• Journal of Animal Reproduction and Biotechnology
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• v.39 no.2
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• pp.67-80
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• 2024

Background: Post-ovulatory aging (POA) of oocytes is related to a decrease in the quality and quantity of oocytes caused by aging. Previous studies on the characteristics of POA have investigated injury to early embryonic developmental ability, but no information is available on its effects on mitochondrial fission and mitophagy-related responses. In this study, we aimed to elucidate the molecular mechanisms underlying mitochondrial fission and mitophagy in in vitro maturation (IVM) oocytes and a POA model based on RNA sequencing analysis. Methods: The POA model was obtained through an additional 24 h culture following the IVM of matured oocytes. NMN treatment was administered at a concentration of 25 μM during the oocyte culture process. We conducted MitoTracker staining and Western blot experiments to confirm changes in mitochondrial function between the IVM and POA groups. Additionally, comparative transcriptome analysis was performed to identify differentially expressed genes and associated changes in mitochondrial dynamics between porcine IVM and POA model oocytes. Results: In total, 32 common genes of apoptosis and 42 mitochondrial fission and function uniquely expressed genes were detected (≥ 1.5-fold change) in POA and porcine metaphase II oocytes, respectively. Functional analyses of mitochondrial fission, oxidative stress, mitophagy, autophagy, and cellular apoptosis were observed as the major changes in regulated biological processes for oocyte quality and maturation ability compared with the POA model. Additionally, we revealed that the activation of NAD⁺ by nicotinamide mononucleotide not only partly improved oocyte quality but also mitochondrial fission and mitophagy activation in the POA porcine model. Conclusions: In summary, our data indicate that mitochondrial fission and function play roles in controlling oxidative stress, mitophagy, and apoptosis during maturation in POA porcine oocytes. Additionally, we found that NAD⁺ biosynthesis is an important pathway that mediates the effects of DRP1-derived mitochondrial morphology, dynamic balance, and mitophagy in the POA model.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.15-15
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• 2015

Aspergillus fumigatus is a major cause of invasive aspergillosis (IA), a significant health issue worldwide with high mortality rates up to 95%. Our lab is interested in how A. fumigatus adapts to low oxygen conditions 'hypoxia', which is one of the important host microenvironments. A. fumigatus SrbA is a basic helix-loop-helix (bHLH) transcriptional regulator and belongs to sterol regulatory element binding protein (SREBP) family members. Loss of SrbA completely blocks growth in hypoxia and results in avirulence in murine models of IA suggesting an essential role of SrbA in hypoxia adaptation and virulence in A. fumigatus. We conducted chromatin immunoprecipitation sequencing (ChIP-seq) with A. fumigatus wild type using a SrbA specific antibody, and 97 genes were revealed as SrbA direct targets. One of the 'SrbA regulons' (AFUB_099590) was a putative bHLH transcriptional regulator whose sequence contained a characteristic tyrosine substitution in the basic portion of the bHLH domain of SREBPs. Therefore, we designated AFUB_099590 SrbB. Further characterization of SrbB demonstrated that SrbB is important for radial growth, biomass production, and biosynthesis of heme intermediates in hypoxia and virulence in A. fumigatus. A series of quantitative real time PCR showed that transcription of several SrbA regulons is coordinately regulated by two SREBPs, SrbA and SrbB in hypoxia. This suggests that SrbA and SrbB have both dependent and independent functions in regulation of genes responsible for hypoxia adaptation in A. fumigatus. Together, our data provide new insights into complicated roles of SREBPs in adaptation of host environments and virulence in pathogenic fungi.

• BMB Reports
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• v.40 no.3
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• pp.315-324
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• 2007

The novel $\alpha$-amylase purified from locally isolated strain, Bacillus sp. KR-8104, (KRA) (Enzyme Microb Technol; 2005; 36: 666-671) is active in a wide range of pH. The enzyme maximum activity is at pH 4.0 and it retains 90% of activity at pH 3.5. The irreversible thermoinactivation patterns of KRA and the enzyme activity are not changed in the presence and absence of $Ca^{2+}$ and EDTA. Therefore, KRA acts as a $Ca^{2+}$-independent enzyme. Based on circular dichroism (CD) data from thermal unfolding of the enzyme recorded at 222 nm, addition of $Ca^{2+}$ and EDTA similar to its irreversible thermoinactivation, does not influence the thermal denaturation of the enzyme and its Tm. The amino acid sequence of KRA was obtained from the nucleotide sequencing of PCR products of encoding gene. The deduced amino acid sequence of the enzyme revealed a very high sequence homology to Bacillus amyloliquefaciens (BAA) (85% identity, 90% similarity) and Bacillus licheniformis $\alpha$-amylases (BLA) (81% identity, 88% similarity). To elucidate and understand these characteristics of the $\alpha$-amylase, a model of 3D structure of KRA was constructed using the crystal structure of the mutant of BLA as the platform and refined with a molecular dynamics (MD) simulation program. Interestingly enough, there is only one amino acid substitution for KRA in comparison with BLA and BAA in the region involved in the calcium-binding sites. On the other hand, there are many amino acid differences between BLA and KRA at the interface of A and B domains and around the metal triad and active site area. These alterations could have a role in stabilizing the native structure of the loop in the active site cleft and maintenance and stabilization of the putative metal triad-binding site. The amino acid differences at the active site cleft and around the catalytic residues might affect their pKa values and consequently shift its pH profile. In addition, the intrinsic fluorescence intensity of the enzyme at 350 nm does not show considerable change at pH 3.5-7.0.

• Molecules and Cells
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• v.41 no.11
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• pp.979-992
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• 2018

Potato (Solanum tuberosum L.) is the third most important food crop, and breeding drought-tolerant varieties is vital research goal. However, detailed molecular mechanisms in response to drought stress in potatoes are not well known. In this study, we developed EMS-mutagenized potatoes that showed significant tolerance to drought stress compared to the wild-type (WT) 'Desiree' cultivar. In addition, changes to transcripts as a result of drought stress in WT and drought-tolerant (DR) plants were investigated by de novo assembly using the Illumina platform. One-week-old WT and DR plants were treated with -1.8 Mpa polyethylene glycol-8000, and total RNA was prepared from plants harvested at 0, 6, 12, 24, and 48 h for subsequent RNA sequencing. In total, 61,100 transcripts and 5,118 differentially expressed genes (DEGs) displaying up- or down-regulation were identified in pairwise comparisons of WT and DR plants following drought conditions. Transcriptome profiling showed the number of DEGs with up-regulation and down-regulation at 909, 977, 1181, 1225 and 826 between WT and DR plants at 0, 6, 12, 24, and 48 h, respectively. Results of KEGG enrichment showed that the drought tolerance mechanism of the DR plant can mainly be explained by two aspects, the 'photosynthetic-antenna protein' and 'protein processing of the endoplasmic reticulum'. We also divided eight expression patterns in four pairwise comparisons of DR plants (DR0 vs DR6, DR12, DR24, DR48) under PEG treatment. Our comprehensive transcriptome data will further enhance our understanding of the mechanisms regulating drought tolerance in tetraploid potato cultivars.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.17 no.3
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• pp.160-180
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• 2012

We have studied the eukaryotic plankton species diversity to compare the community structure of fresh and brackish waters in the lower reaches of the Nakdong River using metagenomic methods. We constructed 18S rDNA clone libraries of total DNAs extracted from environmental water samples collected at Mulgeum (MG100929, fresh) and Eulsukdo bridge (ES, brackish). Through the steps of colony PCR, PCR-RFLP, sequencing and similarity analysis, we discovered the diverse species composition of eukaryotic plankton. Total 338 clones (170 at MG100929 and 168 at ES) were analyzed, and then we found 74 phylotypes (49 for MG100929 and 25 for ES). From the phylogenetic analysis, we confirmed various eukaryotic plankton of broad range of taxonomic groups, including Stramenopiles, Cryptophyta, Viridiplantae, Alveolata, Rhizaria, Metazoa, and Fungi. We also found several unreported species in Korea and candidates of new taxonomic entities at levels higher than genus. Especially, the cryptic species diversity including unreported phylotypes of Pirsonia (Stramenopiles) and Perkinsea (Alveolata) suggests that the molecular monitoring method can produce new informative biological data in monitoring the changes in the Nakdong River Mouth ecosystem.

• Tuberculosis and Respiratory Diseases
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• v.63 no.1
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• pp.52-58
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• 2007

Background: Photodynamic therapy is a viable option for lung cancer treatment, and many studies have shown that it is capable of inducing cell death in lung cancer cells. However, the precise mechanism of this cell death has not been fully elucidated. To investigate the early changes in cancer cell transcription, we treated A549 cells with the photosensitizer DH-I-180-3 and then we illuminated the cells. Methods: We investigated the gene expression profiles of the the A549 lung cancer cell line, using a DEG kit, following photodynamic therapy and we evaluated the cell viability by performing flow cytometry. We identified the genes that were significantly changed following photodynamic therapy by performing DNA sequencing. Results: The FACS data showed that the cell death of the lung cancer cells was mainly caused by necrosis. We found nine genes that were significantly changed and we identified eight of these genes. We evaluated the expression of two genes, 3-phosphoglycerate dehydrogenase and ribosomal protein S29. The expressed level of carbonic anhydrase XII, clusterin, MRP3s1 protein, complement 3, membrane cofactor protein and integrin beta 1 were decreased. Conclusion: Many of the gene products are membrane-associated proteins. The main mechanism of photodynamic therapy with using the photosensitizing agent DH-I-180-3 appears to be necrosis and this may be associated with the altered production of membrane proteins.

• Proceedings of the Korean Society of Crop Science Conference
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• 2004.04a
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• pp.12-27
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• 2004

Thanks to spectacular advances in the techniques for identifying proteins separated by two-dimensional electrophoresis and in methods for large-scale analysis of proteome variations, proteomics is becoming an essential methodology in various fields of plant sciences. Plant proteomics would be most useful when combined with other functional genomics tools and approaches. A combination of microarray and proteomics analysis will indicate whether gene regulation is controlled at the level of transcription or translation and protein accumulation. In this review, we described the catalogues of the rice proteome which were constructed in our program, and functional characterization of some of these proteins was discussed. Mass-spectrometry is a most prevalent technique to identify rapidly a large of proteins in proteome analysis. However, the conventional Western blotting/sequencing technique us still used in many laboratories. As a first step to efficiently construct protein data-file in proteome analysis of major cereals, we have analyzed the N-terminal sequences of 100 rice embryo proteins and 70 wheat spike proteins separated by two-dimensional electrophoresis. Edman degradation revealed the N-terminal peptide sequences of only 31 rice proteins and 47 wheat proteins, suggesting that the rest of separated protein spots are N-terminally blocked. To efficiently determine the internal sequence of blocked proteins, we have developed a modified Cleveland peptide mapping method. Using this above method, the internal sequences of all blocked rice proteins (i. e., 69 proteins) were determined. Among these 100 rice proteins, thirty were proteins for which homologous sequence in the rice genome database could be identified. However, the rest of the proteins lacked homologous proteins. This appears to be consistent with the fact that about 30% of total rice cDNA have been deposited in the database. Also, the major proteins involved in the growth and development of rice can be identified using the proteome approach. Some of these proteins, including a calcium-binding protein that fumed out to be calreticulin, gibberellin-binding protein, which is ribulose-1,5-bisphosphate carboxylase/oxygenase activate in rice, and leginsulin-binding protein in soybean have functions in the signal transduction pathway. Proteomics is well suited not only to determine interaction between pairs of proteins, but also to identify multisubunit complexes. Currently, a protein-protein interaction database for plant proteins (http://genome .c .kanazawa-u.ac.jp/Y2H)could be a very useful tool for the plant research community. Recently, we are separated proteins from grain filling and seed maturation in rice to perform ESI-Q-TOF/MS and MALDI-TOF/MS. This experiment shows a possibility to easily and rapidly identify a number of 2-DE separated proteins of rice by ESI-Q-TOF/MS and MALDI-TOF/MS. Therefore, the Information thus obtained from the plant proteome would be helpful in predicting the function of the unknown proteins and would be useful in the plant molecular breeding. Also, information from our study could provide a venue to plant breeder and molecular biologist to design their research strategies precisely.

• Parasites, Hosts and Diseases
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• v.57 no.4
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• pp.341-357
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• 2019

Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hotspring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological purposes. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technology, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.

• Tuberculosis and Respiratory Diseases
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• v.44 no.2
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• pp.251-263
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• 1997

Background : The emergence of multidrug-resistant strains of Mycobacterium tuberculosis presents a significant challange to the treatment and control of tuberculosis, and there is an urgent need to understand the mechanisms by which strains acquire multidrug resistance. Recent advances in molecular methods for the detection of M. tuberculosis genetic targets have approached the sensitivity of culture. Furthermore the prospect of determining resistance in mycobacteria at the nucleic acid level particulary to first-line drugs like rifampin, isoniazid has provided a glimps of the next generation of sensitivity test for M. tuberculosis. Previous studies in RMP resistant M. tuberculosis have shown that mutation in $\beta$subunit of RNA polymerase is main mechanism of resistance. Method : In this study, rpoB gene for the $\beta$subunit of RNA polymerase from M. tuberculosis of 42 cultured samples (32 were RMP resistant and 10 were sensitive cases) were isolated and characterised the mutations. Direct sequencing data were compared with the results of INNO-LiPA Line Probe Assay (LiPA, Innogenetics, Belgium), commercial RMP resistance detecting kit using reverse hybridization method. Results : All of the RMP resistant samples were revealed the presence of mutation by LiPA. In 22 samples (68.8%) out of 32 RMP resistant cases, the mutation types were confirmed by the positive signal at one of 4 mutation bands in the strip. The most frequent type was R5 (S531L) which were 17 cases (77.3%). Results of direct sequencing were identified the exact characteristics of 8 mutations which were not confirmed by LiPA. S522W type point mutation and 9 base pair deletion at codon 513~515 were new identified mutations for the first time. Conclusion : Mutations in rpoB gene is the main mechanism of RMP resistance in M. tuberculosis and LiPA is a very useful diagnostic tool for the early diagnosis of RMP resistance in M. tuberculosis.

• Journal of Plant Biotechnology
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• v.44 no.4
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• pp.372-378
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• 2017

For comparison of the transcription profiles in apple (Malus domestica L.) cultivars differing in flesh color expression, two cDNA libraries were constructed. Differences in gene expression between red flesh apple cultivar, 'Redfield' and white flesh apple cultivar, 'Granny Smith' were investigated by next-generation sequencing (NGS). Expressed sequence tag (EST) of clones from the red flesh apple cultivar and white flesh apple cultivar were selected for nucleotide sequence determination and homology searches. High resolution melting (HRM) technique measures temperature induced strand separation of short PCR amplicons, and is able to detect variation as small as one base difference between red flesh apple cultivars and white flesh apple cultivars. We applied high resolution melting (HRM) analysis to discover single nucleotide polymorphisms (SNP) based on the predicted SNP information derived from the apple EST database. All 103 pairs of SNPs were discriminated, and the HRM profiles of amplicons were established. Putative SNPs were screened from the apple EST contigs by HRM analysis displayed specific difference between 10 red flesh apple cultivars and 11 white flesh apple cultivars. In this study, we report an efficient method to develop SNP markers from an EST database with HRM analysis in apple. These SNP markers could be useful for apple marker assisted breeding and provide a good reference for relevant research on molecular mechanisms of color variation in apple cultivars.