• Title/Summary/Keyword: Isolation technique

Search Result 392, Processing Time 0.031 seconds

Isolation, Identification and Antagonisms of Rhizospheric Antagonists to Cucumber Wilt Pathogen, Fusarium oxysporum f. sp. cucumerinum Owen (오이 덩굴쪼김병균에 대한 오이 근권길항미생물의 분리, 동정 및 길항작용)

  • Jee Hyeong Jin;Kim Hee Kyu
    • Korean Journal Plant Pathology
    • /
    • v.3 no.3
    • /
    • pp.187-197
    • /
    • 1987
  • Bacteria and fungi antagonistic to Fusarium oxysporum f. sp. cucumerinum Owen were effectively isolated with each of modified Triple Layer Agar (TLA) technique from rhizosphere soil where cucumber had been grown healthily in plastic film house. Three predominant bacterial isolates selected were identified as Pseudomonas fluorescens, and P. putida, Serratia sp. and three fungal isolates were Gliocladium sp. Trichoderma harzianum, and T. viride. Antagonistic bacteria inhibited $26-45\%$ of germination and $41-56\%$ of germ tube elongation of microconidia of F. oxysporum f. sp. cucumerinum on Water Agar (WA). P. fluorescens was the strongest inhibitor. Several my co parasitisms were observed on dual culture of WA between antagonistic fungi and F. oxysporum f. sp. cucumerinum such as coiling, penetration, overgrowing, and lysis. Mycelial lysis of the pathogen was the most severe at pH 4.6, followed by 3.6, 5.6 and 6.6 of the medium in decreasing order. At pH 6.6, mycelia of the pathogen were not conspicuously damaged, however, the antagonistic fungi formed abundant chlamydospores especially Gliocladium sp. T. harzianum revealed the most excellent antagonism in vitro.

  • PDF

The Long-term Results of a Modified Maze Procedure with Using Cryoablation (냉동 절제를 이용한 변형된 Maze술 식의 중장기 결과)

  • Park, Kwon-Jae;Woo, Jong-Soo;Bang, Jung-Hee
    • Journal of Chest Surgery
    • /
    • v.41 no.6
    • /
    • pp.710-717
    • /
    • 2008
  • Background: Atrial fibrillation is associated with several complications such as cerebro-vascular accidents and peripheral arterial embolism. Most of the patients who have this arrhythmia chronically feel their heart beating and so they are frightened; therefore, the quality of a patient's life is decreased. The purpose of this article is to determine the long term results of a modified Maze procedure and the factors that influence the success of the procedure. Material and Method: This study enrolled 88 patients who underwent the modified Maze with using cryoablation between June, 2001 and February, 2007. The 88 consecutive patients were divided into two groups ac cording to how the pulmonary veins were isolated, that is, with or without cryoablation. There were 58 patients who were isolated by cutting and sewing in the right pulmonary veins and by cyroablation in the left pulmonary veins in group 1 (group 1, n=58), and 30 patients who underwent isolation by cryoablation in the right & left pulmonary veins were placed in group 2 (group 2, n=30). The ECG was checked at discharge to determine the sinus conversion rate and we followed up the patients to determine whether or not the patients maintained sinus rhythm. We also checked the ECG at the last visit to determine the patients' heart rhythm. Result: The mean follow up time was $44.3{\pm}19.2$ months. At discharge, 72.4% of the patients in group 1 were in proper sinus rhythm and 66.7% of the patients in group 2 were in proper sinus rhythm. At the last follow up, 81% of the patients in group 1 were in normal sinus rhythm and 60% of the patients in group 2 were in normal sinus rhythm. When we analyzed the data via the Kaplan-Meier method, 86.5% of the patients were free from atrial fibrillation (% free from AF) at 1 year, 80% of the patients were free from atrial fibrillation at 5 year in group 1 and 70% of the patients were free from atrial fibrillation at 1 year and 51 % of the patients in group 2 were free from atrial fibrillation at 5 year. Conclusion: The modified Maze technique using cryoablation was a simple and effective procedure. But the success rate of the Maze technique using cryoblation is lower than that of the standard Maze III. The method using cryoablation shorten the operation time, but we must conduct more studies to get a better result of the modified Maze technique with using cryoablation.

Isolation and characterization of sigH from Corynebacterium glutamicum (Corynebacterium glutamicum의 sigH 유전자의 분리 및 기능분석)

  • Kim Tae-Hyun;Kim Hyung-Joon;Park Joon-Sung;Kim Younhee;Lee Heung-Shick
    • Korean Journal of Microbiology
    • /
    • v.41 no.2
    • /
    • pp.99-104
    • /
    • 2005
  • Corynebacterial clones which exert regulatory effects on the expression of the glyoxylate bypass genes were isolated using a reporter plasmid carrying the enteric lacZ fused to the aceB promoter of Corynebacterium glutamicum. Some clones carried common fragments as turned out by DNA mapping technique. Subcloning analysis followed by the measurement of $\beta-galactosidase$ activity in Escherichia coli identified the region responsible for the aceB-repressing activity. Sequence analysis of the DNA fragment identified two independent ORFs of ORF1 and ORF2. Among them, ORF2 was turned out to be responsible for the aceB-repressing activity. ORF1 encoded a 23,216 Da protein composed of 206 amino acids. Sequence similarity search indicated that the ORF may encode a ECF-type $\sigma$ factor and designated sigH. To identify the function of sigH, C. glutamicum sigH mutant was constructed by gene disruption technique and the sigH mutant showed growth retardation as compared to the wild type strain. In addition, the mutant strain showed sensitivity to oxidative-stress generating agent plumbagin. This result imply that sigH is probably involved in the stress response occurring during normal cell growth.

Immunogenicity of Synthetic Peptide Specific for Major Immunogenic Determinat of Hepatitis B Surface Antigen (B형간염(型肝炎) 표면항원(表面抗原)의 주면역원(主免疫原) 결정기(決定基)에 특이(特異)한 합성(合成) Peptide의 면역원성(免疫原性)에 관한 연구(硏究))

  • Shin, Kwang-soon;Han, Su-nam
    • Korean Journal of Veterinary Research
    • /
    • v.25 no.1
    • /
    • pp.7-17
    • /
    • 1985
  • Many investigators have been pursuing various attempts so far to produce hepatitis B surface antigen(HBsAg) vaccines using the techniques such as isolation from plasma of chronic HBsAg carrier, recombinant DNA technique or preparation of synthetic peptides specific for immunogenic determinants. Hepatitis B virus can not grow on any cell lines by the tissue culture technique at the present time. The plasma of chronic HBsAg carrier is expensive and its source is limited. The HBsAg from the recombinant DNA technique gave still very low yield. Another approach, therefore, has been initiated to develop a synthetic hepatitis B virus vaccine. The possible use of several distinct synthetic vaccines in prophylaxis can be facilitated by availability of full synthetic immunogens. Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins, although the free synthetic peptide can be immunogenic. To understand basic knowledges on the antigenicity and immunogenicity of a synthetic peptide specific for major immunogenic determinant of HBsAg, a nonapeptide, $H_2N^{139}Cys-Thr-Lys-Pro-Thr-Asp-Gly-^{146}Asn-Aba$ COOH, which corresponds to HBsAg amino acid residues 139 to 147, was synthesized by the Merrifield's solid-phase method with a slight modification. The antigenicity and immunogenicity of this specific synthetic peptide were examined comparing with purified plasma-derived natural HBsAg. The results obtained are as follows; 1. The peptide synthesized showed the identical amino acid composition to the theoretical value. The degree of purification and molecular weight were acertained by methods of high performance liquid chromatography and mass spectrometry. 2. Using m-maleimidobenzoyl-N-hydroxysuccinimide ester as a conjugating agent, the synthetic peptide was conjugated to rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin. Their conjugation yields were 8.3, 9.5, 15.8, 13.5, and 11.2%, respectively. 3. The natural HBsAg was purified from plasma of chronic HBsAg carrier. By the electron microscopic observation of the purified natural HBsAg preparation, no Dane particles were observed and the preparation showed negative DNA polymerase activity. 4. Antigenicity of the synthetic peptide and the plasma-derived natural HBsAg was determined by competition radioimmunoassay using $^{125}I$-natural HBsAg. Their 50% inhibitions appeared as $90{\mu}g/ml$ and $0.12{\mu}g/ml$ for the synthetic peptide and the natural HBsAg, respectively. This indicates that the former was about 750-fold less antigenic than the latter. 5. Immunogenicity of the synthetic peptide was determined by administering the peptide-carrier conjugates into rabbits with and without Freund's complete adjuvant. Regardless the carrier proteins and adjuvant, positive immune responses to the synthetic peptide were observed. The higher antibody titers, however, were shown in the groups administered with Freund's complete adjuvant. 6. Immunizing dose 50% in mice of the various peptide-carrier conjugates was 5.47, 6.00, 65.16, 31.25 and $13.03{\mu}g/dose$ for rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin, respectively, while the natural HBsAg showed $0.65{\mu}g/dose$. 7. It was postulated that homologous proteins prefer to heterologous ones as the carriers.

  • PDF

Stability of Human Centromeric Alphoid DNA Repeat during Propagation in Recombination-Deficient Yeast Strains (효모의 재조합 변이주를 이용한 인간 Centromeric Alphoid DNA Repeat의 안정성에 관한 연구)

  • Kim, Kwang-Sup;Shin, Young-Sun;Lee, Sang-Yeop;Ahn, Eun-Kyung;Do, Eun-Ju;Park, In-Ho;Leem, Sun-Hee;SunWoo, Yang-Il
    • Korean Journal of Microbiology
    • /
    • v.43 no.4
    • /
    • pp.243-249
    • /
    • 2007
  • The centromere is a highly differentiated structure of the chromosome that fulfills a multitude of essential mitotic and meiotic functions. Alphoid DNA (${\alpha}$-satellite) is the most abundant family of repeated DNA found at the centromere of all human chromosomes, and chromosomes of primates in general. The most important parts in the development of Human Artificial Chromosomes (HACs), are the isolation and maintenance of stability of centromeric region. For isolation of this region, we could use the targeting hook with alphoid DNA repeat and cloned by Transformation-Associated Recombination (TAR) cloning technique in yeast Saccharomyces cerevisiae. The method includes rolling-circle amplification (RCA) of repeats in vitro to 5 kb-length and elongation of the RCA products by homologous recombination in yeast. Four types of $35\;kb{\sim}50\;kb$ of centromeric DNA repeat arrays (2, 4, 5, 6 mer) are used to examine the stability of repeats in homologous recombination mutant strains (rad51, rad52, and rad54). Following the transformation into wild type, rad51 and rad54 mutant strains, there were frequent changes in inserted size. A rad52 mutant strain showed extremely low transformation frequency, but increased stability of centromeric DNA repeat arrays at least 3 times higher than other strains. Based on these results, the incidence of large mutations could be reduced using a rad52 mutant strain in maintenance of centromeric DNA repeat arrays. This genetic method may use more general application in the maintenance of tandem repeats in construction of HAC.

Survey on Preferences Regarding Rubber Dams in Children, Adolescents and Parents (러버댐에 대한 소아, 청소년 및 보호자에 대한 선호도 조사)

  • Jeong, Hankeul;Lee, Nanyoung;Lee, Sangho
    • Journal of the korean academy of Pediatric Dentistry
    • /
    • v.43 no.1
    • /
    • pp.17-26
    • /
    • 2016
  • The use of a rubber dam is a useful technique for protecting patients and operators, producing good results when treating children and adolescent patients. Despite its many advantages, some children and adolescent patients have negative feelings and sometimes express its discomfort. The purpose of this study was to evaluate the patients' preferences for rubber dam use and the associated factors that may affect it. A survey was conducted targeting patients aged 4-18 years old who visited the pediatric dental clinic, the restorative dental clinic, or the student treatment dental clinic of ${\bigcirc}{\bigcirc}$ University Dental Hospital, and were treated using a rubber dam without sedation. We collected questionnaires through electronic medical records. Most children and adolescent patients showed positive attitudes towards using rubber dams. The group with a short treatment time and the group with past knowledge or experience of rubber dam use showed more positive attitudes towards rubber dams (p < 0.05), while no significant difference was found among factors such as gender, age, procedure, anesthetics, treatment site, and operator (p > 0.05).

Availability of MADLDI-TOF MS for Identification of Gram Positive Bacilli Isolated from Blood Culture

  • Choi, Jin-Un;Kim, Sang-Ha;Hwang, Su-Jeong;Yu, Young-Bin;Kim, Sunghyun;Kim, Young-Kwon
    • Biomedical Science Letters
    • /
    • v.24 no.2
    • /
    • pp.108-115
    • /
    • 2018
  • In the present study, results of the identification of Gram-positive bacilli (GPB) were analyzed by using the MALDI-TOF MS technique to score each 2-year blood culture at a university hospital. In addition, 16S rRNA sequence analyses and MALDI-TOF MS results are compared to targeting strains that had been isolated two or more times within the same patient, to evaluate the usefulness of MALDI-TOF MS in GPB identification. According to the cut-off (${\geq}1.7$) criteria, there were 410 (57.5%) reliable strains and 303 (42.5%) non-identified strains among the GPB identification results of 713 strains, using a microflex MALDI Biotyper (Bruker Daltonik GmbH, Bremen, Germany). The isolation appeared most often in the following order: Corynebacterium striatum, Bacillus cereus, Bacillus subtilis, Paenibacillus urinalis, and Listeria monocytogenes. Nearly three-fourths, 66 out of 89 (74.2%) of the strains for Corynebacterium striatum; 44 out of 60 (73.3%) strains for Bacillus cereus; and all (25 out of 25, 100%) Listeria monocytogenes strains were identified by their high scores of 2.0 or higher. Most (293 strains out of 303) non-identified strains were strains isolated only once and not significant as infectious bacilli. A total of 43 out of 50 (86.0%) strains matched and were able to be identified based on the 16 rRNA sequencing comparison results of strains that were isolated twice or more within the same patient and significant as infection bacilli. Non-matching among 5 out of 7 strains was not identified, even with MALDI-TOF MS. In conclusion, GPB can be identified in blood cultures using MALDI-TOF MS. This can be done accurately with ease, rapidly, and at a low cost. It is also thought to be helpful in GPB diagnosis and treatment.

A Study of the Rn-222 and Ra-226 Analysis in Aqueous Samples with a Low-Level Liquid Scintillation Counter and Pulse-Shape Analysis (저준위 액체섬광계수기와 파형분석법을 이용한 수용액 중 라돈-222 및 라듐-226의 분석법 연구)

  • Shin, Hyun-Sang;Lee, Chang-Woo;Lee, Myung-Ho;Cho, Yung-Hyun;Hong, Kwang-Hee;Choi, Geun-Sik
    • Analytical Science and Technology
    • /
    • v.12 no.5
    • /
    • pp.428-435
    • /
    • 1999
  • A method for measuring Rn-222 and Ra-226 in aqueous sample using liquid scintillation counting technique has been studied. The Rn-222 was extracted easily from the water sample (10 mL) by 12 mL of xylene based organic scintillant. After radioactive equilibrium between Rn-222 and its alpha emitting decay products for three hours, the alpha activity from Rn-222 and its decay products were measured in a scintillation vial using the Wallae $1220^{TM}$ Quantulus liquid scintillation counter. Ra-226 concentration in aqueous sample was determined, after isolation of Ra-226 from the sample matrix, by extraction the ingrowth of the Rn-222 and its alpha emitting decay products with xylene based organic scintillant. The optimum pulse-shape analysis (PSA) value was evaluated by the figure of merit (FM) criterion. Minimum detectable activity (MDA) is about 0.14 Bq/L (3.78 pCi) for the region of Rn-222 and its alpha emitting decay products and 0.06 Bq/L (1.63 pCi) for the region of Po-214 respectively, with 200 min, counting time at PSA level 100 in the low-diffusion polyethylene vial and xylene based cocktail solution. Experiment on the optimum sample-cocktail volume ratio, the influence of agitation and the diffusion of radon from vial were carried out.

  • PDF

Analysis of the Gene Expression by Laser Captured Microdissection (I): Minimum Conditions Required for the RNA Extraction from Oocytes and Amplification for RT-PCR (Laser Captured Microdissection을 이용한 유전자 발현에 대한 연구 (I): RT-PCR을 위한 난자의 RNA 추출 및 증폭을 위한 최소한도의 확립)

  • Park, Chang-Eun;Ko, Jung-Jae;Cha, Kwang-Yul;Lee, Kyung-Ah
    • Clinical and Experimental Reproductive Medicine
    • /
    • v.28 no.3
    • /
    • pp.183-190
    • /
    • 2001
  • Objective: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. Methods : Mouse ovaries were fixed and cut into serial sections (7 im thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell $II^{TM}$ system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). Results: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GA PDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 $\grave{i}$ l with 50 oocytes, thus the resting 19.75 il cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. Conclusion: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

  • PDF

Isolation and Characterization of Humic Acids Present in the Soils at the Vicinity of Domestic Atomic Power Plants(NPPs) (국내 원자력 발전소 주변 토양 휴믹산의 추출 및 특성 규명)

  • Lee, Chang-Hoon;Shin, Hyun-Sang;Chung, Kun-Ho;Cho, Young-Hyun;Lee, Chang-Woo
    • Journal of Radiation Protection and Research
    • /
    • v.28 no.3
    • /
    • pp.165-172
    • /
    • 2003
  • Humic acids present in the soils at the vicinity of domestic atomic power plants(NPPs), located in Yeongkwang(YK), Uljin(UJ), Kori(KR), Koseong(KS), Wolseong(WS) area were isolated, and characterized using elemental analysis and UV/Vis, IR, CPMAS $^{13}C$ NMR spectroscopic methods. The characteristics were compared with one another and with commercial humic acid (Aldrich Co.). Molecular size distributions of the humic acids were determined using a stirred cell ultrafiltration technique. The results of elemental analysis showed that soil humic acid from UJ contains higher oxygen content than humic acids from KR and KS (O/C ratios: 0.51 (UJHA) us. 0.45(KRHA), 0.43(KSHA)). The molecular size distribution revealed that the soil humic acids of UJ and YK contained a higher percentage of larger molecules of > 30,000 daltons, compared to those of KR and KS. The spectral features obtained from UV/vis., IR and CPMAS $^{13}C$ NMR showed that the aromatic character and oxygen containing functional groups in the humic acids from UJ and YK were relatively higher than those of KR and KS. These results indicate that the soil humic acids from UJ and YK were in a higher degree of humification, which may suggest higher affinity of the humic acids with radionuclides released in the soil environments.