• Journal of Microbiology and Biotechnology
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• 제23권1호
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• pp.76-84
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• 2013

A polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) technique followed by sequencing of the 16S rDNA fragments eluted from the bands of interest on denaturing gradient gels was used to monitor changes in the bacterial microflora of two commercial kimchi, salted cabbage, and ingredient mix samples during 30 days of fermentation at $4^{\circ}C$ and $10^{\circ}C$. Leuconostoc (Lc.) was the dominant lactic acid bacteria (LAB) over Lactobacillus (Lb.) species at $4^{\circ}C$. Weissella confusa was detected in the ingredient mix and also in kimchi samples throughout fermentation in both samples at $4^{\circ}C$ and $10^{\circ}C$. Lc. gelidum was detected as the dominant LAB at $4^{\circ}C$ in both samples. The temperature affected the LAB profile of kimchi by varing the pH, which was primarily caused by the temperature-dependent competition among different LAB species in kimchi. At $4^{\circ}C$, the sample variations in pH and titratable acidity were more conspicuous owing to the delayed growth of LAB. Temperature affected only initial decreases in pH and initial increases in viable cell counts, but affected both the initial increases and final values of titratable acidity. The initial microflora in the kimchi sample was probably determined by the microflora of the ingredient mix, not by that of the salted cabbage. The microbial distributions in the samples used in this study resembled across the different kimchi samples and the different fermentation temperatures as the numbers of LAB increased and titratable acidity decreased.

• Proceedings of the National Institute of Ecology of the Republic of Korea
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• 제4권3호
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• pp.104-114
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• 2023

In this study, the distribution, bait preference, hosts, symbionts, habitat status, and cytochrome c oxidase subunit-I sequences of the ant Linepithema humile were analyzed. This species habitat comprised approximately 1.5 km along stone crevices, flower gardens, roadsides, and container yards in the vicinity of Busan station, Korea. Feeding activity of this species was confirmed in 9 species of 7 families of plants. Which was approximately 14% of total flora (63 species of 37 families) in the studied area. Particularly, it was observed that Dendranthema indicum and Camellia japonica were the most frequently visited. Further, we verified that they interacted with four species of aphids. We identified 22 species of ants (17 genera, 4 subfamilies) inhabiting the Busan station area, including L. humile, Tetramorium tsushimae, Nylanderia flavipes and the alien species Paratrechina longicornis. In areas where L. humile was the dominant species, the habitat of local native ants was reduced by approximately 30%. Bait preference was ranked in the following order: jelly, water with glucose (25%), and yogurt, with the highest preference for baits containing sugar and protein. The 630-bp cytochrome c oxidase subunit-I sequences of the local L. humile populations were 99% identical to those of L. humile in the National Center for Biotechnology Information database, and the Argentine ants that invaded Korea had a sequence identical to that of other invasive populations from China, Japan, and New Zealand. As L. humile rapidly increases due to domestic adaptation, continuous monitoring and control strategy for eradication are needed to protect domestic biodiversity.

• 대한수의학회지
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• 제25권1호
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• pp.7-17
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• 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.

• IMMUNE NETWORK
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• 제1권1호
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• pp.77-86
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• 2001

Background: Hepatitis C virus(HCV), a family of Flaviviridae, has a host cell-derived envelope containing a positive-stranded RNA genome, and has been known as the maj or etiological agent for chronic hepatitis, hepatic cirrhosis, and hepatocellular carcinoma. There remains a need to dissect a molecular mechanism of pathogenesis for the development of therapeutic and effective preventive measure for HCV. Identification of cellular receptor is of central importance not only to understand the viral pathogenesis, but also to exploit strategies for prevention of HCV. This study was aimed at identifying peptide mimotopes inhibiting the binding of E2 protein of HCV to MOLT-4 cell. Methods: In this study, phage peptide library displaying a random peptides consisting of 7 or 12 random peptides was employed in order to pan against E2 protein. Free HCV particles were separated from the immune complex forms by immunoprecipitation using anti-human IgG antibody, and used for HCV-capture ELISA. To identify the peptides inhibiting E2-binding to MOLT-4 cells, E2 protein was subj ect to bind to MOLT-4 cells under the competition with phage peptides. Results: Several phage peptides were selected for their specific binding to E2 protein, which showed the conserved sequence of SHFWRAP from 3 different peptide sequences. They were also able to recognize the HCV particles in the sera of HCV patients captured by monoclonal antibody against E2 protein. Two of them, showing peptide sequence of HLGPWMSHWFQR and WAPPLERSSLFY respectively, were revealed to inhibit the binding of E2 protein to MOLT-4 cell efficiently in dose dependent mode. However, few membrane-associated receptor candidates were seen using Fasta3 programe for homology search with these peptides. Conclusion: Phage peptides containing HLGPWMSHWFQR and WAPPLERSSLFY respectively, showed the inhibition of E2-binding to MOLT-4 cells. However, they did not reveal any homologues to cellular receptors from GenBank database. In further study, cellular receptor could be identified through the screening of cDNA library from MOLT-4 or hepatocytes using antibodies against these peptide mimotopes.

• Biomolecules & Therapeutics
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• 제7권4호
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• pp.307-314
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• 1999

The human muscarinic acetylcholine receptor (mAChR) subtypes Hml, Hm2 and Hm3 have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus expression system. Expression of relevant DNA, transcript and receptor proteins was identified by PCR, Northern blotting and [$^{3}H$]QNB binding, respectively. As assessed by [$^{3}H$]QNB binding sites, yields of muscarinic receptors in membrane preparations in this study were as about 5-20 times high as those in mammalian cells reported in previous studies. The [$^{3}H$]QNB competition binding studies with well-known subtype-selective mAChR antagonists showed that the receptors expressed in Sf9 cells retain the pharmacological characteristics expected for the ml , m2 and m3 muscarinic receptors. The ml-selective antagonist, pirenzepine, displayed a considerably higher affinity for Hml by 110-fold and 35-fold than for Hm2 and Hm3, respectively, The m2-selective methoctramine displayed a significantly higher affinity for Hm2 than for Hml and Hm3 (10- and 26-fold, respectively). p-F-HHSiD exhibited high affinity for Hm3 that is not significantly different from those for Hml, but 66-fold higher than its affinity for Hm2. The functional coupling of the recombinant receptors to second messenger systems was also examined. While both Hml and Hm3 stimulated phosphoinositide hydrolysis upon activation by carba-chol, Hm2 produced no response. On the other hand, activation of mAChRs induced the inhibition of forsko-lin-stimulated cyclic AMP formation in Hm2-expressing cells, whereas the significant dose-dependent increase in or poor response on cyclic AMP formation were produced in Hml or Hm3-expressing cells, respectively. These results indicate the differential coupling of recombinant Hml, Hm2 and Hm3 receptors expressed in SF9 cells to intracellular signalling system.

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.199-205
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• 1998

흰쥐 난소에서의 Luteinizing hormonoe (LH) subunit 유전자 발현과 LH polypeptide의 존재를 조사하였다. 이를 위해 LH subunit들에 대한 역전사 중합효소연쇄반응 (RT-PCR)을 시행하고, 난소내 LH 함량을 방사면역측정법으로 정량하였다. 뇌하수체와 정소에서 공통적으로 존재하는 LH-$\beta$ subunit$(LH-\beta})$의 exon에 해당되는 primer를 사용하여 RT-PCR을 시행한 결과 흰쥐 난소에서도 뇌하수체, 정소와 같이 306 bp band가 확인되었고, 정소특이적인 exon에 해당되는 primer를 사용한 결과 정소와 난소에서 예상대로 428 bp band가 검출되었다. 또한 LH, FSH, TSH 그리고 hCG에서 공통적으로 발현되는 common $\alpha$-subunit $(C_\alpha)$의 전사물질도 PCRdml 의해 증폭되었다. 방사면역측정법에서는 LH standard curve와 난소추출물을 사용한 curve가 동일하게 sigmoid 형태를 보임으로서 흰쥐 난소내에 immunoreactive LH가 존재함이 증명되었다. 인위적으로 성적인 성숙을 유도한 PMSG 주사 동물에서 혈중 LH 수준은 주사 후 48시간에 preovulatory LH surge와 유사한 최고 수준을 나타냈으나, 난소내 LH 함량의 경우 주사 24시간 후부터 급격히 감소하여 주사 48, 72시간군까지도 낮은 수준이 유지되었다. 이 결과는 흰쥐 난소의 LH가 생리적으로 조절되고 그 조절방식이 뇌하수체에서와는 다를 가능성을 시사하는 것이다. 본 연구는 흰쥐의 난소에서 LH가 유전자가 발현됨을 최초로 보고한 것이며, LH의 경우 내분비적 경로 (endocrine; 뇌하수체로 부터의 LH)외에도 국부적 경로 (autocrine이나 paracrine; 난소내에서 합성되는 LH)를 통해 난소의 생리와 기능 조절을 담당함을 시사한다.

• Molecules and Cells
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• 제24권2호
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• pp.276-282
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• 2007

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin ($Ca^{2+}/CaM$)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about $Ca^{2+}/CaM$-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative $Ca^{2+}$-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a $Ca^{2+}$-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM $Mn^{2+}$. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other $Ca^{2+}/CaM$-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and $Ca^{2+}/CaM$-dependent protein kinase), increasing the concentration of calmodulin to more than $3{\mu}M$ suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis $Ca^{2+}/CaM$-dependent protein kinase which is presumably involved in CaM-mediated signaling.

• 한국식품위생안전성학회지
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• 제21권2호
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• pp.100-106
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• 2006

ER와 리포터 유전자인 $\beta$-galactosidase가 도입된 효모재조합검색시험법을 이용하여 파라벤류의 내분비계 장애작용을 검색하였다. 양성대조 시험물질로 앞의 시험법들과 동일하게 E2와 BPA를 설정하여 파라벤류의 에스트로젠성을 비교분석 하였다. E2의 경우 $10^{-9}M$에서 가장 활성이 높게 관찰되었고, BPA의 경우 $10^{-7}M$에서 에스트로젠성이 가장 높았다. 파라벤의 경우 이소프로필파라벤이 $10^{-9}M$에서 $10^{-3}M$까지 시험하였을 때, 농도 의존적으로 에스트로젠성이 증가하였으며, $10^{-9}M$의 경우 가장 강력한 에스트로젠성을 보였다. 또한 양성대조군인 E2와 비교하였을 때, $10^{-7}M$에서 $10^{-3}M$까지의 이소프로필파라벤은 오히려 E2보다 높은 내분비계 장애작용이 검색되었다. 이소프로필파라벤을 제외한 나머지 파라벤류의 경우 $10^{-4}M$과 $10^{-5}M$의 프로필파라벤과 $10^{-3}M$과 $10^{-4}M$의 에틸파라벤에서 에스트로젠성이 관찰되었다. 또한 MCF-7세포주는 사람의 유방암 세포이면서 $ER{\alpha}$가 존재하여 에스트로젠 또는 에스트로젠 유사물질이 ER와 반응하여 세포의 성장을 유도하게 된다. 이번 연구에서 파라벤의 시험 이전에 이미 내분비계 장애물질로 널리 알려진 BPA와 체내에 존재하는 강력한 에스트로젠이면서 BPA보다 활성이 1000배정도 높다고 알려진 E2를 양성대조군으로 설정하여 MCF-7세포의 성장을 관찰하였다. 72시간동안 BPA와 E2를 다양한 농도로 MCF-7세포에 노출한 후 DNA 양을 측정하였더니, E2의 경우 $10^{-9}M$에서 대조군보다 약 2.5배의 세포성장을 관찰할 수 있었으며, BPA의 경우 $10^{-8}M$에서 대조군 보다 약 2.2배의 세포성장을 관찰할 수 있었다. 에틸파라벤의 경우 $10^{-7}M$에서 $10^{-4}M$까지 농도 의존적으로 MCF-7세포의 성장을 증가시켰고, $10^{-4}M$이 대조군에 비해 세포성장이 약 2.2배에 달하여 양성대조군과 비슷하면서 높은 에스트로젠 유사반응을 보였다. 프로필파라벤, 부틸파라벤, 이소부틸파라벤과 이소프로필파라벤의 경우 $10^{-5}M$의 농도에서 2배 이상의 세포성장이 유도되었고, 이소프로필파라벤의 경우 RPE값이 약 104%에 이르는 등, 내분비계 장애작용이 검색되었다. 한편, 본 연구팀은 ER에 대한 파라벤의 시험관 내 상경적 결합력을 측정하기 위해 $ER{\alpha}$와 $ER{\beta}$ competition binding assay kit를 사용하여 시험하였다. 이 시험법은 E2와 비교하여 파라벤류의 $ER{\alpha}$와 $ER{\beta}$에 반응하는 시험물질의 RBA(relative binding affinities) 값을 측정하였다. 파라벤의 $ER{\alpha}$ 상경적 결합시험의 경우. E2의 $IC_{50}$의 값이 $4.29{\times}10^{-9}$이었고, 이소부틸파라벤의 경우 $4.5{\times}10^{-7}$에 달하여 RBA값이 0.952가 계산되었다. 이전 연구에 의해 밝혀진 BPA의 경우 RBA값이 0.333인데 반하여, 이소부틸파라벤은 약 3배가 높은 내분비계 장애작용이 검색되었다. 파라벤의 $ER{\beta}$ 상경적 결합시험의 경우. $ER{\alpha}$와 마찬가지로 이소부틸파라벤이 $1.94{\times}10^{-7}M$의 $IC_{50}$값을 가지면서 RBA값이 0.471이 계산되었다. 이것은 파라벤이 $ER{\alpha}$와 $\beta$모두와 결합을 할 수 있고 E2와 경쟁적으로 결합을 할 수 있으며 이는 내분비계를 방해할 수 있다는 것을 다시 한번 뒷받침 해주고 있다. 덧붙여 본 연구팀의 결과는 이소부틸파라벤의 경우 경쟁적으로 결합하는 능력 또한 내분비계교란물질로 잘 알려진 BPA만큼의 능력을 가지고 있는 것으로 나타났다. 결론적으로 in vitro적 방법으로 파라벤류들의 에스트로젠성을 측정한 결과 이미 보고된 연구와 비슷하게 화학 구조적으로 더 길거나 분지된 알킬기를 가지는 파라벤일수록 에스트로젠성이 더 강하게 나타났다. 따라서, 식품이나 화장품 등의 보존제로 사용되는 이 화학물질의 과다한 노출은 정상적인 내분비계에 큰 영향을 미칠 가능성이 있다고 판단된다.