• 한국미생물·생명공학회지
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• 제31권2호
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• pp.140-144
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• 2003

신호펩디드 가수분해는 모든 생물에 필수적인 단백질로 N-말단에 신호서열을 가진 단백질들의 신호서열을 잘라내는 효소로 serine과 lysine을 활성부위로 하는 특이한 dyad serine 효소류로 알려져있다. 최근에 신호펩디드 가수분해효소의 특성으로 새로운 항생제 이용 가능성 때문에 그 활성 메카니즘의 연구가 중요시되고 있다. 본 연구는 E. coli 신호펩디드 효소가 serine/lysine/aspartic acid로 하는 전형적인 triad serine 효소류의 메카니즘을 갖는다는 가정하에 Type Ⅰ 신호펩디드류의 하나인 E. coli에서 aspartic acid 99을 alanine으로 치환하여 효소의 enzymatic activity를 조사하였다. 그 결과 D99잔기는 E. coli Type Ⅰ 신호펩디드 효소의 활성메카니즘에 직접적으로 관여한다기보다는 그 효소를 구조적으로 안정화하는데 중요한 역할을 할 가능성이 있는 것으로 암시되어진다.

• BMB Reports
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• 제44권8호
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• pp.529-534
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• 2011

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.

• Molecules and Cells
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• 제26권1호
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• pp.26-33
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• 2008

The plasmid pJB01, a member of the pMV158 family isolated from Enterococcus faecium JC1, contains three open reading frames, copA, repB, and repC. Plasmids included in this family produce counter-transcribed RNA (ctRNA) that contributes to copy number control. The pJB01 ctRNA, a transcript which consists of 54 nucleotides (nts), is encoded on the opposite strand from the copA/repB intergenic region and partially overlaps an atypical ribosome binding site (ARBS) for repB. The ARBS is integrated by the two underlined conserved regions: 5'-TTTTTGTNNNNTAANNNNNNNNNATG-3', and the ctRNA is complementary only to the 5' conserved sequence 5'-TTTTTGT-3'. This complementary sequence is located at a distance from the terminal loop of the ctRNA secondary structure. The ctRNA structure predicted by the mfold program suggests the possible generation of a terminal and an internal hairpin loop. The amount of in vitro translation product of repB mRNA was inversely proportional to the ctRNA concentration. Mutations in the terminal and internal hairpin loops of the ctRNA had inhibitory effects on its binding to the target mRNA. We propose that the intact structures of the terminal and internal hairpin loops, respectively, play important roles in forming the initial kissing and extending complexes between the ctRNA and target mRNA and that these regulate the copy number of this plasmid.

• The Korean Journal of Physiology and Pharmacology
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• 제10권5호
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• pp.263-271
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• 2006

Since astrocytes were shown to play a central role in maintaining neuronal viability both under normal conditions and during stress such as ischemia, studies of the astrocytic response to stress are essential to understand many types of brain pathology. The micro array system permitted screening of large numbers of genes in biological or pathological processes. Therefore, the gene expression patterns in the in vitro model of astrocytes following exposure to oxygen-glucose deprivation (OGD) were evaluated by using the micro array analysis. Primary astrocytic cultures were prepared from postnatal Swiss Webster mice. The cells were exposed to OGD for 4 hrs at $37^{\circ}C$ prior to cell harvesting. From the cultured cells, we isolated mRNA, synthesized cDNA, converted to biotinylated cRNA and then reacted with GeneChips. The data were normalized and analyzed using dChip and GenMAPP tools. After 4 hrs exposure to OGD, 4 genes were increased more than 2 folds and 51 genes were decreased more than 2 folds compared with the control condition. The data suggest that the OGD has general suppressive effect on the gene expression with the exception of some genes which are related with ischemic cell death directly or indirectly. These genes are mainly involved in apoptotic and protein translation pathways and gap junction component. These results suggest that microarray analysis of gene expression may be useful for screening novel molecular mediators of astrocyte response to ischemic injury and making profound understanding of the cellular mechanisms as a whole. Such a screening technique should provide insights into the molecular basis of brain disorders and help to identify potential targets for therapy.

• 식물병연구
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• 제30권2호
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• pp.114-123
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• 2024

In July 2022, stem rot symptom was found in a peony plant grown in a pot under a greenhouse at Jinju, Gyeongnam Province, South Korea. Two fungal species were isolated from the infected peony stems and cultured on 1/2-strength potato dextrose agar for identification. The morphological characteristics of the fungal isolates were examined, and nucleotide sequences of the internal transcribed spacer region, β-tubulin and translation elongation factor 1-α were analysed. The pathogenicity of the two isolates was confirmed in detached peony leaves, according to Koch's postulates. To our knowledge, this is the report of Neopestalotiopsis clavispora and Sclerotinia sclerotiorum as the causal agents of peony stem rots. Antifungal activity of chemical fungicide propineb and rhizobacterium Bacillus siamensis H30-3 was shown against the two plant pathogenic fungi N. clavispora and S. sclerotiorum.Unidentified diffusible and volatile compounds from B. siamensis H30-3 could suppress in vitro mycelial growths of N. clavispora JJ 8-2-1 and S. sclerotiorum JJ 8-2-2.

• Journal of Plant Biotechnology
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• 제33권2호
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• pp.85-91
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• 2006

Flavanone 3$\beta$-hydroxylase (FHT)는 flavonoid 생합성 경로의 가장 중심부에 작용하는 효소로 flavanone으로부터 dihydroflavonol으로의 변환을 촉매하는 역할을 한다. 본 연구에서는 색소유전자의 전이를 통하여 새로운 색소발현체계를 가진 품종을 육종하기 위한 기초연구로 숙성안개초 (Gypsophila paniculata L.)의 꽃봉오리로부터 cDNA-library를 합성하였고 카네이션의 FHT 유전자를 probe로 사용하여 anthocyanin 합성경로의 중요 효소의 하나인 FHT 유전자를 분리하였다. 염기서열분석을 수행하여 분리유전자의 크기가 1471 bp 이며 이 중 coding region은 1047 bp 임을 확인하였다. 이미 밝혀진 다른 식물체의 FHT 유전자와 서로 염기서열의 일치성을 비교해 본 결과 아라비돕시스, 오렌지, 카네이션, 고구마, 스톡, 페튜니아, 감자 및 포도에서 각각 69% 이상을 나타내었다. 분리유전자의 발현을 확인하기 위하여 Northern blot분석 및 인위적으로 기내에서의 transcription과 translation을 수행하였고, 분리한 유전자의 효소활성을 측정해 본 결과 dihtydrokaempferol의 작은 peak을 확인하였다. Southern blot 분석의 결과 안개초의 FHT 유전자는 다른 대부분의 식물체와 유사하게 한 개가 존재함을 확인하였다

• Applied Biological Chemistry
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• 제38권6호
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• pp.528-533
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• 1995

참비름 (Amaranthus mangostanus) 잎에서 단백질을 추출하여 S-Sepharose, Sephacryl S-200 HR, CM-Sepharose, Blue sepharose column chromatography에 의하여 단백질 합성 저해능이 있는 항바이러스성 단백질 (Amarnanthus antiviral protein, AAP29)을 분리하였다. 정제된 단백질의 분자량은 SDS-PAGE에서 약 29,200이었으며, 등전점은 9.0$50^{\circ}C$에서 20분간 처리한 경우에도 활성의 변화는 없었으며, 이 단백질의 단백질합성 저해능 활성을 in vitro translation system에서 측정한 결과 50% 저해농도 ($IC_{50}$)는 0.18 nM이었다. 담배잎 표면에 AAP29와 cucumber mosaic virus (CMV)를 함께 접종하여 항바이러스 활성을 생물검정한 결과 AAP29는 virus 감염를 현저히 저하시켰다. AAP29의 N-말단 아미노산 서열은 ADLTFTVTKDGTSQSYXTLXNXWRXW이었으며 기존에 알려진 다른 RIP과의 동질성은 없었다.

• International Journal of Industrial Entomology and Biomaterials
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• 제8권2호
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• pp.169-174
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• 2004

Bombyx mori nucleopolyhedrovirus(BmNPV) ecdysteroid UDP-glucosyltransferase gene (egt) promoter fragments of different lengths were amplified from BmNPV ZJ-8 genomic DNA by PCR. Reporter plasmids pBmegt542-luc, pBmegt309-luc and pBmegtl59-luc with luciferase (lue) driven by egt promoters were constructed. Both in vitro and in vivo expressions showed that BmNPV egt promoter activity requires the transactivation of viral factor(s), and expression of luc was detected earliest at 24 hrs post infection (pi). BmNPV ZJ-8 homologous region 3 (hr3) increased the expression of luc by over 1,600-fold. Molting hormone of 1.0 - 2.0 $\mu\textrm{g}$/$m\ell$ can dramatically down regulate expression of luc. Juvenile hormone analogue of 0.5-2.0 ${\mu}g$/$m\ell$ increased expression of luc by 145.8% to 75.7%. Deletion assay revealed that the promoter fragment of 159 bp contains the basal promoter structure; Promoter fragments of 309 bp and 542 bp showed similar but much higher transcriptional activities than that of 159 bp, suggesting that nucleotide from -159 to -309 nt upstream the translation initiation site harbors the main cis-acting elements.

• Journal of Applied Biological Chemistry
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• 제42권4호
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• pp.161-165
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• 1999

The antiviral activity of CAP30 from Chenopodium album, a type1 ribosome-inactivating protein (RIP), was examined against 5 different plant viral pathogens, and its activity against Tobacco mosaic virus was compared to those of well known antiviral proteins such as Pokeweed Antiviral protein from leaves and seeds. When the inoculating concentration of Tobacco mosaic virus was varied from 0.4 to $400{\mu}g/ml$, it was observed that CAP30 at the concentration of $1{\mu}g/ml$ suppressed the viral infection of C. amaranthicolor and C. quinoa almost completely up to $40{\mu}g/ml$ Tobacco mosaic virus. Results from the assays for the inhibitions of in vitro translation of rabbit reticulocyte lysate and the suppression of Tobacco mosaic virus infection ($10{\mu}g/ml$) to C. quinoa indicated that CAP30 is a strong inhibitor of protein synthesis and virus infection. The infection of several viruses other than Tobacco mosaic virus to host plants were also inhibited by $5{\mu}g/ml$ CAP30, suggesting that a gene encoding CAP30 can be used to develop transgenic virus-resistant plants.

• Toxicological Research
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• 제8권2호
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.