• Journal of Periodontal and Implant Science
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• v.44 no.3
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• pp.141-146
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• 2014

Purpose: Peri-implantitis and periodontitis are inflammatory and infectious diseases of implant and tooth-supporting tissues. Recently, the role of gene polymorphisms of immune response components in the relevant pathogenesis has been investigated. The present study was the first to evaluate the relationship between two known single nucleotide polymorphisms (SNPs) of the receptor activator of nuclear factor kappa-${\beta}$ (RANK) gene (rs3018362 and rs35211496) in chronic periodontitis and peri-implantitis patients in an Iranian population. Methods: Eighty-one periodontally healthy patients, 38 patients with peri-implantitis, and 74 patients with chronic periodontitis were enrolled in this study. DNA was extracted from blood arm vein samples by using Miller's salting out technique according to the manufacturer's instructions given in the extraction kit. The concentration of DNA samples was measured using a spectrophotometer. The genetic polymorphisms of the RANK gene were evaluated using a competitive allele specific polymerase chain reaction (KBioscience allele specific PCR) technique. Differences in the frequencies of genotypes and alleles in the diseased and healthy groups were analyzed using chi-squared statistical tests (P<0.05). Results: Analysis of rs35211496 revealed statistically significant differences in the expression of the TT, TC, and CC genotypes among the three groups (P=0.00). No statistically significant difference was detected in this respect between the control group and the chronic periodontitis group. The expression of the GG, GA, and AA genotypes and allele frequencies (rs3018362) showed no statistically significant difference among the three groups (P=0.21). Conclusions: The results of this study indicate that the CC genotype of the rs35211496 RANK gene polymorphism was significantly associated with peri-implantitis and may be considered a genetic determinant for peri-implantitis, but this needs to be confirmed by further studies in other populations.

• Journal of Life Science
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• v.8 no.5
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• pp.604-613
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• 1998

The last enzyme of the sesquiterpen phytoalexin capsidiol synthesis in tobacco cell, 5-epi-aristolochene hydro-xylase which convert 5-epi-aristolochene (EAS) to capsidiol, was cloned by a reverse transcription polymerase chain reaction strategy and cDNA library screening. Cloned CYP-B3 contained high probability amino acid matches to known plant cytochrome P450 sequences and open reading frame with the conserved FxxGxRxCxG heme-binding region. Transcripts of CYP-B3 were not detected in control cells, but induced in elicitor-treated cells. Furthermore, CYP-B3 transcripts were induced by fungal extracts and cellulase but not by other stimuli(chilling, heat shock and 2,4-D). Induction of CYP-B3 transcripts by elicitor treatment was not affected by ancymidol and ketoconazole treat-ments suggesting that an inhibition of hydroxylase activity by Cyt P450 inhibitors resulting from post translational processing event.

• KSBB Journal
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• v.13 no.2
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• pp.214-219
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• 1998

Polymerase chain reaction(PCR) was conducted to obtain the gene encoding glucose oxidase(GOD) from Aspergillus niger(ATCC 2110) and the DNA sequence determined was coincided with published GOD sequence from A. niger. Recombinant transforming vector containing GOD and hygromycin B(hyg.B) resistant gene(hph) was constructed and used for further transformation of A. niger ATCC 2110. Selectivity of hyg.B against A. niger differed depending on which media were used i.e., nutrient-rich media such as potato dextrose agar(PDA) and complete medium(CM) showed only 50% growth inhibition at 400 $\mu$m ml$^-1$ of hyg.B while the minimal media inhibited mycelial growth completely at 200 $\mu$m ml$^-1$ of hyg.B. Twenty to sixty putative transformants were isolated from the hyg.B-containing minimal top agar, transferred successively onto alternating selective and nonselective media for a mitotic stability of hyg.B resistance and, then, single-spored. Among the stable transformants, the transformant(GOD1-6) grown by flask culture showed the considerable increase of extracellular GOD activity, which was estimated to the degree of 50% - 100% comparing to that of wild type. Transformation of tGOD1-6 was resulted from integration of the vectors into heterologous as well as homologous regions of the A. niger genome. Southern blot analysis revealed that there were two independent integrations of vector into fungal genome and one into the GOD gene due to homologous recombination. In addition, GOD activity and sodium gluconate production when tGOD1-6 was fed-batch fermented were enhanced 11 fold and 2.25 fold, respectively, compared to that of the wild type.

• The Journal of Korean Society of Virology
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• v.30 no.1
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• pp.71-81
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• 2000

We have determined and analyzed the full-length cDNA sequence of a coxsackievirus B3 (CVB3) Korean isolate (CVB3-Korea/97) which has been known as a general human pathogen. The whole genome contains 7,400 nucleotides and has a single large open reading frame with 6,555 nucleotides that encodes a potential polyprotein precursor of 2,185 amino acids. The genome also contains a 5' non-coding region (NCR) of 741 bases and a 3' NCR of 104 bases followed by poly(A) tail. Sequence homologies of nucleotides and deduced amino acids between the CVB3-Korea/97 strain and the prototype (Nancy strain) were 81.7% and 91.5%, respectively. The genes encoding the functional proteins including viral protease and RNA dependent RNA polymerase showed higher homology than those encoding the structural proteins. We have further analyzed the sequences of 5' NCR, VP1 and VP2 of CVB3-Korea/97, which are known as cardiovirulent determining factors at the nucleotide and amino acid levels. Although the CVB 3-Korea/97 strain was isolated from an aseptic meningitis patient without cardiomyopathy, its 234th nucleotide and 165th amino acid were uracil and Asn as same as those of other cardiovirulent strains one. However, the 155th amino acid of VP1, which closely associated with cardiovirulence, was replaced with $Arg^{155}$ by single nucleotide substitution from $A^{2916}$ to $T^{2916}$. Moreover, additional amino acid substitutions were observed in the flanking region of $Asp^{155}$. Taken together, amino acid(s) substitution in VP1 may playa critical role in determining cardiovirulence of the CVB3-Korea/97 strain rather than individual nucleotide replacements in the 5' NCR and/or an amino acid substitution in VP2.

• Food Engineering Progress
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• v.15 no.4
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• pp.370-375
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• 2011

In this study the polymerase chain reaction (PCR) combined with denaturing gradient gel electrophoresis (DGGE) was evaluated as a method permitting the rapid detection of pathogens in fresh originally grown vegetables. A universal primer (341GCf/534r) was selected for its ability to amplify the V3 region of 16S-rRNA genes in their target pathogens (Salmonella typhimurium, Pseudomonas fluorescens, Bacillus cereus, Listeria monoytogenes, Staphyloocus aureus, E. coli). The 194 bp fragments in PCR were successfully duplicated as expected. The amplified fragments of the same size from six different pathogens also showed good separation upon DGGE. The detection limit of PCR-DGGE for six pathogens in fresh-cut lettuces were over $10^{5}$ CFU/g when sampled by stomaching. However, when the sampling method was changed from stomaching to shaking, the detection limit of six pathogens in organic vegetables was shown to increase by over $10^{1}$ CFU/g, but only those of B. cereus were over $10^{3}$ CFU/g. Therefore, PCR-DGGE was shown to be a reliable method for the detection of pathogens in fresh-cut vegetables.

• Journal of Applied Biological Chemistry
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• v.55 no.4
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• pp.227-234
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• 2012

We observed that oil began to accumulate at 25 seed days after flowering (DAF) and reached the maximum potential at 35 seed DAF of oilseed rape, and the greatest weight of 100 seeds was obtained at 35 seed DAF. To survey a broad analysis of gene expression in developing embryos of Brassica napus, the Bn 300k microarray have been constructed. The Bn 300k Microarrary was designed from 80,696 unigenes clustered from 543,448 ESTs and 780 cDNA at NCBI. These arrays have been hybridized in a series of experiments with probes derived from seeds and leaf of B. napus. Approximately 8.5% of the 7,000 genes were expressed as ratios 2-fold higher in seed (25 DAF) than leaves and 0.4% at ratios 10. Also we observed that storage and cell differentiation-related genes were highly expressed at 10 DAF, whereas energy-related genes including fatty acid metabolism were increased up depending on seed maturation using Microarray, which was confirmed by reverse transcriptase polymerase chain reaction. These results suggest that B. napus arrays provide a very useful data set of seed-specific expression that can be further analyzed by examination of the promoter regions of these genes and help our understanding of the complex regulatory network in developing seeds.

• Korean Journal of Environment and Ecology
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• v.24 no.3
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• pp.318-324
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• 2010

The aim of this study is to examine feeding habits of the Korean water deer(Hydropotes inermis argyropus) from its rumen contents using a PCR-DGGE method. For this study, rumen contents were collected from water deer causalities by natural death or road-kill in two different sites(Cheorwon, Gangwon province and the Eastern part of Jeonnam province). DNA was extracted from rumen contents of a total of 44 individuals. Two primers, rbcLZ1aF(GC) and rbcL19bR, were used for PCR amplifications of ribulose-1,5-bisphosphate carboxylase large subunit (rbcL) gene. Among 44 samples, twenty-nine samples were successfully amplified by PCRs. The 29 PCR products of partial rbcL gene were applied for PCR-DGGE. Totally, six families of plants were detected from the diet analyses. Five families of plants were found in Cheorwon, Gangwon province, but only three families of plants were found in the Eastern part of Jeonnam province. The PCR-DGGE method will provide us with a potential tool to study feeding habits of ungulates including water deer, even though our results failed to identify the prey plants at the level of species.

• Journal of Life Science
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• v.19 no.8
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• pp.1047-1054
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• 2009

Oral squamous carcinoma (OSC) cells present resistance to chemotherapeutic agents-mediated apoptosis in the late stages of malignancy. Advances in the understanding of bacterial toxins have produced new strategies for the treatment of cancers. It was demonstrated here that Pseudomonas aeruginosa exotoxin A (PEA) significantly decreased the viability of chemoresistant YD-9 cells in the apoptosis mechanism. Apoptotic manifestations were evident through changes in nuclear morphology and generation of DNA fragmentation. PEA treatment induced caspase-3, -6 and -9 cleavage, and activation. These events preceded proteolysis of the caspase substrates poly (ADP-ribose) polymerase (PARP), DNA fragmentation factor 45 (DFF45), and lamin A in YD-9 cells. The reduction of mitochondrial membrane potential, release of cytochrome c and SmacjDlABLO from mitochondria to cytosol, andtranslocation of AlF into nucleus were shown. While p53, p21 and $14-3-3{\gamma}$ were upregulated, cyclin Band cdc2 were downregulated by PEA treatment. Taken together, PEA induces apoptosis in chemoresistant YD-9 cells via activation of caspases, mitochondrial events and regulation of cell cycle genes.

• Advances in Traditional Medicine
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• v.5 no.2
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• pp.117-123
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• 2005

Dyers woad leaf (Daqingye) is a traditional Chinese medicine commonly used as anti-pyretic, anti-bacterial and anti-viral agent against infectious diseases. The Chinese Pharmacopoeia (2005) records that Dyers woad leaf should be derived from the leaves of Isatis indigotica Fort., but the leaves of Polygonum tinctorium Ait., Baphicacanthus cusia (Nees) Bremek. and Clerodendron cyrtophyllum Turcz. have also been used as substitutes of Dyers woad leaf in different regions of China. The leaf morphologies of these four species show a close resemblance, and based on their morphological appearance, it is difficult to identify them. Here, molecular genetic methods were developed as a target to identify different members of Dyers woad leaf. The 5S-rRNA spacer domain was amplified by polymerase chain reaction from genomic DNAs isolated from I. indigotica, P. tinctorium, B. cusia and C. cyrtophyllum, and the nucleotide sequences showed a great diversity. In addition, random amplification of polymorphic DNA analysis was also used to distinguish the members of Dyers woad leaf. These molecular methods could be used as a tool in authentic identification of Dyers woad leaf.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.257-263
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• 1999

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis. 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC ＃289 and the oligo dT$_{11}$-C primer, revealed the highest homology (49.8％) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.a.