• Journal of Life Science
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• v.16 no.1
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• pp.37-43
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• 2006

Gap junction is an ion channel forming between adjacent cells. It also acts as a membrane channel like sodium or potassium channels in a single cell. The amino acid residues up to the $10^{th}$ position in the amino (N)-terminus of gap junction hemichannel affect gating polarity as well as current-voltage (I-V) relation. While wild-type Cx32 channel shows negative gating polarity and inwardly rectifying I-V relation, T8D channel in which threonine residue at $8^{th}$ position is replaced with negatively charged aspartate residue shows reverse gating polarity and linear I-V relation. It is still unclear whether these changes are resulted from the charge effect or the conformational change of the N-terminus. To clarify this issue, we made a mutant channel harboring cysteine residue at the $8^{th}$ position (T8C) and characterized its biophysical properties using substituted-cysteine accessibility method (SCAM). T8C channel shows negative gating polarity and inwardly rectifying I-V relation as wild-type channel does. This result indicates that the substitution of cysteine residue dose not perturb the original conformation of wild-type channel. To elucidate the charge effect two types of methaenthiosulfonate (MTS) reagents (negatively charged $MTSES^-$ and positively charged $MTSET^+$) were used. When $MTSES^-$ was applied, T8C channel behaved as T8D channel, showing positive gating polarity and linear I-V relation. This result indicates that the addition of a negative charge changes the biophysical properties of T8C channel. However, positively charged $MTSET^+$ maintained the main features of T8C channel as expected. It is likely that the addition of a charge by small MTS reagents does not distort the conformation of the N-terminus. Therefore, the opposite effects of $MTSES^-$ and $MTSETT^+$ on T8C channel suggest that the addition of a charge itself rather than the conformational change of the N-terminus changes gating polarity and I-V relation. Furthermore, the accessibility of MTS reagents to amino acid residues at the $8^{th}$ position supports the idea that the N-terminus of gap junction channel forms or lies in the aqueous pore.

• Tuberculosis and Respiratory Diseases
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• v.63 no.2
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• pp.128-138
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• 2007

Backgrounds: Mutations of katG and inhA (ORF and promoter) are known to be related to isoniazid (INH) resistance of Mycobacterium tuberculosis. Because reports on these mutations in Korean isolates are limited (i.e. only the frequency of katG codon 463 was evaluated.), we tried to know the kinds of mutations of two genes and their frequencies in INH resistant Korean M. tuberculosis strains. Methods: PCR was performed to amplify katG (2,223 bp), inhA ORF (-77~897, 975 bp), and inhA promoter (-168~80, 248 bp) from 29 multidrug resistant M. tuberculosis (MDR-TB) DNAs prepared by bead beater-phenol method. Their sequences were determined and analyzed by ABI PRISM 3730 XL Analyzer and MegAlign package program, respectively. Results: All of the isolates had more than one mutation in katG or inhA gene. Twenty seven (93%) of 29 tested strains had katG mutations, which suggests that katG is a critical gene determining INH resistance of M. tuberculosis. Amino acid substitutions, such as Arg463Leu and Ser315Thr, due to point mutations of the katG were the most frequent (62.1% and 55.2%) mutations. In addition, deletion of the katG gene was frequently observed (17.2%). Analyzed Korean MDR-TB isolates also had variable inhA mutations. Point mutation of inhA promoter region, such as -15 ($C{\rightarrow}T$) was frequently found. Substitution of amino acid (Lsy8Asn) due to point mutation ($AAA{\rightarrow}AAC$) of inhA ORF was found in 1 isolate. Interestingly, 14 point mutated types that were not previously reported were newly found. While four types resulted in amino acid change, the others were silent mutations. Conclusions: Although it is not clear that the relationship of these newly found mutations with INH resistance, they show marked diversity in Korean MDR-TB strains. It also suggests their feasibility as a molecular target to supplement determining the INH resistance of clinical isolates because of the possible existence of low-level INH resistant strains.

• Korean Journal of Microbiology
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• v.7 no.4
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• pp.153-158
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• 1969

It is the most important thing to substitute sweet potato for wheat flour as the brewing material of Maggerley(Korean wine) in order to save precious wheat flour which is now consumed as the sole material in it. In this study, the fermenting mash material has been prepared with combination of wheat (KIorean wine) as a first step of sabing wheat flour consuming. The combination ratio of wheat flour and sweet potato starch in mashing, in this experiment, are such as mashing plot of 100% wheat flour (No.3 plot), 50% wheat flour plus 50 % of sweet potato starch (No.2plot), 30% wheat flour plus 70% starch syrup of sweet potato (No. 4 plot), and 60% wheat flour plus sweet potato starch (No. 1 plot). The results of fermentation and chemical components of each mash in experimental plot are as following : 1. Alocholic fermentation was most vigorous in No.4 plost among them, the next are in order to hydrolyze and break down the protein sourece to glucose and amino acid, the new enzymic source were added in experimantal amsh before putting yeasts, those enzymic sources have been developed and prepared in this laboratory No.4 and No.2 plot at same degree. Generally, the activities of alcholic fermentation of all plots are vigorous and fair. The acidity of each fermented mash is in the rangw of 3.7-4.8. 2. Amounts of amino acids in fermented mash are in the range of 145 mg per 100 ml and 158 mg for all experiments, and these amounts are much more than those of present commerical Maggerley. However, the amount of it in the No.4 plots is much less among the m, this is due to the lack of protein source in starch syrup. 3. Amounts of organic acids in fermented mash are in the range of 0.44g per 100ml and 0.62g, these amounts are generally much more than those of the present commercial Maggerley. 4. The result of sensory test for the experimental Maggerley shows good taste and flavour in comparison with those of commercial Maggerely. 5. It is proved that these experimental fermentation of Maggerley is succesful in the partial substitution of sweet potato starch potato starch for wheat flour, and it is also be able to make a good Maggerley in all aspects of quality.

• Korean Journal of Veterinary Research
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• v.39 no.2
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• pp.247-256
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• 1999

A disease of young Holstein calves characterized by recurrent pneumonia, ulcerative and granulomatous stomatitis, enteritis with bacterial overgrowth, periodontitis, delayed wound healing, persistent neutrophilia and death at an early age had been originally described in 1983 and again in 1987. Most of these calves had stunted growth and a persistent, progressive neutrophilia (often exceeding 100,000/ml). By investigation of pedigrees, all of the affected calves have now been traced to a common sire and confirmed by polymerase chain reaction (PCR) diagnostic DNA testing to be homozygous carriers of a defective allele for bovine CD18. Neutrophils from these calves have several functional deficits and, most importantly, fail to adhere in a ${\beta}_2$-integrin dependent manner. The ${\beta}_2$-integrins represent a family of glycoproteins which participate in various leukocyte adhesion reactions during host defense. The presence or absence of ${\beta}_2$-integrin molecules can be demonstrated on the surface of neutrophils, monocytes and lymphocytes from normal or affected calves using specific monoclonal antibodies and flow cytometry, or by colloidal gold immunolabeling and scanning electron microscopy in backscatter mode. Deficiency of the ${\beta}_2$-integrins on all leukocyte types in Holstein calves is analogous to leukocyte adhesion deficiency (LAD) seen in humans. Neutrophils in bovine (BLAD) and human LAD patients are unable to adhere to the endothelial lining of the cardiovascular system thus interrupting egression of neutrophils into infected tissues. Other leukocytes, while still deficient in expression of the ${\beta}_2$-integrins, are still able to efficiently egress from the blood stream due to interactions of other adhesion molecules that are not as highly expressed on neutrophils. Both BLAD cattle and LAD children (who do not receive bone marrow transplants) often die at an early age as a result of the failure of neutrophils to extravasate into infected tissues. In 1991, Shuster, et $al^{27}$, identified two point mutations within the alleles encoding bovine CD18 in a Holstein calf afflicted with leukocyte adhesion deficiency. One mutation causes an aspartic acid to glycine substitution at amino acid 128 (D128G) in an extracellular region of this adhesion glycoprotein that is highly conserved (> 95% identity) between humans, cattle and mice. The other mutation is silent. Numerous calves with clinical symptoms of leukocyte adhesion deficiency have since been tested and all have been found homozygous for the D128G allele. In addition, calves homozygous far the D128G allele have been identified during widespread DNA testing in the United States. All cattle with the mutant allele are related to one bull, who through artificial insemination (A.I.), sired many calves in the 1950's and 1960's. The carrier frequency of the D128G CD18 allele among U.S. Holstein cattle had reached approximately 15% among active A.I. bulls and 8% among cows. By 1993, the organization of the dairy industry and the diagnostic test developed to genotype cattle, enabled virtually complete eradication of bovine leukocyte adhesion deficiency among current and future A.I. bulls.

• IMMUNE NETWORK
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• v.20 no.5
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• pp.41.1-41.11
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• 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is a positive-sense single-stranded RNA (+ssRNA) that causes coronavirus disease 2019 (COVID-19). The viral genome encodes twelve genes for viral replication and infection. The third open reading frame is the spike (S) gene that encodes for the spike glycoprotein interacting with specific cell surface receptor - angiotensin converting enzyme 2 (ACE2) - on the host cell membrane. Most recent studies identified a single point mutation in S gene. A single point mutation in S gene leading to an amino acid substitution at codon 614 from an aspartic acid 614 into glycine (D614G) resulted in greater infectivity compared to the wild type SARS-CoV2. We were interested in investigating the mutation region of S gene of SARS-CoV2 from Korean COVID-19 patients. New mutation sites were found in the critical receptor binding domain (RBD) of S gene, which is adjacent to the aforementioned D614G mutation residue. This specific sequence data demonstrated the active progression of SARS-CoV2 by mutations in the RBD of S gene. The sequence information of new mutations is critical to the development of recombinant SARS-CoV2 spike antigens, which may be required to improve and advance the strategy against a wide range of possible SARS-CoV2 mutations.

• Journal of dental hygiene science
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• v.12 no.2
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• pp.109-113
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• 2012

One hundred seventy four Streptococcus pneumoniae clinical isolates were categorized depending on the types of specimens, the age and the gender, respectively. All isolates were analyzed the characteristics of the multi-drug resistance including levofloxacin antibiotics. In the results of analysis depending on the type of samples, it had been confirmed that sputum was the main source of pneumonia infection because 156 of 174 strains (89.7%) were isolated in sputum samples. The opportunity for isolating the S. pneumoniae that had tolerance to levofloxacin was increased in over 51 age patients group compared with other age and male group. Eight strains of isolates were evaluated higher resistant to levofloxacin, and those also showed multi-drug resistant including penicillin, tetracycline, erythromycin, clindamycin and trimethoprim-sulfamethoxazole. In the results of sequence analysis of quinolone resistance determining region in SP32 (MIC $64{\mu}g/mL$) and SP96 (MIC $8{\mu}g/mL$) which were levofloxacin resistant strains, an amino acid substitutions were found Ser-81$\rightarrow$Phe in both GyrA of SP32 and SP96, and Ser-11$\rightarrow$Gly in only SP96. A Ser-79$\rightarrow$Phe substitution of ParC was found in both.

• Research in Plant Disease
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• v.19 no.2
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• pp.124-127
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• 2013

Black necrotic spots were observed from the fruits of paprika that were cultivating in a vinylhouse. The casual agents of the symptom were identified as several isolates of Pepper mild mottle virus (PMMoV) by responses of indicator plants, electron microscopy, and RT-PCR analysis. Symptoms of the viral disease were mild mottle in the young leaves, necrotic spots on the fruits and the fruit apex of paprika, but the symptoms were not shown on the mature leaves. All of the PMMoV isolates were determined as $P_{1.2.3}$ pathotypes from the biological responses on the chilli pepper lines used for discrimination of tobamovirus pathotypes. Pathogenicity of the PMMoV isolates was also confirmed using mechanical inoculation method to paprika seedlings. The coat protein (CP) genes of the PMMoV isolates were compared at the nucleotide and amino acid levels with the previously published PMMoV isolate. The isolates share 96 to 99% CP nucleotide identity among the isolates. The CP of $P_{1.2}$-pathotype PMMoV-P2 presented Met at position 139, But the CPs of $P_{1.2.3}$-pathotype PMMoVs from paprika showed Met to Asn substitution at the same position. This is the first report of identification of $P_{1.2.3}$-pathotype PMMoV isolates from paprika in Korea.

• Food Science of Animal Resources
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• v.24 no.4
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• pp.355-360
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• 2004

In Korean beef market, one of the major problems is mislabeling or fraudulent distribution of Holstein dairy meat or imported beef as domestic Hanwoo meat. Therefore, there has been a great need for a development of technology to identify beef breeds in meat and meat products. This study was carried out to develop the accurate and reliable method for the identification of beef breed using PCR-RFLP marker of MC1R, MGF and TYRPl genes affecting coat colors in cattle. A single base substitution (G\longrightarrowT transition) at the codon for amino acid position 104 of MC1R gene was identified between Hanwoo and Holstein and Angus breeds. The change at this position creates Msp I restriction site in Holstein and Angus, but not in Hanwoo. When the DNA amplified products (537 bp) was digested with Msp I, Hanwoo meat showed a single band of 537bp, while two fragments of 329bp and 208 bp were observed in Holstein meat and Angus breed, respectively. Thus, breed-specific RFLP marker in the MC1R gene can be used to distinguish between Hanwoo meat and Holstein and Angus meats. In the RFLP genotype of MGF gene, the frequency of r/r type was 75％ in Manwoo, whereas the frequency of R/R was 80％ in Hereford breed. Holstein and Angus breeds showed 100％ for R/r type. Therefore, Hanwoo meat showed significant difference in the MGF genotype frequencies compared with those of Holstein meat and imported beef cattle breeds. However, TYRP1 gene showed the same genotype in all breeds examined. Thus, this TYRP1 gene can not be used as a molecular marker for breed identification. As a consequence, we suggest that RFLP markers of the MC1R and MGF coat color genes could be used as DNA marker for identification of Hanwoo meat from Holstein and imported meats.

• Journal of Microbiology
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• v.44 no.2
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• pp.192-199
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• 2006

Pseudomonas putida KL47 is a natural isolate that assimilates benzene, 1-alkylbenzene $(C_1-C_4)$, biphenyl, p-cumate, and p-cymene. The genetic background of strain KL47 underlying the broad range of growth substrates was examined. It was found that the cym and cmt operons are constitutively expressed due to a lack of the cymR gene, and the tod operon is still inducible by toluene and biphenyl. The entire array of gene clusters responsible for the catabolism of toluene and p-cymene/p-cumate has been cloned in a cosmid vector, pLAFR3, and were named pEK6 and pEK27, respectively. The two inserts overlap one another and the nucleotide sequence (42,505 bp) comprising the cym, cmt, and tod operons and its flanking genes in KL47 are almost identical (>99 %) to those of P. putida F1. In the cloned DNA fragment, two genes with unknown functions, labeled cymZ and cmtR, were newly identified and show high sequence homology to dienelactone hydrolase and CymR proteins, respectively. The cmtR gene was identified in the place of the cmtI gene of previous annotation. Western blot analysis showed that, in strains F1 and KL47, the todT gene is not expressed during growth on Luria Bertani medium. In minimal basal salt medium, expression of the todT gene is inducible by toluene, but not by biphenyl in strain F1; however, it is constantly expressed in strain KL47, indicating that high levels of expression of the todST genes with one amino acid substitution in TodS might provide strain KL47 with a means of adaptation of the tod catabolic operon to various aromatic hydrocarbons.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.721-727
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• 2007

Stability-enhanced mutants, H44, 11-94, 5A2-84, and F8, of L-threonine aldolase(L-TA) from Streptomyces coelicolor A3(2)(SCO1085) were isolated by an error-prone PCR followed by a high-throughput screening. Each of these mutant, had a single amino acid substitution: H177Y in the H44 mutant, A169T in the 11-94 mutant, D104N in the 5A2-84 mutant and F18I in the F8 mutant. The residual L-TA activity of the wild-type L-TA after a heat treatment for 20 min at $60^{\circ}C$ was only 10.6%. However, those in the stability-enhanced mutants were 85.7% for the H44 mutant, 58.6% for the F8 mutant, 62.1% for the 5A2-84 mutant, and 67.6% for the 11-94 mutant. Although the half-life of the wild-type L-TA at $63^{\circ}C$ was 1.3 min, those of the mutant L-TAs were longer: 14.6 min for the H44 mutant, 3.7 min for the 11-94 mutant, 5.8 min for the 5A2-84 mutant, and 5.0 min for the F8 mutant. The specific activity did not change in most of the mutants, but it was decreased by 45% in the case of mutant F8. When the aldol condensation of glycine and 3,4-dihydroxybenzaldehyde was studied by using whole cells of Escherichia coli containing the wild-type L-TA gene, L-threo-3,4-dihydroxyphenylserine(L-threo-DOPS) was successfully synthesized with a yield of 2.0 mg/ml after 20 repeated batch reactions for 100 h. However, the L-threo-DOPS synthesizing activity of the enzyme decreased with increased cycles of the batch reactions. Compared with the wild-type L-TA, H44 L-TA kept its L-threo-DOPS synthesizing activity almost constant during the 20 repeated batch reactions for 100 h, yielding 4.0 mg/ml of L-threo-DOPS. This result showed that H44 L-TA is more effective than the wild-type L-TA for the mass production of L-threo-DOPS.