH was purified by two steps of hydrophobic column chromatography on Phenyl-Sepharose HP with Fast Protein Liquid Chromatography(FPLC). Purification fold and yield of VVH was much improved by changing the elution buffer's pH from 6.0 to 9.8 and adding $1\%$ CHAPS(a zwitter ionic detergent) and $50\%$ ethylene glycol to the 10mM glycine buffer during the repeated hydrophobic column chromatography. Homogeneity of the purified hemolysin was shown by polyacrylamide gel electrophoresis. According to the five times repeated purification results, the specific activity was increased 27500 times and the yield was improved by $23.4\%$ on average. About $250{\mu}g$ of purified hemolysin was harvested from the 2400ml of culture supernatant of V. vulnificus. Molecular weight of VVH was estimated to be 50KDa by the SDS-PAGE and the neutralization scores of the obtained antiserum acting against VVH were $2000{\sim}8500$.

• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.196-203
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• 2011

Staphylococcus spp. is one of the most common bacteria isolated from the lesions of atopic dermatitis (AD) in humans, and their colonization is known to be a possible trigger factor of clinical signs. The aim of this study was to determine the prevalence of Staphylococcus spp. in canine AD (CAD), the types of exotoxins present, and their relation with the clinical severity of CAD. From 79 dogs with AD, 72 samples of Staphylococcus spp. were isolated (91.1%), and 65 (90.3%) were confirmed as Staphylococcus pseudintermedius. Concerning the profile of the exotoxin gene, 50 isolates (69.4%) contained at least one exotoxin gene, and 28 isolates (56%) were found to contain more than 2 different exotoxins. There was a significant difference in clinical severity with the presence of staphylococcal exotoxins (P=0.028), whereas no correlation was found with the presence of Staphylococcus spp. (P=0.598). The clinical severity of CAD increased only in relation to staphylococcal enterotoxin D (SED) and exfoliative toxins (P<0.05). Some clinical evaluation criteria (erythema, papule/pustule) were correlated with the presence of the exotoxin gene (P<0.05). This study showed that the high prevalence of Staphylococcus spp. and staphylococcal exotoxins in lesions from dogs with AD may be regarded as an important trigger factor for exacerbation of the clinical signs of CAD.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.166-174
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• 2003

Microcystin produced by cyanobacteria in surface waters, such as eutrophic lake and river, is a kind of serious environmental problems due to its toxicity to human and wild animals. Microcystin is synthesized nonribosomally by the large modular multi-functional enzyme complex known as microcystin synthetase encoded by the mcy gene cluster. Amplification of mcy genes by PCR from cultures and environmental samples is a simple and efficient method to detect the toxigenic Microcystis. In order to evaluate primers designed to detect toxic microcystin-producing strains, 17 cyanobacterial strains and 20 environmental samples were examined by PCR with 7 pairs of primers. Some microcystin-producing cyanobacteria were not detected with FAA-RAA, TOX4F-TOX4R and FP-RP primers. The fragment of unexpected size was amplified with NSZW2-NSZW1 primers in Microcystis strains isolated from the lakes in Korea. TOX1P-TOX1F primers failed in amplification of toxin-producing strains. Only MSF-MSR and TOX2P- TOX2F primers amplified the fragments of mcy genes from 11 strains of microcystin-producing Microcystis. The water samples taken from 20 lakes in Korea were analyzed by PCR using each of the primers. In all the water samples, cyanobacteria capable of producing microcystin were detected by the PCR with TOX2P-TOX2F primers. These results indicate that TOX2P-TOX2F primers are better than the other primers for detection of microcystin-producing Microcystis strains in Korea. The nucleotide sequences of mcy gene in Microcystis aeruginosa NIER10010 suggest genetic diversity of Korean isolates.

• Development and Reproduction
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• v.10 no.3
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• pp.203-209
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• 2006

Ethane 1,2-Dimethane sulfonate(EDS), a toxin which specifically kills Leydig cells(LC), has been widely used to prepare the reversible testosterone(T) depletion rat model. Previous studies including our own clearly demonstrated that the dramatic weight loss of the T-dependent accessory sex organs such as epididymis and seminal vesicle in this 'LC knock-out' rats. These weight loss could be derived from massive and abrupt death of the cells via apoptotic process. The present study was performed to test the effect of EDS administration on the expression of some apoptotic genes in the rat epididymis. Adult male Sprague-Dawley rats($300{\sim}350$ g B.W.) were injected with single dose of EDS(75 mg/kg, i.p.) and sacrificed on Weeks 0, 1, 2, 3, 4, 5, 6 and 7. Tissue weights and the numbers of the epididymal sperm were measured. The transcriptional activities of the bcl-2, bax, Fas and Fas ligand(Fas-L) were evaluated by semi-quantitative RT-PCR. As expected, the weights and the sperm counts of epididymis declined progressively after the EDS treatment during Week 1 and 2. These decrements were discontinued with a gradual return towards normal during Weeks $5{\sim}7$, although the maximal recoveries of the epididymal weights(71%) and sperm count(38%) were subnormal on Week 7. The initial level of bcl-2 transcripts persisted to Week 6 then elevated significantly on Week 7. The level of bax transcripts significantly decreased on Week 6, and no remarkable change was found in the rest of the experimental period. The transcripts for the Fas in epididymis elevated during Weeks $1{\sim}2$, returned to normal on Week 3, and the level persisted to the Week 7. Similarly, the level of Fas-L transcripts elevated during Weeks $1{\sim}3$ and returned to normal after Week 4. Our results demonstrated the transient T depletion by EDS administration could induce the changes in expression of the apoptotic genes in rat epididymis. The activation of Fas and Fas-L in the epididymis of EDS-treated rats might be responsible for the initial apototic process and consequently the tissue damage and the sperm loss. Future studies will attempt to determine the precise molecular mechanism(s) of apoptosis in the rat epididymis.

• Korean Journal of Ecology and Environment
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• v.33 no.4 s.92
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• pp.366-373
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• 2000

Toxin production and low digestibility of cyanobacteria are known to cause low exploitability of cyanobacteria by zooplankton. In this study, we compared relative tolerance and compatibility of zooplankton taxa in eight eutrophic lakes, exposed to frequent cyanobacterial blooms, uring the summer season of 1999. Microcystis, Anabaena, Oscillatoria and Phormidium were common cyanobacteria in all lakes. with relatively lower $NO_3-N$ concentration (<0.2 mgN/l) and TN/TP ratio (<20), compared with other lakes where colonial cyanobacteria dominated. Rotifers were dominant zooplankton in most lakes, and among them, Keratella, Polyarthra and Hexathra were common. The laboratory feeding experiment showed that relative copepods that greatly decreased (90%) after 4 day when cyanobacteria were used as the food source of zooplankton, while rotifers gradually increased with the change of dominant taxa from Keratella through Pompholyx to Monostyla. These results suggest that rotifers may be capable of coexisting with cyanobacteria by exploiting them for the food source.

• Journal of Life Science
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• v.16 no.5
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• pp.840-849
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• 2006

Severe brain injuries induced by toxin pose one of the most important problems on our health care because of their high morbidity and mortality, are implicated to leucocyte infiltration more premature or immature brain than mature brain. Chemokines are the induction meditators for infiltration of inflammatory cells to the inflammation sites. In order to study the mechanism of leucocyte infiltration, the expression of several chemokines, MCP-1, $MIP-1{\alpha}$ and MIP-2 was studied in lipopolysaccharide(LPS)-stimulated neonatal and adult brain. One week old Sprague-Dawley rats or adult male rats weighing 300-350 g were used for the experiment. After anesthetization, $1\;{\mu}l$ LPS (0.5 mg/ml) subsequently was injected in the right caudate nucleus of the brain with stereotaxic frame. Animals were sacrificed at 6 hours, 24 hours, and 72 hours after injection. The present study was carried out using RT-PCR for the mRNA and immunohistochemistry for the expression of the proteins. In the neonatal rat brain, prominent interstitial edema with significant accumulation of leukocytes was detected at 24 and 72 hours after LPS injection. A semiquantitative analysis of RT-PCR revealed that the MCP-1, $MIP-1{\alpha}$, and MIP-2 mRNA expression peaked at 24 hours in neonatal and adult rat brain. Neonatal rats showed about 2.6, 1.4, and 1.2 times more expression of the MCP-1, $MIP-1{\alpha}$, and MIP-2 than that of the adult rats in the brain tissue. Immunohistochemical analysis also showed that MCP-1 immunoreactivity was paralleled with the RT-PCR results. MCP-1 protein was significantly detected at 24 and 72 hours in the brain parenchyma. $MIP-1{\alpha}$protein was highly expressed at 24 hours. The results of leukocyte infiltration in H&E stain was parallelled with that of the immunohistochemistry. Chemokine proteins were markedly detected at 24 hours after injection of LPS and neutrophil influx into intraparenchymal was prominent at 24 hours. These results suggest that the leukocyte infiltration in the intracranial infection may be controlled by mechanisms influenced by chemokine producing cells in the central nervous system such as microglia, astrocyte and endothelial cell.

• Korean Journal of Microbiology
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• v.54 no.4
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• pp.384-397
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• 2018

The aim of this study is to screen the strains of Bacillus spp. possessing safety, probiotic activity, and so on, which can be utilized as probiotic resource for using the feed and supplement food of companion animal. About 300 isolates were isolated from traditional Korean sauces, four isolates that did not have or produce the six kinds of B. cereus type vomiting and diarrhea toxin genes, ${\beta}$-hemolytic, and three kinds of carcinogenic enzymes were selected. Antibiotic gene retention, cell surface hydrophobicity, antibiotic sensitivity, and glucose utilization were analyzed for four isolates, and finally SRCM 100731 was selected. SRCM 100731 was named as Bacillus amyloliquefaciens SRCM 100731 16S rRNA sequencing analysis, and carried out optimization of cell growth for industrial applications such as pet food and feed. The effects of 14 different components on cell growth were investigated and three significant positive factors, molasses, sodium chloride, and potassium chloride were selected as the main factors based on a Plackett-Burman design. In order to find out optimal concentration on each constituent, we carried out central composite design. The predicted optimized concentrations were 7% molasses, 1.1% sodium chloride, 0.5% potassium chloride. Finally, an overall about 7-fold increase in dry cell weight yield ($12.6625{\pm}0.0658g/L$) was achieved using the optimized medium compared with the non-optimized medium ($1.8273{\pm}0.0214g/L$). This research is expected to be highly utilized in the growing pet industry by establishing optimal cultivation conditions for industrial application as well as screening Bacillus amyloliquefaciens SRCM 100731 as probiotic resource for companion animal.