• Journal of the korean academy of Pediatric Dentistry
• /
• v.32 no.1
• /
• pp.75-88
• /
• 2005

Ameloblastoma is the most commonly occurring odontogenic tumor in oral cavity. Although most are benign epithelial neoplasm, they are generally considered to be locally aggressive and destructive, exhibiting a high rate of recurrence. The biological behavior of this neoplasm is a slowly growing, locally invasive tumor without metastasis, therefore malignant neoplasm, changed its histological appearance to carcinoma or showed distant metastasis, is only defined clinically. In this study, we identified the differentially expressed genes(DEGs) in stages under benign or malignant ameloblastoma compared with normal patient using ordered differential display(ODD) reverse transcription(RT)-PCR and $GeneFishing^{TM}$ technology. ODD RT-PCR is rather effective when the investigation of samples containing very small amounts of total RNA must be accomplished. ODD RT-PCR used the means of amplification with anchored T-primer and adaptor specific primer. bearing definite two bases at their 3' ends and so this method could display differential 3'-expressed sequence taqs(ESTs) patterns without using full-length cDNAs. Compared with standard differential display, ODD RT-PCR is more simple and have enough sensitivity to search for molecular markers by comparing gene expression profiles, However, this method required much effort and skill to perform. $GeneFishing^{TM}$ modified from DD-PCR is an improved method for detecting differentially expressed genes in two or more related samples. This two step RT-PCR method uses a constant reverse primer(anchor ACP-T) to prime the RT reaction and arbitrary primer pairs(annealing control primers, ACPs) during PCR. Because of high annealing specificity of ACPs than ODD RT-PCR, the application of $GeneFishing^{TM}$ to DEG discovery generates reproducible, authentic, and long(100bp to 2kb) PCR products that are detectable on agarose gels. Consequently, various DEGs observed differential expression levels on agarose gels were isolated from normal, benign, and malignant tissues using these methods. The expression patterns of the some isolated DEGs through ODD RT-PCR and $GeneFishing^{TM}$ were confirmed by Northern blot analysis and RT-PCR. The results showed that these identified DEGs were implicated in ameloblastoma neoplasm processes. Therefore, the identified DEGs will be further studied in order to be applied in candidate selection for marker as an early diagnosis during ameloblastoma neoplasm processes.

• KSBB Journal
• /
• v.22 no.5
• /
• pp.356-361
• /
• 2007

Cyanobacteria are a very old group of prokaryotic organisms that produce very diverse secondary metabolites, especially non-ribosomal peptide and polyketide structures. Although some cyanobacteria produce lethal toxins such as microcystins and anatoxins, some may be useful either for development into commercial drugs or as biochemical tools. Detection of unknown secondary metabolites was carried in the present study by a screening of 98 cyanobacterial strains from Cyanobiotech GmbH in order to establish a screening process, isolate pure substances and determine their bioactivities. A degenerated polymerase chain reaction technique as molecular approaches has been used for general screening of NRPS gene and PKS gene in cyanobacteria. A putative PKS gene was detected by DKF/DKR primer in 38 strains (38.8%) and PCR amplicons resulted from a presence of NRPS gene were showed by MTF2/MTR2 primer in 30 strains (30.6%), respectively. A screening of interesting strains was performed by comparing PCR screening results with HPLC analyses of extracts. HPLC analysis for a detection of natural products was performed in extracts from biomass. 5 strains were screened for further scale-up processing. 7 pure substances were isolated from the scale-up cultures and tested for bioactivities under consideration to purity, amount and molecular weight of substances. One substance isolated from CBT 635 showed cytotoxic activity. This substance may be regarded as Microcystin LR.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.38 no.1
• /
• pp.76-82
• /
• 2009

Wheat is the most widely cultivated cereal and an important source of dietary protein worldwide. Wheat allergy, defined as an adverse immunologic reaction to wheat, encompasses a broad spectrum of disorders with different pathomechanisms and clinical manifestation. The Nuruk, a traditional Korean Koji for brewing, was made with wheat flour and fermenting microbes such as bacteria, yeast and mold. The strains grown on Nuruk secrete various enzymes as amylase and protease. By the activation of such enzymes, starch and proteins in Nuruk are hydrolyzed to sugar and amino acid. Therefore, it is supposed to reduce allergic proteins in wheat. To study quality properties and degradation degree of allergenicity in Nuruk by fermentation, we investigated the changes of general ingredients and allergenicity in Nuruk during fermentation. Moisture contents was decreased from 24.2% to 13.6% during fermentation. Crude lipid and protein contents were gradually increased during fermentation. After 15 days of fermentation, reducing sugar and total sugar contents were reached its maximum level, and they were 27.45% and 39.00%, respectively. Acid and neutral protease activity were significantly increased during fermentation, but alkaline protease activity was not detected. ${\alpha}$-amylase activity was gradually increased and showed maximum level about 2,833.00 U/g after 15 days of fermentation. Glucoamylase activity was the highest level about 497.9 U/g after 10 days of fermentation. The increase of these proteolytic and saccharogenic enzyme activities will provide efficient condition for production of rice wine. Also, protein fractions were isolated from Nuruk, and degradation of these proteins during fermentation were confirmed by SDS-PAGE. IgE immunoblotting using patient's sera with wheat allergy was performed to confirm allergenic protein in Nuruk. These results as fermentation of Nuruk will provide a useful tool for developing safer wheat products to prevent wheat allergy.

• Tuberculosis and Respiratory Diseases
• /
• v.55 no.1
• /
• pp.88-97
• /
• 2003

Background : Although smoking is a major cause of chronic obstructive pulmonary disease (COPD), only 10-20% of cigarette smokers develop symptomatic COPD, which suggests the presence of genetic susceptibility. This genetic susceptibility to COPD might depend on variations in the activities of the enzyme that detoxify hazardous chemical products, such as microsomal epoxide hydrolase (mEPHX) and glutathione-S transferase M1 subunit (GSTM1) genes. Methods : The genotypes of 58 patients with COPD, and 79 age matched control subjects, were determined by a polymerase chain reaction, followed by restriction fragment length polymorphism (PCR-RFLP) for the mEPHX, and multiplex PCR for the GSTM1. Results : GSTM1 was deleted in 53.3% of the subjects. There was no difference in GSTM1 deletion rates between the COPD patients (32/58, 55.2%) and the control subjects (41/79, 51.9%). The combination patterns of two polymorphisms of mEPHX showed slow enzyme activity in 29(21.2%), normal in 73(53.3%) and fast in 32(23.4%). The COPD group (7/57, 12.3%) showed a significantly lower incidence of slow enzyme activity compared to the control subjects (22/77, 28.6%, p<0.05). However, when the COPD and control groups were compared with smokers only, there were no significant differences in the genotypes of GSTM1 and mEPHX. Conclusion : The genotypes of GSTM1 and mEPHX were not significant risk factors of COPD in this cohort of study.

• Journal of Genetic Medicine
• /
• v.5 no.1
• /
• pp.15-20
• /
• 2008

Purpose : Large exon deletions in the DMD gene are found in about 60% of DMD/BMD patients. Multiplex PCR has been employed to detect the deletion mutation, which frequently generates noise PCR products due to the presence of multiple primers in a single reaction as well as the stringency of PCR conditions. This often leads to a false-negative or false-positive result. To address this problematic issue, we introduced the dual primer oligonucleotide (DPO) system. DPO contains two separate priming regions joined by a polydeoxyinosine linker that results in high PCR specificity even under suboptimal PCR conditions. Methods : We tested 50 healthy male controls, 50 patients with deletion mutation as deletion-positive patient controls, and 20 patients with no deletions as deletion-negative patient controls using DPO-multiplex PCR. Both the presence and extent of deletion were verified by simplex PCR spanning the promoter region (PM) and 18 exons including exons 3, 4, 6, 8, 12, 13, 17, 19, 43-48, 50-52, and 60 in all 120 controls. Results : DPO-multiplex PCR showed 100% sensitivity and specificity for the detection a deletion. However, it showed 97.1% sensitivity and 100% specificity for determining the extent of deletions. Conclusion : The DPO-multiplex PCR method is a useful molecular test to detect large deletions of DMD for the diagnosis of patients with DMD/BMD because it is easy to perform, fast, and cost-effective and has excellent sensitivity and specificity.

• Journal of Life Science
• /
• v.19 no.9
• /
• pp.1328-1332
• /
• 2009

Anisakidosis is caused by anisakid nematodes (family Anisakidae) larvae which can cause not only direct tissue damage but also a severe allergic response related to excretory-secretion products. Lots of different species of anisakid larvae, including Anisakis simplex, Contracaecum, Goezia, Pseudoterranova, and Hysterothylacium, cause the anisakidosis. But it is difficult to diagnosis the species of larvae since the morphologies of larval anisakid nematodes are almost indistinguishable. In order to diagnosis the differential infections of larval anisakid nematodes, polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) of 18S rDNA - was conducted. Three major species of anisakid larvae including A. simplex, C.ontracaecum spp, and Goezia spp. were collected from mackerel (Scomber japonicus), mullet (Mugil cephalus), founder (Paralichthys olivaceus), eel (Astroconger myriaster) and red sea bream (Pagrus major). PCR amplified 18S rDNA from each species of anisakid larvae was digested with eight restriction enzymes including Taq I, Hinf I, Hha I, Alu I, Dde I, Hae III, Sau96 I, and Sau3A I. The original sizes of PCR amplified 18S rDNA were 2.0Kb in both anisakid larvaes and Goezia. Restrction enzymes including Hinf 1, Alu 1, Hha I, Dde 1 and Hae III cut differently and distinguished the A. simplex and Contracaecum type C'. However, Contracaecum type A showed two different restriction enzyme cutting patterns by Taq 1, Hinf I, Alu 1, and Dde 1. One of the patterns was the same as those of A. simplex, Contracaecum type C' and Goezia and the other was unique. These results suggest that PCR-RFLP pattern by Hinf 1, Alu 1, Hae I, Dde 1 and Hae III can be applied to differential diagnosis of human infection with A. simplex and Contracaecum type C'. Contracaecum type A needs further study of classification by morphological characteristics and genetic analysis.

• Development and Reproduction
• /
• v.10 no.2
• /
• pp.147-154
• /
• 2006

Phthalates such as di(2-ethyl hexyl)phthalate(DEHP) are industrial chemicals with wide-ranging human exposures because of their use in plastics and other common consumer products. Consequently, their adverse effects as endocrine disruptor in the reproductive physiology of both laboratory rodents and human have been studied extensively. The present study was undertaken to examine whether prepubertal exposure to DEHP affects on the onset of puberty and the associated reproductive parameters such as hormone receptor expressions in female rats. DEHP(100mg/kg/day) was administered daily from postnatal day 25(PND 25) through the day when the first vaginal opening(VO) was observed, and the animals were sacrificed on the next day. Gross anatomy and weight of reproductive tissues were compared to test the DEHP's effects on the cell proliferation. Furthermore, histological studies were performed to assess the structural alterations in the tissues. Specific radioimmunoassay was carried out to measure serum LH levels. To determine the transcriptional changes in progesterone receptor(PR), total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). As a result, delayed VO was shown in the DEHP group(PND $37.3{\pm}0.7$) compared to the control group(PND $35.3{\pm}0.7$; p<0.05). DEHP treatment significantly decreased the wet weight of ovaries and uteri compared to the control group(p<0.05). Interestingly, elevation of serum LH levels was shown in the DEHP group(p<0.05). Graafian follicles and corpora lutea were observed only in the ovaries from the control animals. Numerous primary, secondary follicles and small atretic follicles were observed in the ovaries from DEHP-treated animals. Similarly, hypotrophy of luminal and glandular uterine epithelium was found in the DEHP-treated group. These effects were probably due to the inhibitory effects of DEHP on the synthesis and secretion of estrogen from granulosa cells. In the semiquantitative RT-PCR studies, the transcriptional activities of PR in both ovary(p<0.05) and uterus(p<0.01) from DEHP-treated animals were significantly lower than those from the control animals. The present studies demonstrated that the acute exposure to DEHP during the critical period of prepubertal stage could inactivate the reproductive system resulting delayed puberty in female rats.

• Research in Plant Disease
• /
• v.12 no.3
• /
• pp.298-302
• /
• 2006

An isolate of Cucumber mosaic virus(CMV), called as Can-CMV, was originally isolated from Canna generalis showing typical streak mosaic foliar symptoms, and its properties were investigated in this study. Whereas all known isolates of CMV could induce symptoms on their systemic hosts(four kinds of Nicotiana spp and a zucchini squash), Can-CMV induced no symptoms on its systemic hosts tested. Replication and movement of the virus on upper leaves as well as inoculated leaves-were confirmed by RT-PCR suggesting that Can-CMV could only infect systemically on N. benthamiana and N. glutinosa. Size of local lesions on the Can-CMV-inoculated leaves of Chenopodium amaranticolor was much smaller than that of Fny-CMV. Whereas Fny-CMV and LS-CMV could induce distinct necrotic local lesions on Vigna unguiculata 2 to 3 days postinoculation(dpi), chlorotic spots symptom was expressed by Can-CMV 4 to 5 dpi. Virus-specific 4 kinds of dsRNAs were isolated from leaves of N. benthamiana infected with Can-CMV, and these dsRNAs corresponded to the viral genomic RNAs and subgenomic RNAs and their patterns were indistinguishable to those of Fny-CMV and LS-CMV. By restriction mapping analysis of 950 bp of RT-PCR amplified products of coat protein gene of the virus as well as by serological analysis of gel diffusion test, Can-CMV belongs to a typical member of CMV subgroup IA. These results suggest that the Can-CMV isolated from C. generalis possesses unique pathological properties to understand further insight into the various interactions between virus and host.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.41 no.6
• /
• pp.875-880
• /
• 2012

This study was conducted to investigate the level of contamination of pathogenic Escherichia (E.) coli in 50 types of red pepper powders collected domestically. Pathogenic E. coli was confirmed using real-time PCR to confirm the 4 types of EAEC, EPEC, EHEC and ETEC. One sample out of 50 was contaminated with pathogenic E. coli. The type of pathogenic E. coli detected in the sample was EAEC. This study was also conducted to determine the effect of alcohol treatment on the reduction of E. coli populations in red pepper powder. The amount of E. coli in the control was $1.2{\times}10^6$ cfu/mL. The amount of E. coli in 10 minutes immersion treatment with 10% alcohol was $1.1{\times}10^6$ cfu/mL. In samples treated with over 20% alcohol, E. coli was not detected. This showed that 10 minutes of immersion in over 20% alcohol might be effective to reduce E. coli. This study was also conducted to determine the effect of UV irradiation on E. coli reduction. The number of E. coli in the control group was $5.0{\times}10^5$ cfu/mL. However, the number of E. coli in 45 min of the UV irradiated sample decreased to $1.0{\times}10^3$ cfu/mL, by $10^2$ cfu/mL. In contrast, E. coli was not detected in an over 60 min UV irradiated sample in $10^{-2}dilution$. This study showed that over 20% alcohol treatment and UV irradiation for 60 min was effective to control E. coli in red pepper powder.

• Journal of the Korean Society of Food Science and Nutrition
• /
• v.39 no.8
• /
• pp.1179-1186
• /
• 2010

The antiradical property of hot water extract from dried radish (DR) or dried radish roasted with pressure (DRRP) was investigated in vitro and in LLC-PK1 cell system. The contents of total free amino acid and reducing sugar in DR were decreased by 72.86% and 3.17%, respectively, after pressurized roasting. In vitro test, $IC_{50}$ for DR and DRRP for DPPH radical scavenging activity were 646.70 and $135.45\;{\mu}g/mL$, 896.10 and $566.98\;{\mu}g/mL$ for superoxide anion radical, and 722.26 and $531.84\;{\mu}g/mL$ for hydroxy radical, respectively. The radical scavenging effects of DRRP was significantly greater than those for DR (p<0.001). These radical scavenging effects of DR and DRRP were confirmed in LLC-$PK_1$ at which oxidative stresses were induced by superoxide, nitric oxide and peroxynitrite generated in the treatment of pyrogallol, SNP, and SIN-1, respectively. Cell viability was increased in the presence of DR or DRRP, dose dependently (p<0.05), and TBARS formation was decreased. The protective effects of DRRP against oxidative damage in LLC-$PK_1$ were greater than those of DR at the same concentration tested (p<0.05). This superior antiradical activity of DRRP might be due to the products produced during the pressurized roasting in addition to the antioxidative compounds originally present in the radish. 5-hydroxyl methyl furfural (5-HMF) known as an intermediate product of the maillard reaction was detected in DRRP (0.57 mg/g), but not from DR. In conclusion, daily consumption of DRRP may prevent oxidative damage by retarding oxidative stress.