• 대한한의학회지
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• 제40권2호
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• pp.35-50
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• 2019

Objectives: Orostachys japonicas (O. japonicus) has been known for its anti-tumor effect. In the present study, it was investigated whether O. japonicus EtOH extracts could induce apoptosis and autophagy which are part of the main mechanism related to anti-tumor effect in THP-1 cells. Methods: Cells were treated with various concentrations of O. japonicus EtOH extracts ($0-300{\mu}g/ml$) for 24, 48, and 72h. Cell viability was evaluated by MTS/PMS assay and apoptosis rate was examined by flow cytometry and ELISA assay. The mRNA expression of apoptosis-related genes (Bcl-2, Mcl-1, Survivin, Bax) and autophagy-related gene (mTOR) was evaluated using real-time PCR. The protein expression of Caspase-3, Akt, LC3 II, Beclin-1, Atg5, $NF-{\kappa}B$, p38, ERK was evaluated using western blot analysis. Results: O. japonicus EtOH extracts inhibited cell proliferation and apoptosis rate was increased in both flow cytometry and ELISA assay. Bcl-2, Mcl-1, Survivin (anti-apoptosis factors) mRNA expressions were decreased and Bax (pro-apoptosis factor) mRNA level was increased. mTOR mRNA expressions was decreased and LC3 II protein expressions was increased. Activation of $NF-{\kappa}B$ was decreased and phosphorylation of p38 was increased. Conclusion: O. japonicus is regarded to inhibit cell proliferation, to induce apoptosis and to regulate autophagy-related genes in THP-1 cells via $NF-{\kappa}B$ and p38 MAPK signaling pathway. This suggests O. japonicus could be an effective herb in treating acute myeloid leukemia.

• 한국산림과학회지
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• 제90권1호
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• pp.43-54
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• 2001

집단의 유전적 분화는 환경요인과 유전적 소인의 복잡한 상호관계에 의해 이루어지는데, 집단내 요인으로는 종자나 화분 비산, 국소환경변이, 개체의 분포밀도 등을 들 수 있다. 점봉산의 잣나무 임분내 개체 분포와 유전적 구성에 대한 분석을 위하여 신갈나무군락내 잣나무 우접임분에 $100{\times}100m$ 조사구를 설정하였으며, 잣나무 325개체에 대한 동위효소 분석을 실시하고, 공간의 자기상관성을 계산하였다. 11개 동위효소 유전자좌에서 관찰된 평균 다형성 유전자좌 비율(P)은 72.7%, 이형접합도의 관찰치(Ho)는 0.200, 기대치(He)는 0.251로 이형접합체 과소현상을 나타내어 근연가계의 개체들이 다수 분포하는 것으로 조사되었다. 동위효소의 유전자형에 따라 325개체는 총 147 개의 그룹으로 구분되었으며, 한 개 유전자형에 최대 34개체가 포함되었다. 동위효소 유전자형의 이성성이 24~32m 구간(거리간격 8m)을 기점으로 증가하는 양상을 보였으며, 생장특성(수고와 직경)에 대한 simple block distance도 24~32m 구간에서 임의분포를 보였다. 높은 근연교배의 고정계수(F=0.204), 유전적 자기상관성, 생장특성의 분석, 동일 유전자형의 분포범위 등을 고려할 때 점봉산 잣나무 임분은 화분비산이나 숲틈 형성에 따른 종자의 집중 투입에 따른 유전자 이동(gene flow)보다는 유전적 근연(近緣)개체의 밀도(密度)에 의존(density dependent)해서 유전적 구조를 유지하는 것으로 생각되며, 유전적 동질성을 갖는 군락의 크기는 24~32m에서 결정할 수 있었다. 본 연구의 결과 유전자원보존을 위한 개체의 선정은 최소한 37m 이상의 거리를 띄어야 바람직한 것으로 조사되었다.

• Asian-Australasian Journal of Animal Sciences
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• 제32권11호
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• pp.1789-1800
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• 2019

Objective: Although alveolar macrophages play a key role in the respiratory immunity of livestock, studies on the mechanism of differentiation and survival of alveolar macrophages are lacking. Therefore, we undertook to investigate changes in the lipid metabolism and survival rate, using 3D4/31 macrophages and Dudleya brittonii which has been used as a traditional asthma treatment. Methods: 3D4/31 macrophages were used as the in vitro porcine alveolar macrophages model. The cells were activated by exposure to phorbol 12-myristate 13-acetate (PMA). Dudleya brittonii extraction was performed with distilled water. For evaluating the cell survival rate, we performed the water-soluble tetrazolium salt cell viability assay and growth curve analysis. To confirm cell death, cell cycle and intracellular reactive oxygen species (ROS) levels were measured using flow cytometric analysis by applying fluorescence dye dichlorofluorescein diacetate and propidium iodide. Furthermore, we also evaluated cellular lipid accumulation with oil red O staining, and fatty acid synthesis related genes expression levels using quantitative polymerase chain reaction (qPCR) with SYBR green dye. Glycolysis, fatty acid oxidation, and tricarboxylic acid (TCA) cycle related gene expression levels were measured using qPCR after exposure to Dudleya brittonii extract (DB) for 12 h. Results: The ROS production and cell death were induced by PMA treatment, and exposure to DB reduced the PMA induced downregulation of cell survival. The PMA and DB treatments upregulated the lipid accumulation, with corresponding increase in the acetyl-CoA carboxylase alpha, fatty acid synthase mRNA expressions. DB-PMA co-treatment reduced the glycolysis genes expression, but increased the expressions of fatty acid oxidation and TCA cycle genes. Conclusion: This study provides new insights and directions for further research relating to the immunity of porcine respiratory system, by employing a model based on alveolar macrophages and natural materials.

• Asian Pacific Journal of Cancer Prevention
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• 제15권24호
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• pp.10819-10824
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• 2015

Aims: To explore the effect and probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethylphenyl ester (ATPR) on apoptosis of MDA-MB-231 breast cancer cells. Materials and Methods: MTT assays were performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all-trans retinoic acid (ATRA) and ATPR. Morphologic changes were observed by microscopy. The apoptosis rates and cell cycling of MDA-MB-231 cells treated with ATRA or ATPR were assessed using flow cytometry analysis. Expression of retinoic acid receptor and phosphorylation of ERK, JNK, p38 proteins were detected by Western blotting. Results: Treatment of the cells with the addition of $15{\mu}mol/L$ ATPR for 48 h clearly demonstrated reduced cell numbers and deformed cells, whereas no changes in the number and morphology were observed after treatment with ATRA. The apoptosis rate was 33.2% after breast cancer MDA-MB-231 cells were treated by ATPR ($15{\mu}mol/L$) whereas ATRA ($15{\mu}mol/L$) had no apoptotic effect. ATPR inhibited the phosphorylation of ERK, JNK, and p38 while ATRA had no significant effect. ATPR inhibited the expression of BiP and increased the expression of Chop at the protein level compared with control groups, ATRA and ATPR both decreased the protein expression of $RXR{\alpha}$, ATPR reduced the protein expression of $RAR{\beta}$ and $RXR{\beta}$ while ATRA did not decrease $RAR{\beta}$ or $RXR{\beta}$. Conclusions: ATPR could induce apoptosis of breast cancer MDA-MB-231 cells, possible mechanisms being binding to $RAR{\beta}/RXR{\beta}$ heterodimers, then activation of ER stress involving the MAPK pathway.

• Journal of Acupuncture Research
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• 제17권2호
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• pp.169-186
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• IMMUNE NETWORK
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• 제1권3호
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• pp.236-243
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• 2001

Background: An immunological approach for aging mechanism appears to be important. Lymphocyte subsets analysis in peripheral blood is widely performed to assess the immune status and to diagnose and monitor various diseases. Some lymphocyte subsets are known to change with age, but only few data about age-related reference ragnes for these subsets in healthy individuals have been reported. So we attempted to report reference ranges for these subsets in each age group and review changes of the results with age for the secondary studies about immune cell function as lymphocyte blast transformation and immunoglobulin gene rearrangement (VDJ) including recombination activating genes (RAG-1 and RAG-2). Methods: Lymphocyte subset analysis was performed on 302 subjects, 189 males and 113 females with age group of all decades of life. Two color direct immunofluorescene flow cytometry (FCM) was done using $Simultest^{TM}$ IMK-Lymphocyte kit (Becton Dickinson, USA), $FACScan^{TM}$ (Becton Dickinson, USA) and $FACSCalibur^{TM}$ (Becton Dickinson, USA). Lymphocyte subsets analysed were T ($CD3^+$) and B cells ($CD19^+$), helper/inducer T ($CD4^+$) and suppressor/cytotoxic T cells ($CD8^+$), helper/suppressor ($CD4^+/CD8^+$) ratio and natural killer (NK) cells ($CD3^-CD16^+/CD56^+$). The absolute numbers of each subset were calculated from total lymphocyte counts. Data collected was analysed using SAS 6.12. A P-value of < 0.05 was considered significant. Results: We reported the counts and percentages of lymphocyte and these subsets in each age group. There were no statistically significant differences between male and female subjects. The percentage of $CD4^+$ T cells, and the count of NK cells did not show the significant difference among the various age groups. The age-related changes observed in our study were as following: 1) a decrease in the percentages of T cells, B cells and $CD8^+$ T cells ; 2) a decrease in the counts of B cells and $CD8^+$ T cells ; 3) an increase in the percentage and count of NK cells ; and 4) an increase in the $CD4^+/CD8^+$ ratio. Conclusion: The characteristics of aging process appeared to be showing a marked decrease of lympocyte subsets T and B cells as well as T8 ($CD8^+$). The age-related increase of the percentage of cells bearing NK marker can be interpreted as a compensatory consequence to cope with the decrease of T cells related to the thymic involution. These changes with age appeared to be for the secondary study about immune cell function as lymphocyte blast transformation and immunoglobulin gene rearrangement.

• Biomolecules & Therapeutics
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• 제20권1호
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• pp.81-88
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• 2012

Histone deacetylases (HDACs) are enzymes involved in the remodelling of chromatin, and have a key role in the epigenetic regulation of gene expression. Histone deacetylase (HDAC) inhibitors are emerging as an exciting new class of potential anti-cancer agents. In recent years, a number of structurally diverse HDAC inhibitors have been identifi ed and these HDAC inhibitors induce growth arrest, differentiation and/or apoptosis of cancer cells in vitro and in vivo. However, the underlying molecular mechanisms remain unclear. This study aimed at investigating the anti-tumor activity of various HDAC inhibitors, IN-2001, using T47D human breast cancer cells. Moreover, the possible mechanism by which HDAC inhibitors exhibit anti-tumor activity was also explored. In estrogen receptor positive T47D cells, IN-2001, HDAC inhibitor showed anti-proliferative effects in dose-and time-dependent manner. In T47D human breast cancer cells showed anti-tumor activity of IN-2001 and the growth inhibitory effects of IN-2001 were related to the cell cycle arrest and induction of apoptosis. Flow cytometry studies revealed that IN-2001 showed accumulation of cells at $G_2$/M phase. At the same time, IN-2001 treatment time-dependently increased sub-$G_1$ population, representing apoptotic cells. IN-2001-mediated cell cycle arrest was associated with induction of cdk inhibitor expression. In T47D cells, IN-2001 as well as other HDAC inhibitors treatment significantly increased $p21^{WAF1}$ and $p27^{KIP1}$ expression. In addition, thymidylate synthase, an essential enzyme for DNA replication and repair, was down-regulated by IN-2001 and other HDAC inhibitors in the T47D human breast cancer cells. In summary, IN-2001 with a higher potency than other HDAC inhibitors induced growth inhibition, cell cycle arrest, and eventual apoptosis in human breast cancer possibly through modulation of cell cycle and apoptosis regulatory proteins, such as cdk inhibitors, cyclins, and thymidylate synthase.

• Animal Systematics, Evolution and Diversity
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• 제12권4호
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• pp.331-347
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• 1996

한국산 밀어(R. brunneus complex)에 대한 지리적 변이 및 분포를 조사한 결과 반 문 및 체색 등에 뚜렷한 차이가 있는 3type이 채집되었다. 이들은 유전적으로도 typerks에 총 genome의 26-30%(A-B type : 30%, A-C type : 30%, B-C type : 26%) 차이가 있어 유전적 근연치(Rogers' S)가 S=0.628-0.661(A-B type : 0.631, A-C type : 0.628, B-C type : 0.661)로 뚜렷한 유전적 차이를 나타냈다. 각 type의 유전적 변이 정도를 비교한 결과 A-type은 평균 P=25.90%, Ho=0.081, He=0.092로 B-type(P=12.33%, Ho=0.037, He=0.044)과 C-type(P=11.10%, Ho=0.032, He=0.048)에 비하여 약 2배 이상 높은 특징을 보였다. 이들 3 type의 분류학적 위치를 명확히 구명하고자 3type이 모두 서식하는 고성의 북천에서 유전적 표식인자를 이용하여 각 typerks의 생식적 격리 수준을 분석하였다. 이들 3 type은 동서(同 棲)하천에서 하구로부터의 거리에 따라 parapatric 하게 서식하면서 각 typerks에 유전자 교 환이 전혀없었다. 이러한 결과로부터 3type은 이미 생식적 격리가 완성되고 미세분포역에 차이가 있는 별종으로 판단되었다.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.129-129
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• 2017

The first key point to the successful pollination and fertilization in plants is the pollen pistil interaction, referring to the cellular and molecular levels, which mainly play active roles in limiting gene flow among maize populations and between maize and teosinte. This study was carried out to identify proteins and investigate the mechanism of gametophytic factors using protein analysis. W22 (ga1); which didn't carry a gametophytic factor and W22 (Ga1), a near iso-genic line were used for the proteome investigation. SDS-PAGE was executed to investigate proteins in the pollen and pistil of W22 (ga1) and W22 (Ga1). A total of 44 differentially expressed proteins were identified in the pollen and pistil on SDS-PAGE using LTQ-FTICR MS. Among the 44 proteins, a total of 24 proteins were identified in the pollen of W22 (ga1) and W22 (Ga1) whereas 20 differentially expressed proteins were identified from the pistil of W22 (Ga) and W22 (Ga1). However, in pollen, 2 proteins were identified only in the W22 (ga1) and 12 proteins only in the W22 (Ga1) whereas 10 proteins were confirmed from the both of W22 (ga1) and W22 (Ga1). In contrary, 10 proteins were appeared only in the pistil of W22 (ga1) and 7 proteins from W22 (Ga1) while 3 proteins confirmed in the both of W22 (ga1) and W22 (Ga1). Moreover, the identified proteins were generally involved in hydrolase activity, nucleic acid binding and nucleotide binding. These results help to reveal the mechanism of gametophytic factors and provide a valuable clue for the pollen and pistil research in maize. In addition, it might provide a comprehensive insight on the proteins that were involved in the regulation of pollen-pistil interaction.

• IMMUNE NETWORK
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• 제20권5호
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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.