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Mass Spectrometry-based Comparative Analysis of Membrane Protein: High-speed Centrifuge Method Versus Reagent-based Method (질량분석기를 활용한 막 단백질 비교분석: High-speed Centrifuge법과 Reagent-based법)

  • Lee, Jiyeong;Seok, Ae Eun;Park, Arum;Mun, Sora;Kang, Hee-Gyoo
    • Korean Journal of Clinical Laboratory Science
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    • v.51 no.1
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    • pp.78-85
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    • 2019
  • Membrane proteins are involved in many common diseases, including heart disease and cancer. In various disease states, such as cancer, abnormal signaling pathways that are related to the membrane proteins cause the cells to divide out of control and the expression of membrane proteins can be altered. Membrane proteins have the hydrophobic environment of a lipid bilayer, which makes an analysis of the membrane proteins notoriously difficult. Therefore, this study evaluated the efficacy of two different methods for optimal membrane protein extraction. High-speed centrifuge and reagent-based method with a -/+ filter aided sample preparation (FASP) were compared. As a result, the high-speed centrifuge method is quite effective in analyzing the mitochondrial inner membranes, while the reagent-based method is useful for endoplasmic reticulum membrane analysis. In addition, the function of the membrane proteins extracted from the two methods were analyzed using GeneGo software. GO processes showed that the endoplasmic reticulum-related responses had higher significance in the reagent-based method. An analysis of the process networks showed that one cluster in the high-speed centrifuge method and four clusters in the reagent-based method were visualized. In conclusion, the two methods are useful for the analysis of different subcellular membrane proteins, and are expected to assist in selecting the membrane protein extraction method by considering the target subcellular membrane proteins for study.

Adjunctive hyperbaric oxygen therapy for irradiated rat calvarial defects

  • An, Heesuk;Lee, Jung-Tae;Oh, Seo-Eun;Park, Kyeong-mee;Hu, Kyung-Seok;Kim, Sungtae;Chung, Moon-Kyu
    • Journal of Periodontal and Implant Science
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    • v.49 no.1
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    • pp.2-13
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    • 2019
  • Purpose: The aim of this study was to conduct a histologic evaluation of irradiated calvarial defects in rats 4 weeks after applying fibroblast growth factor-2 (FGF-2) with hyaluronan or biphasic calcium phosphate (BCP) block in the presence or absence of adjunctive hyperbaric oxygen (HBO) therapy. Methods: Twenty rats were divided into HBO and non-HBO (NHBO) groups, each of which was divided into FGF-2 and BCP-block subgroups according to the grafted material. Localized radiation with a single 12-Gy dose was applied to the calvaria of rats to simulate radiotherapy. Four weeks after applying this radiation, 2 symmetrical circular defects with a diameter of 6 mm were created in the parietal bones of each animal. The right-side defect was filled with the materials mentioned above and the left-side defect was not filled (as a control). All defects were covered with a resorbable barrier membrane. During 4 weeks of healing, 1 hour of HBO therapy was applied to the rats in the HBO groups 5 times a week. The rats were then killed, and the calvarial specimens were harvested for radiographic and histologic analyses. Results: New bone formation was greatest in the FGF-2 subgroup, and improvement was not found in the BCP subgroup. HBO seemed to have a minimal effect on new bone formation. There was tendency for more angiogenesis in the HBO groups than the NHBO groups, but the group with HBO and FGF-2 did not show significantly better outcomes than the HBO-only group or the NHBO group with FGF-2. Conclusions: HBO exerted beneficial effects on angiogenesis in calvarial defects of irradiated rats over a 4-week healing period, but it appeared to have minimal effects on bone regeneration. FGF-2 seemed to enhance new bone formation and angiogenesis, but its efficacy appeared to be reduced when HBO was applied.

Influence of (E)-2-hexenyl (Z)-3hexenoate on the Occurrence of Egg Parasitoids, Ooencyrtus nezarae (Hymenoptera: Encyrtidae) and Gryon japonicum (Hymenoptera: Scelionidae), in Apple Orchards (사과원에서 (E)-2-hexenyl (Z)-3hexenoate 성분이 노린재깡충좀벌(Ooencyrtus nezarae)(Hymenoptera: Encyrtidae)과 노린재기생알벌(Gryon japonicum) (Hymenoptera: Scelionidae) 발생에 미치는 영향)

  • Baek, Seonghoon;Park, Chung Gyoo;Seo, Bo Yoon;Cho, Jum Rae;Park, Chang-Gyu
    • Korean journal of applied entomology
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    • v.60 no.1
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    • pp.91-98
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    • 2021
  • The populations of Riptortus pedestris (Fabricius) cause serious economic damages in local apple orchards. However, there was no study to manage its populations with environment friendly tactics. Thus, this study was conducted to analyze its attraction effects of one of R. pedestris aggregation pheromone compounds, (E)-2-hexenyl (Z)-3hexenoate (E2HZ3H), to its parasitoids, Ooencyrtus nezarae (Ishii) and Gryon japonicum (Ashmead). This compound did not increase the number of these two parasitoids and change its spatial distribution. However, this compound could attract two parasitoids within limited distance. These results indicates that one compound of R. pedestris aggregation pheromone, E2HZ3H, could be a good candidate to manage R. pedestris populations in conditions that its populations locally occurs in apple orchards.

Inhibitory effects of the atypical antipsychotic, clozapine, on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

  • Kang, Minji;Heo, Ryeon;Park, Seojin;Mun, Seo-Yeong;Park, Minju;Han, Eun-Taek;Han, Jin-Hee;Chun, Wanjoo;Ha, Kwon-Soo;Park, Hongzoo;Jung, Won-Kyo;Choi, Il-Whan;Park, Won Sun
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.4
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    • pp.277-285
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    • 2022
  • To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentration-and use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

Masticatory Performance and the Related Factors in Korean Children and Adolescents (한국 소아청소년의 저작 능력 평가 및 관련 요인)

  • Minah, Lee;Taeyang, Lee;Baek-il, Kim;Je Seon, Song
    • Journal of the korean academy of Pediatric Dentistry
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    • v.49 no.2
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    • pp.170-179
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    • 2022
  • This study aimed to evaluate the masticatory performance of Korean children and adolescents and to investigate related factors. Early loss of primary molars, degree of occlusion of first molars, occlusal relationship of first molars, and presence of anterior crossbite were considered. From March 2020 to July 2021, 56 children and adolescents between the ages of 6 and 12 were included. The mixing ability index (MAI) was calculated to evaluate the masticatory efficiency of children and adolescents. The subjects were classified into three groups according to the number of early lost primary molars; normal dentition, 1 - 2 teeth lost, more than 3 teeth lost. The number of participants are 23, 18, and 15, respectively. There was no difference in the MAI values between the normal dentition group and the group has 1 - 2 teeth lost. However, when 3 or more primary molars were lost, the MAI value decreased and a significant difference was observed. This study is the first study to evaluate the masticatory performance of children and adolescents in Korea, and it will be helpful to pediatric dentists who evaluate the masticatory performance of children and adolescents and strive to improve the masticatory efficiency of children and adolescents in clinical practice.

Receptor binding motif surrounding sites in the Spike 1 protein of infectious bronchitis virus have high susceptibility to mutation related to selective pressure

  • Seung-Min Hong;Seung-Ji Kim;Se-Hee An;Jiye Kim;Eun-Jin Ha;Howon Kim;Hyuk-Joon Kwon;Kang-Seuk Choi
    • Journal of Veterinary Science
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    • v.24 no.4
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    • pp.51.1-51.17
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    • 2023
  • Background: To date, various genotypes of infectious bronchitis virus (IBV) have co-circulated and in Korea, GI-15 and GI-19 lineages were prevailing. The spike protein, particularly S1 subunit, is responsible for receptor binding, contains hypervariable regions and is also responsible for the emerging of novel variants. Objective: This study aims to investigate the putative major amino acid substitutions for the variants in GI-19. Methods: The S1 sequence data of IBV isolated from 1986 to 2021 in Korea (n = 188) were analyzed. Sequence alignments were carried out using Multiple alignment using Fast Fourier Transform of Geneious prime. The phylogenetic tree was generated using MEGA-11 (ver. 11.0.10) and Bayesian analysis was performed by BEAST v1.10.4. Selective pressure was analyzed via online server Datamonkey. Highlights and visualization of putative critical amino acid were conducted by using PyMol software (version 2.3). Results: Most (93.5%) belonged to the GI-19 lineage in Korea, and the GI-19 lineage was further divided into seven subgroups: KM91-like (Clade A and B), K40/09-like, QX-like (I-IV). Positive selection was identified at nine and six residues in S1 for KM91-like and QX-like IBVs, respectively. In addition, several positive selection sites of S1-NTD were indicated to have mutations at common locations even when new clades were generated. They were all located on the lateral surface of the quaternary structure of the S1 subunits in close proximity to the receptor-binding motif (RBM), putative RBM motif and neutralizing antigenic sites in S1. Conclusions: Our results suggest RBM surrounding sites in the S1 subunit of IBV are highly susceptible to mutation by selective pressure during evolution.

GRIM-19 Ameliorates Multiple Sclerosis in a Mouse Model of Experimental Autoimmune Encephalomyelitis with Reciprocal Regulation of IFNγ/Th1 and IL-17A/Th17 Cells

  • Jeonghyeon Moon;Seung Hoon Lee;Seon-yeong Lee;Jaeyoon Ryu;Jooyeon Jhun;JeongWon Choi;Gyoung Nyun Kim;Sangho Roh;Sung-Hwan Park;Mi-La Cho
    • IMMUNE NETWORK
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    • v.20 no.5
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    • pp.40.1-40.15
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    • 2020
  • The protein encoded by the Gene Associated with Retinoid-Interferon-Induced Mortality-19 (GRIM-19) is located in the mitochondrial inner membrane and is homologous to the NADH dehydrogenase 1-alpha subcomplex subunit 13 of the electron transport chain. Multiple sclerosis (MS) is a demyelinating disease that damages the brain and spinal cord. Although both the cause and mechanism of MS progression remain unclear, it is accepted that an immune disorder is involved. We explored whether GRIM-19 ameliorated MS by increasing the levels of inflammatory cytokines and immune cells; we used a mouse model of experimental autoimmune encephalomyelitis (EAE) to this end. Six-to-eight-week-old male C57BL/6, IFNγ-knockout (KO), and GRIM-19 transgenic mice were used; EAE was induced in all strains. A GRIM-19 overexpression vector (GRIM19 OVN) was electrophoretically injected intravenously. The levels of Th1 and Th17 cells were measured via flow cytometry, immunofluorescence, and immunohistochemical analysis. IL-17A and IFNγ expression levels were assessed via ELISA and quantitative PCR. IL-17A expression decreased and IFNγ expression increased in EAE mice that received injections of the GRIM-19 OVN. GRIM19 transgenic mice expressed more IFNγ than did wild-type mice; this inhibited EAE development. However, the effect of GRIM-19 overexpression on the EAE of IFNγ-KO mice did not differ from that of the empty vector. GRIM-19 expression was therapeutic for EAE mice, elevating the IFNγ level. GRIM-19 regulated the Th17/Treg cell balance.

Morroniside Protects C2C12 Myoblasts from Oxidative Damage Caused by ROS-Mediated Mitochondrial Damage and Induction of Endoplasmic Reticulum Stress

  • Hyun Hwangbo;Cheol Park;EunJin Bang;Hyuk Soon Kim;Sung-Jin Bae;Eunjeong Kim;Youngmi Jung;Sun-Hee Leem;Young Rok Seo;Su Hyun Hong;Gi-Young Kim;Jin Won Hyun;Yung Hyun Choi
    • Biomolecules & Therapeutics
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    • v.32 no.3
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    • pp.349-360
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    • 2024
  • Oxidative stress contributes to the onset of chronic diseases in various organs, including muscles. Morroniside, a type of iridoid glycoside contained in Cornus officinalis, is reported to have advantages as a natural compound that prevents various diseases. However, the question of whether this phytochemical exerts any inhibitory effect against oxidative stress in muscle cells has not been well reported. Therefore, the current study aimed to evaluate whether morroniside can protect against oxidative damage induced by hydrogen peroxide (H2O2) in murine C2C12 myoblasts. Our results demonstrate that morroniside pretreatment was able to inhibit cytotoxicity while suppressing H2O2-induced DNA damage and apoptosis. Morroniside also significantly improved the antioxidant capacity in H2O2-challenged C2C12 cells by blocking the production of cellular reactive oxygen species and mitochondrial superoxide and increasing glutathione production. In addition, H2O2-induced mitochondrial damage and endoplasmic reticulum (ER) stress were effectively attenuated by morroniside pretreatment, inhibiting cytoplasmic leakage of cytochrome c and expression of ER stress-related proteins. Furthermore, morroniside neutralized H2O2-mediated calcium (Ca2+) overload in mitochondria and mitigated the expression of calpains, cytosolic Ca2+-dependent proteases. Collectively, these findings demonstrate that morroniside protected against mitochondrial impairment and Ca2+-mediated ER stress by minimizing oxidative stress, thereby inhibiting H2O2-induced cytotoxicity in C2C12 myoblasts.

Effects of Elevated Temperature after the Booting Stage on Physiological Characteristics and Grain Development in Wheat (밀에서 출수 후 잎의 생리적 특성 및 종실 생장에 대한 수잉기 이후 고온의 효과)

  • Song, Ki Eun;Choi, Jae Eun;Jung, Jae Gyeong;Ko, Jong Han;Lee, Kyung Do;Shim, Sang-In
    • KOREAN JOURNAL OF CROP SCIENCE
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    • v.66 no.4
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    • pp.307-317
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    • 2021
  • In recent years, global warming has led to frequent climate change-related problems, and elevated temperatures, among adverse climatic factors, represent a critical problem negatively affecting crop growth and yield. In this context, the present study examined the physiological traits of wheat plants grown under high temperatures. Specifically, the effects of elevated temperatures on seed development after heading were evaluated, and the vegetation indices of different organs were assessed using hyperspectral analysis. Among physiological traits, leaf greenness and OJIP parameters were higher in the high-temperature treatment than in the control treatment. Similarly, the leaf photosynthetic rate during seed development was higher in the high-temperature treatment than in the control treatment. Moreover, temperature by organ was higher in the high-temperature treatment than in the control treatment; consequently, the leaf transpiration rate and stomatal conductance were higher in the control treatment than in the high-temperature treatment. On all measuring dates, the weight of spikes and seeds corresponding to the sink organs was greater in the high-temperature treatment than in the control treatment. Additionally, the seed growth rate was higher in the high-temperature treatment than in the control treatment 14 days after heading, which may be attributed to the higher redistribution of photosynthates at the early stage of seed development in the former. In hyperspectral analysis, the vegetation indices related to leaf chlorophyll content and nitrogen state were higher in the high-temperature treatment than in the control treatment after heading. Our results suggest that elevated temperatures after the booting stage positively affect wheat growth and yield.

Characterization of a Korean Domestic Cyanobacterium Limnothrix sp. KNUA012 for Biofuel Feedstock (토착 남세균 림노트릭스 속 KNUA012 균주의 바이오연료 원료로서의 특성 연구)

  • Hong, Ji Won;Jo, Seung-Woo;Kim, Oh Hong;Jeong, Mi Rang;Kim, Hyeon;Park, Kyung Mok;Lee, Kyoung In;Yoon, Ho-Sung
    • Journal of Life Science
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    • v.26 no.4
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    • pp.460-467
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    • 2016
  • A filamentous cyanobacterium, Limnothrix sp. KNUA012, was axenically isolated from a freshwater bloom sample in Lake Hapcheon, Hapcheon-gun, Gyeongsangnam-do, Korea. Its morphological and molecular characteristics led to identification of the isolate as a member of the genus Limnothrix. Maximal growth was attained when the culture was incubated at 25℃. Analysis of its lipid composition revealed that strain KNUA012 could autotrophically synthesize alkanes, such as pentadecane (C15H32) and heptadecane (C17H36), which can be directly used as fuel without requiring a transesterification step. Two genes involved in alkane biosynthesis-an acyl-acyl carrier protein reductase and an aldehyde decarbonylase-were present in this cyanobacterium. Some common algal biodiesel constituents-myristoleic acid (C14:1), palmitic acid (C16:0), and palmitoleic acid (C16:1)-were produced by strain KNUA012 as its major fatty acids. A proximate analysis showed that the volatile matter content was 86.0% and an ultimate analysis indicated that the higher heating value was 19.8 MJ kg−1. The isolate also autotrophically produced 21.4 mg g−1 phycocyanin-a high-value antioxidant compound. Therefore, Limnothrix sp. KNUA012 appears to show promise for application in cost-effective production of microalga-based biofuels and biomass feedstock over crop plants.