• Plant Biotechnology Reports
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• 제5권4호
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• pp.331-344
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• 2011

The well-conserved NBS domain of resistance (R) genes cloned from many plants allows the use of a PCR-based approach to isolate resistance gene analogs (RGAs). In this study, we isolated an RGA (CapRGC) from Capsicum annuum "CM334" using a PCR-based approach. This sequence encodes a protein with very high similarity to Rx genes, the Potato Virus X (PVX) R genes from potato. An evolutionary analysis of the CapRGC gene and its homologs retrieved by an extensive search of a Solanaceae database provided evidence that Rx-like genes (eight ESTs or genes that show very high similarity to Rx) appear to have diverged from R1 [an NBS-LRR R gene against late blight (Phytophthora infestans) from potato]-like genes. Structural comparison of the NBS domains of all the homologs in Solanaceae revealed that one novel motif, 14, is specific to the Rx-like genes, and also indicated that several other novel motifs are characteristic of the R1-like genes. Our results suggest that Rx-like genes are ancient but conserved. Furthermore, the novel conserved motifs can provide a basis for biochemical structural. function analysis and be used for degenerate primer design for the isolation of Rx-like sequences in other plant species. Comparative mapping study revealed that the position of CapRGC is syntenic to the locations of Rx and its homolog genes in the potato and tomato, but cosegregation analysis showed that CapRGC may not be the R gene against PVX in pepper. Our results confirm previous observations that the specificity of R genes is not conserved, while the structure and function of R genes are conserved. It appears that CapRGC may function as a resistance gene to another pathogen, such as the nematode to which the structure of CapRGC is most similar.

• 한국미생물·생명공학회지
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• 제37권3호
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• pp.248-257
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• 2009

Vibrio harveyi 쿼럼 센싱 (quorum sensing; QS) 신호전달에 대한 저해제들이 주 신호물질인 N-3-hydroxybutanoyl-L-homoserine lactone(3-OH-$C_4$-HSL)의 분자 구조를 변형함에 의해 개발되었다. 일련의 구조 변형체들인 N-(3-hyoxysulfonyl)-L-homoserine lactones(HSHLs)들은 고체상 유기합성법 (solid-phase organic synthesis method)으로 합성되었다. 이 물질들의 생체내 쿼럼 센싱 저해능이 V. harveyi 발광을 이용한 bloassay를 system에 의해 측정되었을 때, 모두 의미있는 저해효과를 보여주었다. 이 물질들과 3-OH-$C_4$-HSL 수용체 단백질인 LuxN 사이의 상호작용을 분석하기 위하여 LuxN의 신호 결합 부위를 다른 acyl-HSL 결합 단백질들과의 유사성에 기초하여 시험적으로 결정하였다. 이 추정 신호결합 부위의 부분적 삼차구조를 ORCHESTRA program을 이용하여 예측하였으며, 이 부위 내에서 3-OH-$C_4$-HSL와 HSHLs의 결합 형태와 에너지를 계산하였다. 이렇게 모델링을 통해 얻어진 결과와 생체 내 bioassay를 통해 얻어진 결과의 비교를 통해, 수용체 단백질과 그 리간드 사이의 상호 작용에 관한 in silica 해석이 특히 단백질의 삼차 구조에 대한 정보가 제한적인 경우에 보다 나은 저해제 개발을 위한 유용한 방법이 될 수 있음을 제안한다.

• International Journal of Industrial Entomology and Biomaterials
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• 제20권1호
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• pp.29-35
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• 2010

To compare the quality of manufactured goods distributed in the domestic markets, 6 isolates of Dongchongxiacao products, namely, 4 Paecilomyces tenuipes specimens (J2P, 901A, 901B, and 901C), 1 Cordyceps militaris specimen (901D), and 1 Cordyceps sinensis specimen (CI-1), were analyzed for their physicochemical properties and sequence-structure relationships. P. tenuipes (J2P), a kind of Dongchongxiacao, was successfully inoculated on silkworms by percutaneous infection of Rural Development AdminstraionNam et al., 1999); fruiting bodies were then formed on the complete surface of the pupa. Since P. tenuipes (J2P) from silkworm larva was also proved to have remarkable pharmacological activities, it has been produced in bulk and has been successfully sold to buyers in the Korean market. Additionally, imitation products such as 901A, 901B, 901C, 901D, and CI-1 were sold simultaneously, resulting in deterioration of product quality. This research focuses on establishing quality standards to discriminate between the original and imitation products circulating in the market. The products obtained for the experiments included J2P, 901A, 901B, 901C, 901D, and CI-1; proximate analysis was performed for these products. The hosts and methods of conidia inoculation for proliferation of mycelia differed among the products. P. tenuipes (J2P) was proliferated in live silkworm larvae, and dead silkworm pupae were used to produce 901A, 901B, and 901C. On the other hand, 901D was produced on hulled rice medium. Quality analysis of C. sinensis revealed that CI-1, which was imported from China, smelled bad and proved to be a counterfeit with the fruiting body glued to the insect by twigs. The results of the proximate analysis of 901A, 901B, 901C, and 901D were similar to those of J2P with respect to the moisture content. Otherwise, J2P contained higher crude protein than 901A, 901B, 901C, and 901D, but contained very low fat. C. militaris (901D) and C. sinensis (CI-1) had low crude protein content-12.79% and 9.78% respectively-as compared to that of J2P, which was 62.38%. In contrast to the crude ash content of 6.4% in J2P, the crude ash content of CI-1 was 18.51% and this specimen was found to contain many impurities. phylogenetic analysis of P. tenuipes revealed that the sequence similarity of J2P, 901B, and 901C was in the range of 92.3~92.7%. Additionally, differences in the sequences were found at the positions 65 bp, 436 bp, 441 bp, 463 bp, etc.

• Journal of Microbiology and Biotechnology
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• 제26권4호
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• pp.730-738
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• 2016

est19 is a gene from Bacillus sp. K91 that encodes a new esterase. A comparison of the amino acid sequence showed that Est19 has typical Ser-Gly-Asn-His (SGNH) family motifs and could be grouped into the SGNH hydrolase family. The Est19 protein was functionally cloned, and expressed and purified from Escherichia coli BL21(DE3). The enzyme activity was optimal at 60℃ and pH 9.0, and displayed esterase activity towards esters with short-chain acyl esters (C₂-C₆). A structural model of Est19 was constructed using phospholipase A1 from Streptomyces albidoflavus NA297 as a template. The structure showed an α/β-hydrolase fold and indicated the presence of the typical catalytic triad Ser49-Asp227-His230, which were further investigated by site-directed mutagenesis. To the best of our knowledge, Est19 is a new member of the SGNH hydrolase family identified from thermophiles, which may be applicable in the industrial production of semisynthetic β-lactam antibiotics after modification.

• 미생물학회지
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• 제52권3호
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• pp.269-277
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• 2016

Erm 단백질은 미생물의 23S rRNA의 특정 nucleotide ($A_{2058}$)에 methylation 시킴으로써 $MLS_B$ (macrolide-lincosamide-streptogramin B) 항생제에 대하여 내성을 나타내는 항생제 내성인자 단백질이다. 이 단백질들을 계통수분석을 하였을 때 두 개의 주된 집단 즉 항생제 생성균과 병원균으로 각각 구성된 Actinobacteria와 Firmicutes에서 유래된 단백질로 구분이 된다. 두 집단을 각각 대표하는 2개의 단백질을(항생제 생성균 유래 ErmS와 ErmE, 병원균 유래 ErmC'와 ErmB) 선택하여 그 활성을 비교하였다. 전체적으로 항생제 생성균에서 비롯된 Erm 단백질이 병원균에서 비롯된 단백질에 비해 높은 활성을 보였다: ErmC'와 ErmE 비교시 9배, ErmB와 ErmS 비교시 13배의 차이가 남. ErmS에서 59개의 아미노산이 제거된 NT59TE 단백질의 활성이 야생형 보다 22.5% 정도에 머물렀기 때문에 이러한 활성의 현격한 차이는 ErmS에서는 N-terminal에 붙어있는 가외의 아미노산에 의한 것으로 관찰되었고 ErmE에서는 C-terminal의 가외의 아미노산에 의한 것으로 추정되었다. 그러나 NT59TE가 ErmC'와 ErmB에 비하여 2.2, 3배의 높은 활성을 보이는 것으로 관찰되어 단백질의 핵심부위에서도 높은 활성을 도와주는 부위가 있음을 알 수 있다. 다중 아미노산 배열 정렬로부터 이 부위는 197RWS199 (ErmS와 ErmE 모두로부터 유래), 261GVGGSLY267 (ErmS로부터 유래) 그리고 261GVGGNIQ267 (ErmE로부터 유래)와 291SVV293 (ErmS로부터 유래) 그리고 291GAV293 (ErmE로부터 유래)으로 추정되었다.

• 미생물학회지
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• 제52권2호
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• pp.230-235
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• 2016

본 연구는 Streptomyces subrutilus P5의 천연 Fe superoxide dismutase (FeSOD)와 유전자 재조합 기술로 생산된 6xHis-태그가 결합된 Fe superoxide dismutase (6xHis- FeSOD)의 활성을 비교하여 6xHis-태그의 효소에 대한 영향을 알아보기 위하여 수행되었다. 두 효소 모두 최적 pH는 7로 동일하였으나 6xHis-태그에 의해서 pH 범위는 축소되었다. 천연 효소는 pH 4-9의 범위에서 안정성을 보인 반면 6xHis-FeSOD는 pH 9에서 안정성이 상실되었다. 두 효소의 최적 온도는 차이가 없으나 열 안정성에 있어서는 천연 효소는 $40^{\circ}C$ 이하에서 720분까지 안정성을 유지하였으나 6xHis-FeSOD는 $20^{\circ}C$에서도 360분 이내에 활성을 잃는 것으로 나타났다. $H_2O_2$의 6xHis-FeSOD에 대한 저해는 0.5 mM에서 나타났다. 따라서 6xHis-FeSOD는 효소활성은 유지되더라도 열 안정성이 크게 감소되는 결과를 얻었다. 이것은 6xHis-태그가 활성부위 보다는 단백질 전체 구조에 더 많은 영향을 미친 결과라고 생각되었다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2000년도 국제심포지움
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• pp.10-12
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$-subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$ -subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was. efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to consist of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t63I or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632-653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors ~2- and ~17-fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• 한국가축번식학회지
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• 제24권4호
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• pp.357-364
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• 2000

Members of the glycoprotein family, which includes CG, LH, FSH and TSH, comprise two noncovalently linked $\alpha$- and $\beta$-subunits. Equine chorionic gonadotropin (eCG), known as PMSG, has a number of interesting and unique characteristics since it appears to be a single molecule that possesses both LH- and FSH-like activities in other species than the horse. This dual activity of eCG in heterologous species is of fundamental interest to the study of the structure-function relationships of gonadotropins and their receptors. CG and LH $\beta$ genes are different in primates. In horse, however, a single gene encodes both eCG and eLH $\beta$ -subunits. The subunit mRNA levels seem to be independently regulated and their imbalance may account for differences in the quantities of $\alpha$ - and $\beta$-subunits in the placenta and pituitary. The dual activities of eCG could be separated by removal of the N-linked oligosaccharide on the $\alpha$-subunit Asn 56 or CTP-associated O-linked oligosaccharides. The tethered-eCG was efficiently secreted and showed similar LH-like activity to the dimeric eCG. Interestingly, the FSH-like activity of the tethered-eCG was increased markedly in comparison with the native and wild type eCG. These results also suggest that this molecular can implay particular models of FSH-like activity not LH-like activity in the eCG/indicate that the constructs of tethered molecule will be useful in the study of mutants that affect subunit association and/or secretion. A single-chain analog can also be constructed to include additional hormone-specific bioactive generating potentially efficacious compounds that have only FSH-like activity. The LH/CG receptor (LH/CGR), a membrane glycoprotein that is present on testicular Leydig cells and ovarian theca, granulosa, luteal, and interstitial cells, plays a pivotal role in the regulation of gonadal development and function in males as well as in nonpregnant and pregnant females. The LH/CGR is a member of the family of G protein-coupled receptors and its structure is predicted to of a large extracellular domain connected to a bundle of seven membrane-spanning a-helices. The LH/CGR phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. The truncated form of LHR also was down-regulated normally in response to hCG stimulation. In contrast, the cell lines expressing LHR-t631 or LHR-628, the two phosphorylation-negative receptor mutant, showed a delay in the early phase of hCG-induced desensitization, a complete loss of PMA-induced desensitization, and an increase in the rate of hCG-induced receptor down-regulation. These results clearly show that residues 632~653 in the C-terminal tail of the LHR are involved in PMA-induced desensitization, hCG-induced desensitization, and hCG-induced down-regulation. Recently, constitutively activating mutations of the receptor have been identified that are associated with familial male-precocious puberty. Cells expressing LHR-D556Y bind hCG with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation enhances the internalization of the free and agoinst-occupied receptors ~2- and ~17- fold, respectively. We conclude that the state of activation of the LHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing LHR-L435R is due to the fast rate of internalization of the bound hCG. This statement is supported by the finding that hCG responsiveness is restored when the cells are lysed and signal transduction is measured in a subcellular fraction (membranes) that cannot internalize the bound hormone.

• 생명과학회지
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• 제28권3호
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• pp.307-313
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• 2018

탈모증상은 겉으로 보이는 모습으로 인해 정신적인 스트레스로 작용한다. 그래서 탈모 방지관련 제품의 글로벌 시장 규모는 지속적으로 성장하고 있다. Timosaponin A III는 지모 추출물에서 발견되는 대표적인 saponin 계열의 생리 활성 효능 성분이다. 본 연구에서는 5-beta reductase 단백질 길항제(antagonist) finasteride, androgen receptor 단백질 길항제 minoxidil, 그리고 지모 추출물의 효능 성분 timosaponin A III의 각각의 타깃 단백질 활성 부위에 대한 친화도 분석 실험을 in silico 컴퓨터 분자결합 분석 방법을 통해 비교하였다. 5-beta reductase 및 androgen receptor 의 3차원 구조 정보는 PDB database (5-beta reductase PDB ID: 3G1R / androgen receptor PDB ID:4K7A)를 활용하였다. In silico 결합 분석을 수행하기 위해 PyRx, Autodock Vina, Discovery Studio Version 4.5, and NX-QuickPharm 프로그램을 각 분석 조건에 따라 활용하였다. 5-beta reductase 활성 부위에 대한 timosaponin A III의 최대 결합친화도는 -12.20 kcal/mol으로 나왔으며 이는 -11.70 kcal/mol으로 분석된 finasteride의 5-beta reductase 활성부위에 대한 결합 친화도 보다 훨씬 더 높고 효율적인 것으로 분석되었다. Androgen receptor 활성 부위에 대한 timosaponin A III의 최대결합친화도 또한 -9.00 kcal/mol으로 -7.40 kcal/mol의 minoxidil에 비하여 훨씬 우수한 결합친화도 값을 나타내었다. Finasteride와 timosaponin A III의 5-beta reductase 단백질 활성 부위에 대한 X,Y,Z Grid 값은 유사한 좌표로 분석되었으나 minoxidil과 timosaponin A III의 androgen receptor 활성 부위에 대한 X,Y,Z centroid grid 좌표는 상당한 거리를 두고 떨어져 있음이 확인 되었다. 즉, timosaponin A III는 minoxidil이 androgen receptor에 결합하는 부위와는 다른 부위에 결합하여 단백질 활성에 영향을 주는 것으로 사료되었다. 이상의 연구 결과들을 바탕으로 분석해 볼 때, 5-beta reductase 길항제 finasteride와 androgen receptor 길항제 minoxidil보다 지모 추출물 생리 활성 물질인 timosaponin A III가 보다 더 효율적인 길항제로 작용할 수 있음을 확인하였다. 결론적으로 지모 추출물 또는 timosaponin 계열이 함유된 효능 성분은 탈모 방지 효능 및 모발 건강 개선을 위한 의약품, 의약외품 및 신물질 연구 개발 분야에 효율적으로 활용할 수 있을 것으로 사료된다.

• 한국식품영양과학회지
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• 제29권1호
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• pp.174-179
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• 2000

우리 나라에서 활발히 양식되고 있는 양직산과 자연산 넙치의 맛성분 조성을 비교하기 위하여 맛과 관계가 깊은 함질소 엑스성분을 분석하여 비교한 결과는 다음과 같다. 일반성분 중 수분은 양식산이 자연산보다 다소 낮은 반면 단백질과 지질은 다소 높았다. 엑스분 질소는 자연산이 다소 높았다. 유리아미노산은 양식산에서 31종, 자연산에서 36종이 검출되어 자연산이 더 다양한 아미노산 조성을 보였으며, 그 총량은 양식산 자연산 모두 전반적으로 낮은 수준이었다. 넙치의 taurine 함량은 양식산과 자연산에서 각각 115 mg과 91 mg으로 유리아미노산 중 가장 높았으며, 우리아미노산 총량의 각각 57.0%와 37.0%를 차지하였다. 양식산에서 함량이 많은 유리아미노산으로서는 taurine, alanine, asparagine, glutamine, glutamic acid, lysine 등이었고, 자연산에서는 taurine, lysine, alanine, carnosine, hydroxyproline 등이었다. 넙치는 oligopeptide류 함량이 869 mg과 1,063 mg으로서 유리아미노산 총량의 4.3배에 달하였다. ATP관련 화합물의 총량은 각각 $9.56\;\mumol과\;6,58\;\mumol$로서 양식산이 다소 높았으며 IMP가 각각 68.2%와 78.6%로서 대부분을 차지하였다. TMAO는 각각 273 mg과 316 mg으로서 자연산이 높고, creatine은 각각 522 mg와 487 mg으로서 양식산이 높았다. Betaine류, TMA, creatinine은 모두 미량이었다. 엑스분 중의 질소분포는 creatine과 creatinine 질소가 가장 높고, 다음은 oligopeptide류 질소, TMAO와 TMA질소, ATP관련 물질 질소, 유리아미노산 질소의 순이었고 질소의 회수율은 95% 이상이었다.취상태를 평가받고 과부족이 되기 쉬운 영양소의 급원 식품과 음식에 대한 영양정보도 제공받을 수 있도록 할 뿐 아니라 식이요법을 필요로 하는 질환의 영양상담을 위한 기본 프로그램으로 활용될 수 있도록 하는데 그 의의가 있다.사만을 선택하여 성별과 결혼여부에 따라 환경친화적 음식소비 행동의 차이를 알아보기 위한 결과 역시 차이를 보이지 않았다. 환경친화적 음식소비행동은 연령, 월평균소득 및 엥겔계수와 의미 있는 상관관계를 보이지 않았다. 조리사와 주부는 음식을 소비하는 전 과정에서 가장 많이 관여하고 있는 집단이다. 그런데 조리사와주부 모두 전반적으로 환경친화적 음식소비행동의 수준이 높지 않음으로 합리적인 식생활문화를 생활화 할 수 있도록 교육과 홍보가 필요하다고 본다. 주부의 경우 유치원이나 어린이집, 초중고둥학교에서 하는 부모교육과 반상회의 홍보자료에 반상회의 홍보자료에 반드시환경친화적 음식소비행동에 관한 교육이 포함되어야 한다. 조리사의 경우 정기교육과정에 환경친화적 음식소비행동에 관한 홍보를 함께 함으로써 외식이나 단체급식에서 발행하는 음식물 쓰레기의 양을 줄 일 수 있을 것이다.m its genes controlling host specificity to its population sturctures and dynamics, have begun to provide new insights into the potential mechanisms underlying race variation. In this review we aim to provide an overview on (a) the molecular basis of host specificity of M. grisea, (b) the population structure and dynamics of rice pathogens, and (c) the nature and mechanisms