• Parasites, Hosts and Diseases
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• 제61권4호
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• pp.397-404
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• 2023

Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.

• BMB Reports
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• 제41권10호
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• pp.739-744
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• 2008

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding $\beta$-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing $\beta$-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

• 한국응용곤충학회지
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• 제56권2호
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• pp.153-164
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• 2017

이중나선형 RNA (dsRNA)를 이용한 유전자 발현 억제기술이 다양한 생명체에서 기능 유전체학을 연구하는 데 이용되고 있다. 이 기술의 원리는 전사후 단계에서 유전자 발현을 조절하는 RNA 간섭에 기인된다. dsRNA를 이용하여 특정 유전자의 발현 억제는 심각한 치사효과를 줄 수 있다. 이러한 분자기작을 해충 방제에 적용하여 특정 dsRNA를 이용한 새로운 살충제를 개발하고 있다. 본 종설은 dsRNA를 이용한 RNA간섭 원리를 설명하고 이를 이용한 해충 방제를 구현한 여러 예를 살펴본다. 그리고 해충방제를 실현시키기 위해 현재 이 기술이 담고 있는 한계를 고찰하고 이에 대한 대응 방안을 본 종설에서 제공하고자 한다.

• 대한임상검사과학회지
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• 제43권3호
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• pp.89-97
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• 2011

The Human Papilloma Virus (HPV) infection has been strongly associated with pathogenesis of uterine cervix carcinoma. HPV type 16, a causative agent of uterine cervix carcinoma, encodes the E6 and E7 oncogenes, expression of which is pivotal for malignant transformation and maintenance of malignant phenotypes. To develop a gene therapy for HPV-related carcinoma, We investigated the effect of E6 short-interfering RNA (E6 siRNA) on the expression of this oncogene and on the growth of HPV 16-related uterine cervix carcinoma cells. SiHa cells, a uterine cervix carcinoma cell line, which contain a single copy of HPV 16 integrated in the chromosome and express the E6 and E7 oncogenes. Before 24 hr of transfection, cells were seeded and transfected with control plasmid or E6 siRNA-expressing plasmid. The mRNA was analysed by reverse transcriptase polymerase chain reaction (RT-PCR). The cell growth rate was investigated by MTT method. The E6 mRNA level in SiHa cells was decreased in HPV 16 E6 siRNA-expression vector transfected cells and a decrease in the growth of these cells was also observed. From these results. it is evident that E6 siRNA played a role in suppression of growth of SiHa cells and has a fair chance as a candidate for gene specific therapy for HPV related uterine cervix carcinoma.

• Molecules and Cells
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• 제28권1호
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• pp.57-65
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• 2009

CDC48 is a member of the AAA ATPase superfamily. Yeast CDC48 and its mammalian homolog p97 are implicated in diverse cellular processes, including mitosis, membrane fusion, and ubiquitin-dependent protein degradation. However, the cellular functions of plant CDC48 proteins are largely unknown. In the present study, we performed virus-induced gene silencing (VIGS) screening and found that silencing of a gene encoding a tobacco CDC48 homolog, NgCDC48, resulted in severe abnormalities in leaf and shoot development in tobacco. Furthermore, transgenic tobacco plants (35S:anti-NgCDC48), in which the NgCDC48 gene was suppressed using the antisense RNA method, exhibited severely aberrant development of both vegetative and reproductive organs, resulting in arrested shoot and leaf growth and sterile flowers. Approximately 57-83% of 35S:anti-NgCDC48 plants failed to develop mature organs and died at early stage of development. Scanning electron microscopy showed that both adaxial and abaxial epidermal pavement cells in antisense transgenic leaves were significantly smaller and more numerous than those in wild type leaves. These results indicate that NgCDC48 is critically involved in cell growth and development of tobacco plants. An in vivo targeting experiment revealed that NgCDC48 resides in the endoplasmic reticulum (ER) in tobacco protoplasts. We consider the tantalizing possibility that CDC48-mediated degradation of an as-yet unidentified protein(s) in the ER might be a critical step for cell growth and expansion in tobacco leaves.

• Biomolecules & Therapeutics
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• 제26권5호
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• pp.446-457
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• 2018

Adiponectin, a hormone predominantly originated from adipose tissue, has exhibited potent anti-inflammatory properties. Accumulating evidence suggests that autophagy induction plays a crucial role in anti-inflammatory responses by adiponectin. However, underlying molecular mechanisms are still largely unknown. Association of Bcl-2 with Beclin-1, an autophagy activating protein, prevents autophagy induction. We have previously shown that adiponectin-induced autophagy activation is mediated through inhibition of interaction between Bcl-2 and Beclin-1. In the present study, we examined the molecular mechanisms by which adiponectin modulates association of Bcl-2 and Beclin-1 in macrophages. Herein, we demonstrated that globular adiponectin (gAcrp) induced increase in the expression of AUF1 and ZFP36L1, which act as mRNA destabilizing proteins, both in RAW 264.7 macrophages and primary peritoneal macrophages. In addition, gene silencing of AUF1 and ZFP36L1 caused restoration of decrease in Bcl-2 expression and Bcl-2 mRNA half-life by gAcrp, indicating crucial roles of AUF1 and ZFP36L1 induction in Bcl-2 mRNA destabilization by gAcrp. Moreover, knock-down of AUF1 and ZFP36L1 enhanced interaction of Bcl-2 with Beclin-1, and subsequently prevented gAcrp-induced autophagy activation, suggesting that AUF1 and ZFP36L1 induction mediates gAcrp-induced autophagy activation via Bcl-2 mRNA destabilization. Furthermore, suppressive effects of gAcrp on LPS-stimulated inflammatory mediators expression were prevented by gene silencing of AUF1 and ZFP36L1 in macrophages. Taken together, these results suggest that AUF1 and ZFP36L1 induction critically contributes to autophagy induction by gAcrp and are promising targets for anti-inflammatory responses by gAcrp.

• Parasites, Hosts and Diseases
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• 제46권1호
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• pp.1-15
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• 2008

Introduction of double-stranded RNA (dsRNA) into some cells or organisms results in degradation of its homologous mRNA, a process called RNA interference (RNAi). The dsRNAs are processed into short interfering RNAs (siRNAs) that subsequently bind to the RNA-induced silencing complex (RISC), causing degradation of target mRNAs. Because of this sequence-specific ability to silence target genes, RNAi has been extensively used to study gene functions and has the potential to control disease pathogens or vectors. With this promise of RNAi to control pathogens and vectors, this paper reviews the current status of RNAi in protozoans, animal parasitic helminths and disease-transmitting vectors, such as insects. Many pathogens and vectors cause severe parasitic diseases in tropical regions and it is difficult to control once the host has been invaded. Intracellularly, RNAi can be highly effective in impeding parasitic development and proliferation within the host. To fully realize its potential as a means to control tropical diseases, appropriate delivery methods for RNAi should be developed, and possible off-target effects should be minimized for specific gene suppression. RNAi can also be utilized to reduce vector competence to interfere with disease transmission, as genes critical for pathogenesis of tropical diseases are knockdowned via RNAi.

• KSBB Journal
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• 제31권1호
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• pp.52-57
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• 2016

Mutant p53 (R280K) is highly expressed in MDA-MB-231 triple-negative human breast cancer cells. Currently, we reported the role of mutant p53-R280K in mediating the survival of MDA-MB-231 cells in vitro. The present study was undertaken to determine whether mutant p53-R280K affects breast cancer cell growth in vivo. To this end, we used small interfering RNA to knockdown the level of mutant p53-R280K in MDA-MB-231 cells. Silencing of mutant p53-R280K in MDA-MB-231 cells causes substantial tumor regression of established xenografts in vivo. In xenograft model for breast cancer, silencing of mutant p53-R280K in MDA-MB-231 cells significantly inhibited the tumor growth. Moreover, TUNEL assay showed more occurrence of apoptotic cells in mutant p53-R280K silenced tumors compared to control. Our data indicate that mutant p53-R280K has an important role in mediating tumor growth of MDA-MB-231 cells in vivo. Taken together, this study suggests that endogenous mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.

• The Plant Pathology Journal
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• 제36권3호
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• pp.280-288
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• 2020

RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) in its vector, Laodelphax striatellus, using RNAi, dsRNAs against L. striatellus genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/μl and, possibly compromising viral replication. L. striatellus genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in L. striatellus. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-L. striatellus interactions.

• 대한의생명과학회지
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• 제28권4호
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• pp.247-259
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• 2022

Immunotherapy, which uses an immune mechanism in the body, has received considerable attention for cancer treatment. Suberoylanilide hydroxamic acid (SAHA), also known as a histone deacetylase inhibitor (HDACi), is used as a cancer treatment to induce active immunity by increasing the expression of T cell-induced chemokines. However, this SAHA treatment has the disadvantage of causing PD-L1 overexpression in tumor cells. In this study, we prevented PD-L1 overexpression by blocking the PD-1/PD-L1 pathway using PD-L1 siRNA. We designed two types of liposomes, the neutral lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholin (POPC) for SAHA, and 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) for siRNA. To effectively target PD-L1 in cancer cells, we conjugated PD-L1 antibody with liposomes containing SAHA or PD-L1 siRNA. These immunoliposomes were also evaluated for cytotoxicity, gene silencing, and T-cell-induced chemokine expression in human non-small cell lung cancer A549 cells. It was confirmed that the combination of the two immunoliposomes increased the cancer cell suppression efficacy through Jurkat T cell induction more than twice compared to SAHA alone treatment. In conclusion, this combination of immunoliposomes containing a drug and nucleic acid has promising therapeutic potential for non-small-cell lung carcinoma (NSCLC).