• Title/Summary/Keyword: gel electrophoresis

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Structural Analysis of Class I Integron Gene Cassette and Assessment of Genetic Relationships by PFGE of Salmonella enterica Serovar Typhimurium Isolated in Gyeongbuk Area (경북지역에서 분리된 Salmonella enterica Serovar Typhimurium의 Class I Integron Gene Cassette 특성과 PEGE 유형분석)

  • Sohn, Chang-Kyu;Lee, Jung-A;Lee, Do-Young;Hun, Wan;Jung, Jung-Kyo
    • Korean Journal of Microbiology
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    • v.42 no.1
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    • pp.12-18
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    • 2006
  • Thirty five Salmonella enterica serovar Typhimurium strains were isolated from diarrheic patients and pigs in Gyeongbuk area from 2003 to 2004. All 35 strains (17 strains from diarrheic patients and 18 from pigs) were resistant to more than one drug and most of strains isolated from pigs were resistant to ampicillin, ohloram-phenicol, streptomycin, sulfamethoxazole-trimethoprime, tetracyclin and nalidixic acid. Each isolate was also screened or the presence of class I, II and III integron gene cassettes. Among 35 strains,3 out of 17 strains isolated from diarrheic patients, carried dhfrX-orfF-aadA2 integron gene cassette and among 18 strains isolated from diseased pigs, 11 strains carried dhfrX-orfF-aadA2 integron gene cassette and 1 strain carried aadA2 integron only. But any class II and class II integron gene cassette were not detected in 35 strains. Thirty five strains were divided by five pulsotypes. Thirty one strains out of thirty five were pulsotype A. Among the remaining 4 strains, one each strain belonged to pulsotype B, C, D and pulsotype E. This data of pulsotypes showed that the widespread of pulsotype A, Salmonella enterica serovar Typhimurium in human and pigs in Gyeongbuk area may have been caused by the dissemination of a few epidemic strains in this area. Thirteen strains contain dhfrX-orfF-aadA2 integron gene cassette showed pulsotype A and one strain contains dhfrX-orfF-aadA2 integron gene cassette showed pulsotype B. One strain contains aadA2 integron showed pulsotype E. But fifteen strains do not contain any integron showed pulsotype A.

Biological Activity and Chemical Characteristics of Fermented Silkworm Powder by Mold (유용곰팡이 균주에 의한 발효 누에분말의 이화학적 특성 및 생리활성)

  • Cha, Jae-Young;Kim, Yong-Soon;Kang, Pil-Don;Ahn, Hee-Young;Eom, Kyung-Eun;Cho, Young-Su
    • Journal of Life Science
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    • v.20 no.2
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    • pp.237-244
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    • 2010
  • Five mold strains - Mucor racemousus, Rhizopus oryzae, Aspergillus oryzae, Aspergillus kawachii, and Monascus purpureus - were used for the fermentation of both freeze-dried and air-dried silkworm powders. The concentrations of proteins and minerals, electrophoretical protein patterns, fatty acid composition and the activities of fibrinolytic and antioxidation with freeze-dried silkworm (FDSW) or air-dried silkworm powders (ADSW) fermented by the five molds were investigated. The concentrations of major minerals in fermented FDSW and ADSW powders were K by 72.0-76.3 and 77.1-78.9 ppm, Mg by 29.6-49.7 and 44.3-58.7 ppm, Ca by 1.9-14.9 and 9.8-21.6 ppm and Zn by 0.64-0.70 and 4.17-4.52 ppm, respectively. Major fatty acids in fermented FDSW and ADSW powders were linolenic acid, oleic acid, and palmitic acid. There were slightly varietal differences in electrophoretical protein patterns when total protein patterns of fermented FDSW and ADSW powders were analyzed by native-and SDS-polyacrylamide gel electrophoresis (PAGE). DPPH radical scavenging activity was slightly stronger in fermented ADSW powders than that in fermented FDSW. Fibriolytic activity was only detected in the FDSW fermented by Aspergillus kawachi and Monascus purpureus. These results may provide the basic data to understand the biological activities and chemical characteristics of silkworm fermented by mold, which can be used for the development of functional foods.

Rapid Extraction of DNA using Ion Exchange Resin for Early Detection of Mycobacterium tuberculosis by the Polymerase Chain Reaction (결핵균 PCR에서 이온교환수지를 이용한 신속한 DNA 분리)

  • Kim, Cheol-Min;Park, Seung-Kyu;Shon, Mal-Hyun;Song, Sun-Dae;Kim, Young;Jun, Eun-Sook;Son, Han-Chul;Jung, Byung-Sun
    • Tuberculosis and Respiratory Diseases
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    • v.43 no.1
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    • pp.30-37
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    • 1996
  • Background: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. Methods: We used the $InstaGene^{TM}$ DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1,245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2,536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. Results: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value. Conclusion: Even though both methods had lower possibility of cross contamination, shorter time requirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usefulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.

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Effect of the magnetism(neodymium magnet) on growth factor receptors of osteoblasts (희토류 자석의 자성이 골모세포 성장인자 수용체의 증가에 미치는 영향에 관한 연구)

  • Lee, Sang-Min;Lee, Sung-Bok;Choi, Boo-Byung
    • Journal of Dental Rehabilitation and Applied Science
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    • v.19 no.2
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    • pp.87-96
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    • 2003
  • The purposes of this study were to find out the optimum intensity of magnetic field where magnetism could promote the activity of osteoblast, and to discover the possibility of clinical application in the areas of dental implants and bone grafts by confirming the effect of clinically increasing bone formation. In this experiment, we used the Neodymium magnet, which had magnetic power six times as strong as the current ones and enabled the resistances against the demagnetization up to 20 to 50 times to be minimized with the size of 1mm in sight. In order to culture cells, a specially designed device was used. It was made to adjust the distance and accordingly to control the intensity of the magnetic field, by placing the cell culture plate in the center with a magnet of 1mm long and thick installed on the both ends. Using MC3T3-E1 cell, a kind of osteoblast-like cell, we cultured, for 24 hours, not only the test group which had been cultured under the magnetic fields with different intensity of 5, 10, 50, 100, 500, and 1000 Gauss, but also the control group excluding the influences of the magnetic field. After observing the cell's form and the density of the culture medium through an inverted microscope, we made a series of proceedings needed for the immunofluoroscence staining, such as fixation, normal serum reaction, primary antibody reaction, and secondary antibody reaction. And with a fluorescence microscope, we observed those-above and compared the frequency of expression of IFG-1 receptor. To make a Western immunoblotting analysis, the cells cultured under the same condition as the above had the procedure of the lysis buffer and the acrylamide gel electrophoresis was carried out. Protein transferred into the nitrocellulose membrane and tested on the primary and the secondary antibody reactions was observed and compared. The results were as follows: When observed through an inverted microscope, the nuclear divisions of the cells under the magnetic field of 10 Gauss were the most active, and the density of the cells could be observed the most enormously. As the result of an immunofluoroscence staining of IGF-1 receptor, the expression of IFG-1 was the most frequently observed under the magnetic field of 10 Gauss. On the other hand, few differences of consideration were made between the test group cultured under the magnetic fields of 5, 500, and 1000 Gauss and the control group. In respect of the expression of IFG-1 receptor, the test group cultured under the magnetic fields of 50 and 100 Gauss were higher than the control group, and lower than that cultured under the magnetic field of 10 Gauss.(p<0.05) According to the Western immunoblotting analysis, the band of IFG-1 receptor which had 85KDa of molecular weight was the darkest. Judging from the above-mentioned results, the growth factor receptor of an osteoblast cell which was an important criterion for the bone formation was increased in maximum under the magnetic field of 10 Gauss. Moreover it was observed that the optimum intensity of magnetic field in which magnetism made the activity of the osteoblast cell increase was about 10 Gauss.

Physicochemical Properties and Microbial Analysis of Korean Solar Salt and Flower of Salt (한국산 꽃소금과 천일염의 이화학적 특성 및 미생물 분석)

  • Lee, Hye Mi;Lee, Woo Kyoung;Jin, Jung Hyun;Kim, In Cheol
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.42 no.7
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    • pp.1115-1124
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    • 2013
  • The present study was conducted to ensure the diversity of domestic solar salt by analyzing the composition and microbiological characteristics of solar salt (from Docho island: DS) and the flower of salt produced in different Korean salt flats (Sinui island: SF, Bigum island: BF, and Docho island: DF). The analyses showed that the moisture content of the three types of flower of salt and solar salt ranged from 10.54~13.82% and NaCl content ranged from 78.81~84.61%. The mineral content of those salts ranged from 3.57~5.51%. The content of insoluble matter in these salts was $0.01{\pm}0.00{\sim}0.05{\pm}0.00%$. The sand content of these salts was $0.01{\pm}0.01{\sim}0.03{\pm}0.01%$. By Hunter's color value analysis, the color of the flower of salt was brighter and whiter than solar salt. The salinity of the flower of salt was a little higher than solar salt as well. The magnesium and potassium ion content of DF was $9,886.72{\pm}104.78mg/kg$ and $2,975.23{\pm}79.73mg/kg$, respectively, which was lower than the content in SF, BF, and DS. The heavy metal content of all salts was acceptable under the Korean Food Sanitation Law. The flower of salt was confirmed to be sweeter and preferable to solar salt. More than 80% of the solar salt crystals were 2~3 mm in size, whereas crystals from the flower of salt were 0.5~2 mm in size. The bacterial diversity of DF and DS were investigated by culture and denaturing gradient gel electrophoresis (DGGE) methods. The number of cultured bacteria in flower of salt was approximately three times more than solar salt. By DGGE analysis, major microbes of DF were Maritimibacter sp., Cupriavidus sp., and unculturable bacteria, and those of DS were Cupriavidus sp., Dunalidella salina and unculturable bacteria. The results of DGGE analysis showed that major microorganisms in solar salts were composed of unidentified and unculturable bacteria and only a few microorganisms were culturable.

Cellular Analysis and Measurement of Mucin in Sputum of Chronic Airway Disease (만성기도질환의 객담세포분석과 mucin의 측정)

  • Kim, Ki-Up;Kim, Yang-Ki;Shin, Chan-Young;Kim, Do-Jin;Uh, Soo-Taek;Kim, Yong-Hoon;Ko, Kwang-Ho;Park, Choon-Sik
    • Tuberculosis and Respiratory Diseases
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    • v.49 no.1
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    • pp.82-92
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    • 2000
  • Background : In chronic airway disease, mucus secretion is increased, but extraction of mucin, which is the main component of mucus secretion, is a very complicated and limited in clinical use. Recently, monoclonal antibody for mucin was developed for possible clinical use. In this study, cellular analysis and quantification of respiratory mucin in sputum of patients with chronic airway diseases were performed. Method : Sputum was collected from patients with asthma(n=33), bronchiectasis(n=8) or chronic bronchitis (n=13) by spontaneous expectoration or by hypertonic saline induction. Collected sputums was treated by 0.1% dithiotreitol to dissociate the disulfide bond of the mucus and filtered through a nylon gauze. Total cell count, viability and differential count were measured. For detection of mucin, collected samples were treated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and then with monoclonal antibody(HMO2), as the primary antibody, and PAS stain. The amount of mucin was measured with ELISA by HMO2. Correlation with clinical information, cellular analysis, and amount of measured mucin were analyzed. Results : Total cell counts of sputum were significantly increased in patients with bronchiectasis but viability remained the same. Eosinophils were significantly increased in patients with asthma, neutrophils in bronchiectasis chronic bronchitis, respectively (p<0.05). The results of Western blotting and PAS staining confirmed the presence of glycoproteins and matched? with mucin. The amounts of mucin measured by ELISA were not significantly different among the disease groups. Significant correlation was identified between the amount of mucin and viability(r=-0.482, p<0.05). Conclusion : Inflammatory cells in the sputum of those with chronic airway disease were different for each disease type. Measurement of mucin by ELISA via monoclonal antibodies may be a simple method for the evaluation of chronic airway disease.

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Antigen analysis of Toxoplasma gondii Iysate and excretory-secretory materials by enzyme-linked immunoelectrotransfer blot (EITB) (효소면역 전기영동이적법에 의한 톡소포자충 용해물 및 분비 항원의 분석)

  • 안명희;손혁진
    • Parasites, Hosts and Diseases
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    • v.32 no.4
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    • pp.249-258
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    • 1994
  • Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplosmo Iysate (RH strain) , frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain) . Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1 :512 of 10 days after primary immunization, 1 : 2,048 of 10 days after secondary immunization, 1: 1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplcsma major antigens corresponding to n of 24 kDa, 27 kDa,30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T gondii, the IgG antibody bud of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory- secretory materials and Iysate was important major antigen of T gondii (RH).

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Production of Amylase by a Thermophi1ic Fungus, Mucor Sp. (고온성(高溫性) 사상균(絲狀菌) Mucor Sp.에 의(依)한 Amylase의 생산(生産))

  • Lee, Sang Ho;Park, Yoon Joong
    • Korean Journal of Agricultural Science
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    • v.15 no.2
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    • pp.153-163
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    • 1988
  • This experiment was carried out to obtain the thermophilic fungus producing amylase and to investigate properties of the amylase. The selected strain, L-11 was obtained from soil in the vicinity of a hot spring and identified as Mocor sp.. And then the conditions for enzyme production in wheat bran cultures and properties of the crude enzyme were investigated. Furthermore, the enzyme was purified and the characteristics of purified enzyme were studied. The results obtained were as follows: 1. On the wheat bran medium added 80-100% water, amylase was effectively produced by the selected strain, L-11 for 48 hrs incubation at $50^{\circ}C$. 2. When the crude enzyme solution of the strain L-11 was passed through DEAE-Sephadex A-50 column chromatography, two peaks having amylase activity were obtained, and one peak was that of the main enzyme (enzyme of B peak). 3. The purified enzyme (enzyme of B peak) was recognized as single protein band on polyacrylamide disc gel electrophoresis. 4. In the hydrolysis reaction of soluble starch by the enzyme of main amylase, oligosaccharides produced at early stage were maltose and maltotriose mainly and procedure of the reaction maltose amount of maltose and glucose was increased. 5. The strain L-11 was recognized as a special strain producing ${\alpha}-amylase$ mainly and scarcely glucoamylase. 6. The optimum pH, optimum temperature, pH stability, and temperature stability of ${\alpha}-amylase$ were pH 4.0, $60-65^{\circ}C$, pH 4.0-9.0, and below$70^{\circ}C$.

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Genetic Analysis of Some Polymorphic Isozymes in Pinus densiflora (I) -Inheritance of Glutamate-Oxalate Transaminase and Leucine Aminopeptidase, and Linkage Relationship among Allozyme Loci- (소나무의 몇가지 다형적(多形的) 동위효소(同位酵素)의 유전분석(遺傳分析)(I) -Glutamate-Oxalate Transaminase 와 Leucine Aminopeptidase의 유전(遺傳)과 동위효소(同位酵素) 유전자좌(遺傳子座) 간(間)의 연관관계(連關關係)-)

  • Kim, Zin Suh;Hong, Yong Pyo
    • Journal of Korean Society of Forest Science
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    • v.58 no.1
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    • pp.1-7
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    • 1982
  • Megagametophyte and embryo tissue of Pinus densiflora were subjected to study the inheritance of glutamate-oxalate transaminase(GOT) and leucine aminopeptidase(LAP), and linkage relationship among isozyme loci coding both enzymes by starch gel zone-electrophoresis. Four zones of activity were observed for GOT. No variation was found in the fastest migrating zone (GOT-A). Electrophoretic phenotypes of the other two zones (GOT-B and GOT-C) showed 1:1 segregation ration, suggesting that each zone is controlled by a single locus. Foru and three alleles were identified at both loci respectively. The isozyme pattern of the fourth zone(GOT-D), migrated cathodally, coincided precisely with that of GOT-C. Whether the two zones are controlled by the same locus or by two tightly linked loci remained unknown. In all three variant GOT zones, heterozygoes embryos produced triple band patterns, indicating that GOT isozyme in Pinus densiflora is a dimer. Two zones of activity stained for LAP were found. The segregation of the two zones (LAP-A and LAP-B) suggested that tow loci control each of both isozymes. Two and three alleles were identified at both loci. GOT-B and LAP-B were found to be tightly linked, showing an average recombination frequency of 12.5 percent. Slight deviation from independent assortment was observed between GOT-B and GOT-C, with recombination frequency of 41 percent.

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The Effects of Monoamine Oxidase A CA Repeat Polymorphism on Behavioral Trait and Clinical Characteristics in Korean Male Alcoholics (단가아민 산화제 A CA 반복 유전자 다형성이 한국 알코올의존 남자환자의 행동특성과 임상양상에 미치는 영향)

  • Lee, Jung-Sik;Yang, Byung-Hwan;Lee, Kyung-Mee;Kim, Hong-Kwan;Kim, Yong-In;Chai, Young-Gyu
    • Korean Journal of Biological Psychiatry
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    • v.7 no.1
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    • pp.64-73
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    • 2000
  • There are several candidate genes in genetic study of alcoholism. Among them, allelic associations have been reported between MAOA CA repeat polymorphism and alcohol dependence, recently. And also, several studies have been investigated genotype-phenotype relationships between MAOA CA repeat polymorphism and clinical manifestations. The authors tried to identify differences in allelic frequency of MAOA CA repeat polymorphism between alcohol dependence and controls, and in behavioral trait and clinical characteristics according to MAOA CA repeat polymorphism. We also tried to investigate genotype-phenotype relationships between MAOA CA repeat polymorphism and behavioral trait such as aggression. We examined 49 male patients with alcohol dependence(DSM-IV) who had been admitted in Yong-In Mental Hospital from June 1st 1998 to October 31th 1998. We performed semistructured interview for demographic and clinical characteristics. Self-report questionnaire for BDHI(Buss-Durkey Hostility Inventory) was given to all subject at least 4weeks later after admission. Using polymerase chain reaction and polyacrylamide gel electrophoresis, MAOA CA repeat polymorphism were observed in 52 male controls and 49 male patients with alcohol dependence. We devided alcoholic patients into two groups according to allelic length of MAOA CA repeat polymorphism ; alcoholics with short alleles(${\leq}$119bp, N=20) and alcoholics with long alleles(${\geq}$123bp, N=29). T-test, ${\chi}^2$-test and Fisher exact probability test were used for statistical analysis. There were no significant differences in frequency of each allele and short and long alleles of MAOA CA repeat polymorphism between alcoholics and controls. But there were significant differences in clinical symptoms and behavioral trait between alcoholics with short and long alleles. In clinical symptoms, alcoholics with long alleles used alcohol more frequently during one month before admission, had much more maximum amount of beer drinking and reported withdrawal seizure more frequently than with short alleles. In contrary, alcoholics with short alleles expressed depressed mood and guilty feeling more frequently and wanted complete abstinence as a treatment goal more frequently than with long alleles. In behavioral trait, alcoholics with long alleles had higher total aggression score and showed much more self-assertive attitude(subscale of expression of aggression) than with short alleles. Allelic length of MAOA CA repeat polymorphism was correlated with self-assertive attitude and accounted for 9% of the variance of self-assertive attitude. And also, predictable variables of allelic length of MAOA CA repeat polymorphism were drinking frequency and self-assertive attitude. Our findings suggest that MAOA CA repeat polymorphism may provide some behavior modifying role especially in self-assertive attitude and indirect symptom modifying role in Korean male alcoholics.

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