• 한국콘텐츠학회논문지
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• 제10권4호
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• pp.19-27
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• 2010

최근에 고속 genome-wide RNA 간섭 스크리닝 기술은 복잡한 세포 기능을 이해하는 생명공학 연구의 핵심적인 도구로 자리 잡고 있다. 그러나 관련 연구에서 발생되는 수많은 영상을 수작업을 통해 분석하는 것은 많은 시간과 노력이 요구된다. 따라서 세포영상의 자동분석 기술은 매우 시급히 확보되어야 하는 기술이며, 그 중 영상 분할은 자동분석을 위한 첫 단계로서 가장 중요한 과정이라 할 수 있다. 세포영상의 자동분할에서는 영역의 겹침 현상과 영역별 모양의 다양성 및 영상 특성의 불균일성 등이 정확한 세포 분할을 어렵게 만드는 주원인으로 작용한다. 본 논문에서는 이러한 문제점을 극복하기 위해 영상 특징들의 국부적인 연속성과 특징 벡터 기반의 워터쉐드 알고리즘을 적용한 새로운 자동 세포 분할 알고리즘을 제안한다. 영상 특징들의 연속성을 국부적인 영역으로 제한함으로써 영역별 모양의 다양성 및 영상 특성의 불균일성에 따른 문제점을 극복할 수 있으며, 특징벡터의 사용을 통해 하나의 영상특징만을 고려한 경우 발생되는 겹침 영역에서의 분할 성능 저하를 개선할 수 있다. 세포영상 분석을 위한 소프트웨어 패키지인 Cellprofiler와의 비교/분석 실험을 통해 제안 알고리즘의 효율성을 입증하였다.

• The Plant Pathology Journal
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• 제21권4호
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• pp.334-342
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• 2005

The prototype Chlorella virus PBCV-l encodes 11 tRNA genes and over 350 protein-encoding genes in its 330 kbp genome. Initial attempts to overexpress the recombinant A189/192R protein, a putative virus attachment protein, in E. coli strain BL21(DE3) SI were unsuccessful, and multiple protein bands were detected on Western blots. However, the full-length A189/192R recombinant protein or fragments derived from it were detected when they were expressed in E. coli BL21 CodonPlus (DE3) RIL, which contains extra tRNAs. Codon usage analysis of the a189/192r gene showed highly biased usage of the AGA and AVA codons compared to genes encoded by E. coli and Chlorella. In addition, there were biases of XXA/U($56\%$) and XXG/ C($44\%$) in the codons recognized by the viral tRNAs, which correspond to the codon usage bias in the PBCV-1 genome of XXA/U ($63\%$) over those ending in XXC/G ($37\%$). Analysis of the codon usage in the major capsid protein and DNA polymerase showed preferential usage of codons that can be recognized by the viral tRNAs. The Asn (AAC) and Lys (AAG) codons whose corresponding tRNA genes are duplicated in the tRNA gene cluster were the most abundant (i.e., preferred) codons in these two proteins. The tRNA genes encoded in the PBCV-l genome seem to play a very important role during the synthesis of viral proteins through supplementing the tRNAs that are frequently used in viral proteins, but are rare in the host cells. In addition, these tRNAs would help the virus to adapt to a wide range of hosts by providing tRNAs that are rare in the host cells.

• Molecules and Cells
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• 제45권3호
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• pp.148-157
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• 2022

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.

• Asian Pacific Journal of Cancer Prevention
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• 제13권9호
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• pp.4827-4834
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• 2012

Tumor suppressor genes have received much attention for their roles in the development of human malignancies. Gelsolin has been found to be down-regulated in several types of human cancers, including leukemias. It is, however, expressed in macrophages, which are the final differentiation derivatives for the monocytic myeloid lineage, implicating this protein in the differentiation process of such cells. In order to investigate the role of gelsolin in leukaemic cell differentiation, stable clones over-expressing ectopic gelsolin, and a control clone were established from U937 leukaemia cells. Unlike the control cells, both gelsolin-overexpressing clones displayed retarded growth, improved monocytic morphology, increased NADPH and NSE activities, and enhanced surface expression of the ${\beta}$-integrin receptor, CD11b, when compared with the parental U937 cells. Interestingly, RT-PCR and western blot analysis also revealed that gelsolin enhanced p21CIP1 mRNA and protein expression in the overexpressing clones. Moreover, transient transfection with siRNA silencing P21CIP1, but not the control siRNA, resulted in a reduction in monocytic differentiation, accompanied by an increase in proliferation. In conclusion, our work demonstrates that gelsolin, by itself, is capable of inducing monocytic differentiation in U937 leukaemia cells, most probably through p21CIP1 activation.

• Asian Pacific Journal of Cancer Prevention
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• 제14권6호
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• pp.3897-3901
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• 2013

Background: Optimal treatment for prostate cancer remains a challenge worldwide. Recently, T cell immunoglobulin mucin-3 (TIM-3) has been implicated in tumor biology but its contribution prostate cancer remains unclear. The aim of this study was to investigate the role of TIM-3 as a prognostic marker in patients with prostate cancer. Methods: TIM-3 protein expression was determined by immunohistochemistry and Western blotting in 137 prostate cancer tumor samples and paired adjacent benign tissue. We also performed cell proliferation assays using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl- 2H tetrazolium bromide (MTT) and cell invasion assays. The effects of small interfering RNA (siRNA)-mediated knockdown of TIM-3 (TIM-3 siRNA) in two human prostate cancer cell lines were also evaluated. Results: TIM-3 expression was higher in prostate cancer tissue than in the adjacent benign tissue (P<0.001). High TIM-3 expression was an independent predictor of both recurrence-free survival and progression-free survival. TIM-3 protein was expressed in both prostate cancer cell lines and knockdown suppressed their proliferation and invasion capacity. Conclusions: TIM-3 expression is associated with a poor prognosis in prostate cancer. Taken together, our resutlts indicate that TIM-3 is a potential prognostic marker in prostate cancer.

• The Korean Journal of Physiology and Pharmacology
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• 제17권2호
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• pp.157-162
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• 2013

Insulin-like growth factor binding proteins (IGFBPs) are important components of insulin growth factor (IGF) signaling pathways. One of the binding proteins, IGFBP-5, enhances the actions of IGF-1, which include the enhanced proliferation of smooth muscle cells. In the present study, we examined the expression and the biological effects of IGFBP-5 in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). The levels of IGFBP-5 mRNA and protein were found to be higher in the VSMC from SHR than in those from WKY. Treatment with recombinant IGFBP-5-stimulated VSMC proliferation in WKY to the levels observed in SHR. In the VSMCs of WKY, incubation with angiotensin (Ang) II or IGF-1 dose dependently increased IGFBP-5 protein levels. Transfection with IGFBP-5 siRNA reduced VSMC proliferation in SHR to the levels exhibited in WKY. In addition, recombinant IGFBP-5 significantly up-regulated ERK1/2 phosphorylation in the VSMCs of WKY as much as those of SHR. Concurrent treatment with the MEK1/2 inhibitors, PD98059 or U0126 completely inhibited recombinant IGFBP-5-induced VSMC proliferation in WKY, while concurrent treatment with the phosphatidylinositol-3 kinase inhibitor, LY294002, had no effect. Furthermore, knockdown with IGFBP-5 siRNA inhibited ERK1/2 phosphorylation in VSMC of SHR. These results suggest that IGFBP-5 plays a role in the regulation of VSMC proliferation via ERK1/2 MAPK signaling in hypertensive rats.

• Molecules and Cells
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• 제25권2호
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• pp.184-195
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• 2008

We examined the role of c-FLIP in the motility of HeLa cells. A small interfering RNA (siRNA) directed against c-FLIP inhibited the adhesion and motility of the cells without affecting their growth rate. The long form of c-FLIP ($c-FLIP_L$), but not the short form ($c-FLIP_S$), enhanced adhesion and motility. Downregulation of $c-FLIP_L$ with siRNA decreased phosphorylation of FAK and ERK, while overexpression of $c-FLIP_L$ increased their phosphorylation. Overexpression of FAK activated ERK, and enhanced the motility of HeLa cells. FRNK, an inhibitory fragment of FAK, inhibited ERK and decreased motility. Inhibition of ERK also significantly suppressed $c-FLIP_L$-promoted motility. Inhibition of ROCK by Y27632 suppressed the $c-FLIP_L$-promoted motility by reducing phosphorylation of FAK and ERK. Overexpression of $c-FLIP_L$ increased the expression and secretion of MMP-9, and inhibition of MMP-9 by Ilomastat reduced $c-FLIP_L$- promoted cell motility. A caspase-like domain (amino acids 222-376) was found to be necessary for the $c-FLIP_L$-promoted cell motility. We conclude that $c-FLIP_L$ promotes the motility of HeLa cells by activating FAK and ERK, and increasing MMP-9 expression.

• Asian Pacific Journal of Cancer Prevention
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• 제13권3호
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• pp.821-825
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• 2012

Purpose: Centromere protein H (CENP-H) and Ki67 are overexpressed in some malignancies, but whether they are predictors of survival after primary resection for hypopharyngeal squamous cell carcinoma (HSCC) remains unknown. Methods: We assessed immunohistochemical expression of CENP-H and Ki67 in 112 HSCC specimens collected between March 2003 and March 2005 for analysis by clinical characteristics. The Kaplan-Meier method was used to analyze relapse-free survival and logistic multivariate regression to determine risk factors of relapse-free survival. Cholecystokinin octapeptide assays and flow cytometry were used to examine cell proliferation and apoptosis after siRNA inhibition of CENP-H in HSCC cells. Results: Overall, 50 (44.6%) HSCC specimens showed upregulated CENP-H expression and 69 (61.6%) upregulated Ki67. An increased CENP-H protein level was associated with advanced cancer stage and alcohol history (P=0.012 and P=0.048, respectively) but an increased Ki67 protein level only with advanced cancer stage (P=0.021). Increased CENP-H or Ki67 were associated with short relapse-free survival (P<0.001 or P=0.009, respectively) and were independent predictors of relapse-free survival (P=0.001 and P=0.018, respectively). siRNA knockdown of CENP-H mRNA inhibited cell proliferation and promoted cancer cell apoptosis in vitro. Conclusions: Upregulated CENP-H and Ki67 levels are significantly associated with short relapse-free survival in HSCC. These factors may be predictors of a relapsing phenotype in HSSC cases.

• Biomolecules & Therapeutics
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• 제26권5호
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• pp.464-473
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• 2018

Cripto is a small glycosylphosphatidylinositol-anchored signaling protein that can detach from the anchored membrane and stimulate proliferation, migration, differentiation, vascularization, and angiogenesis. In the present study, we demonstrated that Cripto positively affected proliferation and survival of mesenchymal stem cells (MSCs) without affecting multipotency. Cripto also increased expression of phosphorylated janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), 78 kDa glucose-regulated protein (GRP78), c-Myc, and cyclin D1. Notably, treatment with an anti-GRP78 antibody blocked these effects. In addition, pretreatment with STAT3 short interfering RNA (siRNA) inhibited the increase in p-JAK2, c-Myc, cyclin D1, and BCL3 levels caused by Cripto and attenuated the pro-survival action of Cripto on MSCs. We also found that incubation with Cripto protected MSCs from apoptosis caused by hypoxia or $H_2O_2$ exposure, and the level of caspase-3 decreased by the Cripto-induced expression of B-cell lymphoma 3-encoded protein (BCL3). These effects were sensitive to down-regulation of BCL3 expression by BCL3 siRNA. Finally, we showed that Cripto enhanced expression levels of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). In summary, our results demonstrated that Cripto activated a novel biochemical cascade that potentiated MSC proliferation and survival. This cascade relied on phosphorylation of JAK2 and STAT3 and was regulated by GRP78. Our findings may facilitate clinical applications of MSCs, as these cells may benefit from positive effects of Cripto on their survival and biological properties.

• The Plant Pathology Journal
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• 제36권6호
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• pp.618-627
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• 2020

Although limited progress have been made about pathogen system of Hibiscus rosa-sinensis and Hibiscus latent Fort Pierce virus (HLFPV), interaction between plant host and pathogen remain largely unknown, which led to deficiency of effective measures to control disease of hibiscus plants caused by HLFPV. In this study, infection of HLFPV in Hibiscus rosa-sinensis was firstly confirmed for the first time by traditional electron microscopy, modern reverse transcription polymerase chain reaction and RNA-seq methods in China (HLFPV-Ch). Sequence properties analyzing suggested that the full-length sequences (6,465 nt) of HLFPV-Ch had a high sequence identity and a similar genomic structure with other tobamoviruses. It includes a 5'-terminal untranslated region (UTR), followed by four open reading frames encoding for a 128.5-kDa replicase, a 186.5-kDa polymerase, a 31-kDa movement protein, 17.6-kDa coat protein, and the last a 3'-terminal UTR. Furthermore, HLFPV-Ch-derived virus-derived siRNAs (vsiRNAs) ant its putative target genes, reported also for the first time, were identified and characterized from disease Hibiscus rosa-sinensis through sRNA-seq and Patmatch server to investigate the interaction in this pathogen systems. HLFPV-Ch-derived vsiRNAs demonstrated several general and specific characteristics. Gene Ontology classification revealed predicted target genes by vsiRNAs are involved in abroad range of cellular component, molecular function and biological processes. Taken together, for first time, our results certified the HLFPV infection in China and provide an insight into interaction between HLFPV and Hibiscus rosa-sinensis.