• The Korean Journal of Zoology
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• v.38 no.3
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• pp.417-425
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• 1995

The present study was carried out to investigate the effect of microwave fixation in comparison with that of chemical fixation in preparing the microscopic samples. The microwave fixator was equipped with infrared-temperature sensor, and that was designed to compensate air temperature in the microwave fixator. In the microwave fixation, rat tongue was well preserved in terms of muscular fasciculus and pancreas stained by Feulgen reagents showed clear reaction products in the nucleus. Reaction products by PAS method in duodenal villi appeared specifically at the goblet cells. In electron microscopy, pancreatic cellular components such as secretory granules and collagen bundles were well preserved in both fixations. In aspect of histochemical reaction and electron microscopy, high quality was due to the protein content of microwave fixed specimen. The microwave fixation method saved total duration engaging microscopic preparation.

• Korean Journal of Veterinary Service
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• v.37 no.2
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• pp.85-96
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• 2014

Total 99 strains of Campylobacter spp. were isolated from 117 cases of duck's fecal samples. Among 99 strains of Campylobacter spp. isolates, 93 strains (93.9%) were C. jejuni and 6 strains (6.1%) were C. coli. Prevalence of virulence and GBS associated genes of 72 C. jejuni isolates was determined by m-PCR. Among the 10 kinds of virulence associated genes, cadF, dnaJ, flaA and ceuE genes were detected in all of C. jejuni isolates from ducks, racR, pldA, iamA, ciaB, virB11 and docC genes were 87.5%, 84.7%, 77.8%, 48.6%, 13.9% and 11.1%, respectively. Antimicrobial susceptibility test was performed on 72 C. jejuni isolates. The rate of resistance were 62.5% for oxytetracycline, 55.6% for kanamycin, 54.2% for enrofloxacin, 50% for ciprofloxacin, 37.5% for tetracycline and nalidixic acid, 18.1% for ampicillin, 15.3% for streptomycin, and 6.9% for ofloxacin. All isolates were susceptible to erythromycin. The adherence (intracellular and extracellular bacteria) abilities of the 20 isolates to INT-407 cells were between $4.21{\pm}1.27{\times}10^4$ CFU/well and $1.053{\pm}0.451{\times}10^6$ CFU/well from the isolates of cj-55 and cj-52, respectively, and that can be expressed as 0.1033% to 5.2655% to the infecting inoculum. The invasion (intracellular bacteria) abilities of the 20 isolates to INT-407 were between $1.00{\pm}1.73{\times}10^3$ CFU/well and $8.47{\pm}5.16{\times}10^4$ CFU/well from the isolates of cj-13 and cj-47, respectively, and that can be expressed as 0.0050% to 0.4235% to the infecting inoculums. The average CFU/well of 20 campylobacters isolated from ducks for adherence to and invasion were $2.646{\pm}2.886{\times}10^5$ and $3.03{\pm}2.7{\times}10^4$ respectively, and that was $1.3230{\pm}1.2139%$ and $0.1516{\pm}0.1343%$ of the starting viable inoculum. There was considerable correlation ($R^2$=0.627) between the adherence and invasion ability of C. jejuni isolates for INT-407 cell.

• Journal of Life Science
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• v.26 no.10
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• pp.1113-1120
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• 2016

Two bacterial strains producing extracellular man nanase were isolated from doenjang, a traditionally fermented soybean paste in Korea. The isolates, WL-6 and WL-11, were identified as Bacillus subtiis on the basis of their 16S rRNA gene sequences, morphological, and biochemical properties. Two genes encoding the mannanase of both B. subtilis WL-6 and B. subtilis WL-11 were each cloned into Escherichia coli, and their nucleotide sequences were determined. Both mannanase genes consisted of 1,086 nucleotides, encoding polypeptides of 362 amino acid residues. The deduced amino acid sequences of the two WL-6 and WL-11 mannanases, designated Man6 and Man11, respectively, differed from each other by eight amino acid residues, and they were highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The 26 amino acid stretch in the N-terminus of Man6 and Man11 was a predicted signal peptide. Both Man6 and Man11 were localized at the level of 94–95% in an intracellular fraction of recombinant E. coli cells. The enzymes hydrolyzed both locust bean gum and mannooligosaccharides, including mannotriose, mannotetraose, mannopentaose, and mannohexaose, forming mannobiose and mannotriose as predominant products. The optimal reaction conditions were 55°C and pH 6.0 for Man6, and 60°C and pH 5.5 for Man11. Man11 was more stable than Man6 at high temperatures.

• Molecules and Cells
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• v.24 no.2
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• pp.276-282
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• 2007

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

• Molecules and Cells
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• v.24 no.2
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• pp.200-209
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• 2007

We generated gene expression data from the tissues of 50 gastric cancer patients, and applied meta-analysis and gene set analysis to this data and three other stomach cancer gene expression data sets to define the gene expression changes in gastric tumors. By meta-analysis we identified genes consistently changed in gastric carcinomas, while gene set analysis revealed consistently changed biological themes. Genes and gene sets involved in digestion, fatty acid metabolism, and ion transport were consistently down-regulated in gastric carcinomas, while those involved in cellular proliferation, cell cycle, and DNA replication were consistently up-regulated. We also found significant differences between the genes and gene sets expressed in diffuse and intestinal type gastric carcinoma. By gene set analysis of cytogenetic bands, we identified many chromosomal regions with possible gross chromosomal changes (amplifications or deletions). Similar analysis of transcription factor binding sites (TFBSs), revealed transcription factors that may have caused the observed gene expression changes in gastric carcinomas, and we confirmed the overexpression of one of these, E2F1, in many gastric carcinomas by tissue array and immunohistochemistry. We have incorporated the results of our meta- and gene set analyses into a web accessible database (http://human-genome.kribb.re.kr/stomach/).

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.

• Journal of Microbiology and Biotechnology
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• v.27 no.7
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• pp.1276-1280
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• 2017

The mcr-1 gene is a new "superbug" gene discoverd in China in 2016 that makes bacteria highly resistant to the last-resort class of antibiotics. The mcr-1 gene raised serious concern about its possible global dissemination and spread. Here, we report a potential anti-resistant strategy using the CRISPR/Cas9-mediated approach that can efficiently induce mcr-1 gene knockout in Escherichia coli. Our findings suggested that using the CRISPR/Cas9 system to knock out the resistance gene mcr-1 might be a potential anti-resistant strategy. Bovine myeloid antimicrobial peptide-27 could help deliver plasmid pCas::mcr targeting specific DNA sequences of the mcr-1 gene into microbial populations.

• Proceedings of the Ginseng society Conference
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• 1998.06a
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• pp.231-243
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• 1998

In the present study, we provide evidence that ginsenoside $Rh_2$ (G-$Rh_2$) as well as staurosporine induces apoptosis of human hepatoma SK-HEP-1 cells by caspase 3-mediated processing of $p21^{WAFI/CIPI}$ in the early stage of apoptosls. Immunoblottings showed that G-$Rh_2$ as well as statrosporine induced the processing of caspase-3 to an active form, pl7. In stable Bcl-2 transfectants however, G-$Rh_2$ induced DNA fragmentation, while staurosporine did not. In the early stage of apoptosis, $p21^{WAFI/CIPI}$ was detected to undergo proteolytic processing specifically conducted by caspase-3. $p21^{WAFI/CIPI}$ translated in vitro was cleaved into a p14 fragment, when incubated with cell extracts obtained from either G-$Rh_2$- or staurosporine-treated cells. Cleavage was equally inhibited in both cases by adding Ac-DEVD-cho, a specific caspase-3 inhibitor, but not by Ac-YVkD-cho, a specific caspase-l inhibitor. Similarly, $p21^{WAFI/CIPI}$ was efficiently leaved by recombinant caspase-3 overexpressed in E. coli. Moreover, the endogenous $p21^{WAFI/CIPI}$ of untreated-cell extracts was also cleaved by recombinant caspase-3. Mutation analysis allowed identification of two caspase-3 cleavage sites, $DHVD^{112}$/L and $SMTD^{149}$/F, which are located within, or near the interaction domains for cyclins, Cdks, and PCNA. Taken together, these results show that G-$Rh_2$ as well as staurosporine increases caspase-3 activity, which in turn directly cleaves $p21^{WAFI/CIPI}$ resulting in elevation of Cdk kinase activity in the early stages of apoptosis. We propose that proteolytic cleavage of $p21^{WAFI/CIPI}$ is a functionally relevant event that allows unleashing the cyclin/Cdk activity from the inhibitor seen in the earlier stage of apoptosis, the event of which may be associated with the triggering mechanism for the execution of apoptosis.

• Korean Journal of Veterinary Service
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• v.31 no.1
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• pp.1-16
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• 2008

Salmonella infections cause the disease in pigs but also some zoonotic Salmonella serotypes can be transmitted to human through swine products, resulting in food poisoning. The objective of this study was to investigate the bacteriological prevalence and detection of invA gene using Salmonella specific polymerase chain reaction (PCR), the epidemiological characteristics related to Salmonella strains cultured from pig samples in Gangwon areas using serotyping, random amplified polymorphic DNA (RAPD) and pulsed field gel electrophoresis (PFGE) methods. During the period of November 2001 through April 2002, 1,174 ileocecal lymph node were collected from the slaughtered pigs raised in 38 farms located in Gangwon province. The samples were submerged in boiling water and macerated in saline and lymph node homogenates were inoculated into Tetrathionate broth with iodine (TTB, Difco, 0.5% iodine was added) for enrichment growth. Then additional tests were performed using several mediums, and suspects were identified by API 20E kit (BioMerieux) and PCR. Of total 1,174 samples from 38 farms, 44 (3.7%) were isolated as Salmonella spp from 13 farms (34.2%). Of 44 isolates, 31 were in Yangyang region, followed by 9 in Goseong, 2 in both Gangneung and Sokcho. However, there was no difference in regional isolation frequency. All isolates have a 521bp amplified product in Salmonella specific PCR with primer invA which encodes in proteins for invasion of epithelial cells. Of 44 recovered serotypes, 23 (52.3%) were S Eingedi, 10 (22.7%) S Schwarzengrund, 9 (20.5%) S Typhimurium, and 2 (4.5%) S Mbandaka. In RAPD analysis, there appeared to be unique bands distinguishing each serotype, although similarities exist between the different serotypes. Four serotypes of 44 Salmonella isolates appeared to fall into 14 different RAPD types. In PFGE analysis, 9 S Typhimurium were tested with XbaI enzyme and SpeI enzyme. The combination of results obtained with two enzymes subdivided the 9 S Typhimurium into 4 PFGE types.

• Journal of Periodontal and Implant Science
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• v.34 no.3
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• pp.607-622
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• 2004

Recently epidemiologic studies have indicated that the patients with periodontitis may have increased risk of ischemic cardiovascular events, and have suggested the important roles of blood cytokines and acute reactant proteins in the systemic infection and inflammatory response. Periodontitis and coronary heart disease (CHD) may share the common risk factors and the genetic mechanism associated with interleukin(IL)-1A, B and RA genotype may be involved in the production of IL-1. This study was aimed to investigate the relationship between angiographically defined CHD and periodontitis as chronic Gram-negative bacterial infection and to determine whether the IL-1 gene polymorphism is associated in both diseases. Patients under the age of 60 who had undergone diagnostic coronary angiography were enrolled in this study. Subjects were classified as positive CHD (+CHD, n=37) with coronary artery stenosis more than 50% in at least one of major epicardial arteries, and negative CHD (-CHD, n=30) without significant stenosis. After recording the number of missing teeth, periodontal disease severity was measured by means of plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD), clinical attachment level (CAL), and radiographic bone loss around all remaining teeth. Gingival crevicular fluid (GCF) was collected from the 4 deepest periodontal pockets and assessed for cytokine ($IL-1{\beta}$, IL-6, IL-1ra, tumor necrosis $factor-{\alpha}$, and prostaglandin $E_2$). Additionally, blood CHD markers, lipid profile, and blood cytokines were analyzed. IL-1 gene cluster genotyping was performed by polymerase chain reaction and enzyme restriction using genomic DNA from buccal swab, and allele 2 frequencies of IL-1A(+4845), IL-1B(+3954), IL-B(-511), and IL-1RA(intron 2) were compared between groups. Even though there was no significant difference in the periodontal parameters between 2 groups, GCF level of $PGE_2$ was significantly higher in the +CHD group(p<0.05). Correlation analysis showed the positive relationship among PD, CAL and coronary artery stenosis(%) and blood $PGE_2$. There was also significant positive relationship between the periodontal parameters (PI, PD, CAL) and the blood CHD markers (leukocyte count, C-reactive protein, and lactic dehyrogenase). IL-1 gene genotyping showed that IL-1A(+3954) allele 2 frequency was significantly higher in the +CHD group compared with the -CHD group (15% vs. 3.3%, OR 5.118,p=0.043). These results suggested that periodontal inflammation is related to systemic blood cytokine and CHD markers, and contributes to cardiovascular disease via systemic inflammatory reaction. IL-1 gene polymorphism might have an influence on periodontal and coronary heart diseases in Korean patients.