• Molecules and Cells
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• v.40 no.11
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• pp.828-836
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• 2017

Eukaryotic cells consist of a complex network of thousands of proteins present in different organelles where organelle-specific cellular processes occur. Identification of the subcellular localization of a protein is important for understanding its potential biochemical functions. In the post-genomic era, localization of unknown proteins is achieved using multiple tools including a fluorescent-tagged protein approach. Several fluorescent-tagged protein organelle markers have been introduced into dicot plants, but its use is still limited in monocot plants. Here, we generated a set of multicolored organelle markers (fluorescent-tagged proteins) based on well-established targeting sequences. We used a series of pGWBs binary vectors to ameliorate localization and co-localization experiments using monocot plants. We constructed different fluorescent-tagged markers to visualize rice cell organelles, i.e., nucleus, plastids, mitochondria, peroxisomes, golgi body, endoplasmic reticulum, plasma membrane, and tonoplast, with four different fluorescent proteins (FPs) (G3GFP, mRFP, YFP, and CFP). Visualization of FP-tagged markers in their respective compartments has been reported for dicot and monocot plants. The comparative localization of the nucleus marker with a nucleus localizing sequence, and the similar, characteristic morphology of mCherry-tagged Arabidopsis organelle markers and our generated organelle markers in onion cells, provide further evidence for the correct subcellular localization of the Oryza sativa (rice) organelle marker. The set of eight different rice organelle markers with four different FPs provides a valuable resource for determining the subcellular localization of newly identified proteins, conducting co-localization assays, and generating stable transgenic localization in monocot plants.

• Journal of Ginseng Research
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• v.43 no.1
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• pp.86-94
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• 2019

Background: Ginseng is believed to have antitumor activity. Autophagy is largely a prosurvival cellular process that is activated in response to cellular stressors, including cytotoxic chemotherapy; therefore, agents that inhibit autophagy can be used as chemosensitizers in cancer treatment. We examined the ability of Korean Red Ginseng extract (RGE) to prevent autophagic flux and to make hepatocellular carcinoma (HCC) cells become more sensitive to doxorubicin. Methods: The cytotoxic effects of total RGE or its saponin fraction (RGS) on HCC cells were examined by the lactate dehydrogenase assay in a dose- or time-dependent manner. The effect of RGE or RGS on autophagy was measured by analyzing microtubule-associated protein 1A/1B-light chain (LC)3-II expression and LC3 puncta formation in HCC cells. Late-stage autophagy suppression was tested using tandem-labeled green fluorescent protein (GFP)-monomeric red fluorescent protein (mRFP)-LC3. Results: RGE markedly increased the amount of LC3-II, but green and red puncta in tandem-labeled GFP-mRFP-LC3 remained colocalized over time, indicating that RGE inhibited autophagy at a late stage. Suppression of autophagy through knockdown of key ATG genes increased doxorubicin-induced cell death, suggesting that autophagy induced by doxorubicin has a protective function in HCC. Finally, RGE and RGS markedly sensitized HCC cells, (but not normal liver cells), to doxorubicin-induced cell death. Conclusion: Our data suggest that inhibition of late-stage autophagic flux by RGE is important for its potentiation of doxorubicin-induced cancer cell death. Therapy combining RGE with doxorubicin could serve as an effective strategy in the treatment of HCC.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• Tuberculosis and Respiratory Diseases
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• v.43 no.6
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• pp.862-870
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• 1996

Background : Multidrug-resistant tuberculosis(MDR-Tb) has been increased not only in Asia but also in Western society, which may cause public health problems and reduce the efficacy of treatment of tuberculosis. In Western society HIV infection is believed to do a central role in increasing incidence of MDR tuberculosis, but MDR-Tb in Korea may be somewhat different about clinical features, underlying disorders, and prognosis. Goble et al reponed that overall treatment failure rate in MDR-Tb including resistance to isoniazid(INH) and rifampin (RFP) was 44 %. The aim of this study is to find the treatment result in Korea and the factors determining the prognosis. Methods: A retrospective study of pulmonary tuberculosis cultured M. tuberculosis from sputum or bronchial washing fluid between 1986 through 1992 was conducted in the Seoul Paik Hospital, Inje University. We reviewed clinical courses of 141 patients, who had a tuberculosis with resistance to 2 or more drugs including isoniazid(INH) and rifampin(RFP). One hundred and 4 patients of 141 patients had completed treatment and followed up for more than one year. Results: Of 104 (mean age $43.6{\pm}16.7$, M: F=63 : 41) patients with sufficient follow-up data, 73(84.6%) patients responded which is defined as negative Sputum cultures for at least 3 consecutive months. Seven patients(6.7%) had a failure in negative conversion and 9(8.7%) of the patients who initially responded relapsed. Overall treatment failure rate was 15.4%, Patients who were treated for less than 12 months had a higher relapse rate(12.3%) than 18 months(4.9%). And there was a statistically significant correlation between the relapse rate and the number of drugs to which isolates wera resistant(p<0.05). Conclusion : The treatment failure rate of MDR-Tb in Korea was lower than previous studies in western Country and the major determining factor of prognosis was the number of resistant drugs to M. tuberculosis at drug sensitivity test. For reducing the relapse rate, we recommend more than 12 months of treatment for MDR tuberculosis.

• Proceedings of the Korea Information Processing Society Conference
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• 2023.11a
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• pp.1193-1195
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• 2023

전 세계적으로 PQC migration 을 위한 암호 표준화, 로드맵 발표 등 많은 관심이 집중되고 있다. 그 중, 인터넷 환경에 필수적으로 사용되는 TLS 는 PQC 전환이 필수적이다. 하지만 NIST 표준으로 선정된 PQC 서명 알고리즘들은 기존 서명 알고리즘에 비해 공개키와 서명의 크기가 크다. 따라서 TLS 통신에 필요한 인증서를 PQC 서명알고리즘을 통해 생성하게 되면 통신 오버헤드가 급증하는 문제가 있다. 이에, 본 논문에서는 TLS 에 사용되는 인증서에 RFP8879 에 정의된 3 가지 압축알고리즘(Zlib, Z-standard, Brotli)과, CBOR 인코딩 기법을 사용하여 인증서 압축성능을 비교해본다.

• Journal of Ginseng Research
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• v.47 no.2
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• pp.337-346
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• 2023

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-I to LC3-II and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.7
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• pp.936-940
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• 2008

The PCR-amplified prion protein (PrP) gene was sequenced to determine the frequency of scrapie-associated as well as novel PrP genotypes in 72 healthy goats representing five breeds. A total of six genotypes were detected, resulting from the three reported 143 (H/R), 154 (R/H) and 240 (S/P) and the two novel 39 (S/R) and 185 (I/F) amino acid polymorphisms. Of the four silent mutations 42 (a$\rightarrow$g), 138 (c$\rightarrow$t), 231 (c$\rightarrow$a) and 237 (g$\rightarrow$c) detected in this study, 237 (g$\rightarrow$c) is novel. A genotype (SIP/RFP) harboring three amino acid polymorphisms 39 (S/R), 185 (I/F) and 240 (S/P) was found in few goats. Although both scrapie-associated genotypes with 143 (H/R) and 154 (R/H) polymorphisms and others with 39 (S/R), 185 (I/F) and 240 (S/P) polymorphisms were present in the studied Pakistani goats, their frequency was lower than that of the wild-type genotype SHRIS/SHRIS (34.7%). These results emphasize the need for further sequencing of the PrP gene in a large number of goats representing the five studied breeds, so that overall PrP variability can be assessed in these breeds in research addressing future concerns about scrapie.

• Molecules and Cells
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• v.27 no.4
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• pp.391-396
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• 2009

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events - for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5$^{TM}$ and CyTRAK Orange$^{TM}$. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.

• The Plant Pathology Journal
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• v.35 no.1
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• pp.19-31
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• 2019

Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.

• Tuberculosis and Respiratory Diseases
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• v.62 no.4
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• pp.270-275
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• 2007

The QuantiFERON-TB Gold assay and tuberculin skin test (TST) have been useful test for diagnosing latent tuberculosis infections (LTBI). However, there are few reports on the efficacy of the QuantiFERON-TB Gold assay and TST in evaluating the response after the treatment of LTBI. This study examined the changes in the TST and QuantiFERON-TB Gold assay before and after a treatment for latent tuberculosis in health care workers (HCWs) at a local tertiary hospital. Methods: A cohort of volunteers working as nurses and doctors who underwent a TST and QuantiFERON-TB Gold assay was established. The volunteers positive for the QuantiFERON-TB Gold assay had been treated with 3 months of isoniazid (INH) and rifampin (RFP). After completing treatment, the TST and QuantiFERON-TB Gold assay were repeated. Results: Of the 48 participants (14 doctors, 34 nurses, M: F=11:37, mean $age=29.9{\pm}5.5$ years, mean employment $period=74.9{\pm}64.3$ months), 19 (39.6%) tested positive to the TST (mean induration=$19.1{\pm}9.7mm$) and 8 (16.7%) were QuantiFERON-TB Gold assay. Among them, one had active pulmonary tuberculosis. Seven volunteers were consistently positive to both the TST and QuantiFERON-TB Gold assay after being medicated with INH and RFP for 3 months. Conclusion: TST and QuantiFERON-TB Gold assay are unsuitable for evaluating the treatment response of LTBI because they were consistently positive both before and after the anti-tuberculosis medication.