• Journal of Chest Surgery
• /
• v.44 no.6
• /
• pp.406-412
• /
• 2011

Background: Development of thoracic aortic aneurysms and aortic dissections (TAAD) is attributed to unbearable wall tension superimposed on defective aortic wall integrity and impaired aortic repair mechanisms. Central to this repair mechanisms are well-balanced and adequately functional cellular components of the aortic wall, including endothelial cells, smooth muscle cells (SMCs), inflammatory cells, and adventitial fibroblasts. Adventitial fibroblasts naturally produce aortic extracellular matrix (ECM), and, when aortic wall is injured, they can be transformed into SMCs, which in turn are involved in aortic remodeling. We postulated the hypothesis that adventitial fibroblasts in patients with TAAD may have defects in ECM production and SMC transformation. Materials and Methods: Adventitial fibroblasts were procured from the adventitial layer of fresh aortic tissues of patients with TAAD (Group I) and of multi-organ donors (Group II), and 4-passage cell culture was performed prior to the experiment. To assess ECM production, cells were treated with TNF-${\alpha}$ (50 pM) and the expression of MMP-2/MMP-3 was analyzed using western blot technique. To assess SMC transformation capacity, cells were treated with TGF-${\beta}1$ and expression of SM ${\alpha}$-actin, SM-MHC, Ki-67 and SM calponin was evaluated using western blot technique. Fibroblasts were then treated with TGF-${\beta}1$ (10 pM) for up to 10 days with TGF-${\beta}1$ supplementation every 2 days, and the proportion of transformed SMC in the cell line was measured using immunofluorescence assay for fibroblast surface antigen every 2 days. Results: MMP-3 expression was significantly lower in group I than in group II. TGF-${\beta}1$-stimulated adventitial fibroblasts in group I expressed less SM ${\alpha}$-actin, SM-MHC, and Ki-67 than in group II. SM-calponin expression was not different between the two groups. Presence of fibroblast was observed on immunofluorescence assay after more than 6 days of TGF-${\beta}1$ treatment in group I, while most fibroblasts were transformed to SMC within 4 days in group II. Conclusion: ECM production and SMC transformation are compromised in adventitial fibroblasts from patients with TAAD. This result suggests that functional restoration of adventitial fibroblasts could well be a novel approach for the prevention and treatment of TAAD.

• Molecules and Cells
• /
• v.41 no.5
• /
• pp.413-422
• /
• 2018

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from $T_2$ seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than ${\underline{n}}on-{\underline{t}}ransgenic$ (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants ($T_2$) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

• Molecules and Cells
• /
• v.26 no.5
• /
• pp.462-467
• /
• 2008

TPA is known to cooperate with an activated Ras oncogene in the transformation of rodent fibroblasts, but the biochemical mechanisms responsible for this effect have not been established. In the present study we used c-fos promoter-luciferase constructs as reporters, in transient transfection assays, in NIH3T3 cells to assess the mechanism of this cooperation. We found a marked synergistic interaction between TPA and a transfected v-Ha-ras oncogene in the activation of c-fos promoter and SRE. SRE has binding sites for TCF and SRF. A dominant-negative Ras (ras-N17) inhibited the TPA-Ras synergy by blocking the PKC-MAPK-TCF pathway. Dominant-negative RhoA and Rac1 (but not Cdc42Hs) inhibited the TPA-Ras synergy by blocking the Ras-Rho-SRF signaling pathway. Constitutively active $PKC{\alpha}$ and $PKC{\varepsilon}$ showed synergy with v-Ras. These results suggest that the activation of two distinct pathways such as Ras-Raf-ERK-TCF pathway and Rho-SRF pathway are responsible for the induction of c-fos by TPA and Ras in mitogenic signaling pathways.

• Molecules and Cells
• /
• v.24 no.3
• /
• pp.358-363
• /
• 2007

Swine endothelial cells are commonly used as an in vitro model for studying features of the blood-brain barrier and some hemorrhagic diseases. However, primary cultures of swine cells have finite lifespans. To establish immortalized swine umbilical vein endothelial cells (SUVECs) using human telomerase reverse transcriptase (hTERT), the plasmid pCI-neo-hTERT was transfected into SUVECs by lipofection. Clones were selected for G418 resistance, and positive clones were amplified. One of the clones was cultured for up to 50 passages. Factor VIII-related antigen and CD34 were detected. The immortalized cells shared the properties of normal cells, such as contact inhibition, serum requirement and anchorage dependence. Karyotype analysis revealed that the immortalized cells were in the diploid range. In addition, both in vivo and in vitro assays of tumorigenicity showed no neoplastic transformation. Furthermore, NO, $PGI_2$, and ET-1 concentrations in the transfected cells were normal. These results suggest that the SUVECs immortalized by hTERT retain their original characteristics.

• The Korean Journal of Cytopathology
• /
• v.8 no.2
• /
• pp.115-119
• /
• 1997

Cytologic diagnosis of reactive or malignant effusion is sometimes difficult. Especially, differentiation of benign reactive mesothelial cells from malignant cells in body effusion is more difficult. Recently, immunohistochemistry has been used to diagnose difficult cases. Phospholipase $C(PLC)-{\gamma}1$ is one of the isoenzyme of the PLC which plays central role in signal transduction involving cellular growth, differentiation and transformation by phosphorylating many protein component. Increased expression of $PLC-{\gamma}1$ in human breast carcinoma, colorectal carcinoma and stomach cancers are reported. To evaluate the efficacy of positive $PLC-{\gamma}1$ immunostaining in the diagnosis of malignancy in effusions, paraffin-embedded cell blocks of pleural fluid and ascites from 10 patients(5 metastatic adenocarcinomas, and 5 reactive mesothelial cells) were immunostained with a monoclonal antibody to $PLC-{\gamma}1$. $PLC-{\gamma}1$ immuostained all the adenocarcinomas in cell block(5/5) with intense membrane pattern, however, none of the reactive mesothelial proliferations stained with the diagnostic membrane pattern. Thus, our study strongly supports the conclusion that $PLC-{\gamma}1$ immunopositivity is likely to become a useful adjunct for the diagnosis of malignancy in effusions.

• Microbiology and Biotechnology Letters
• /
• v.25 no.3
• /
• pp.258-265
• /
• 1997

The effects of medium pH and culture temperature on the expression and secretion of inulinase and invertase were investigated with recombinant Saccharomyces cerevisiae cells. These cells were obtained by transformation of 2$\mu$-based plasmids pYI10 and pYS10 which contain Kluyveromyces marxianus inulinase gene (INU1A) and S. cerevisiae invertase gene (SUC2), respectively, in the downstream of GAL1 promoter. The expression level and localization of inulinase and invertase were not affected significantly by the initial medium pH: secretion efficiencies of inulinase and invertase into the medium were about 90% and 60%, respectively, in the pH ranges of 4.0 to 6.5. However, the expression and secretion of both enzymes were strongly dependent on the culture temperature. The highest expression (7.7 units/mL) and secretion (6.7 units/mL) of inulinase were observed at 28$\circ$C and 30$\circ$C. As a consequence of decreased localization of inulinase in the periplasmic space, the secretion efficiency increased from 68% at 20$\circ$C, to 95% at 35$\circ$C,. The total expression level and secretion efficiency of invertase increased from 19 units/mL and 55% at 20$\circ$C to 25 units/mL and 68% at 35$\circ$C, respectively. Irrespective of the culture temperature, the invertase activity in the cellular fraction (periplasmic space and cytoplasmic fractions) was kept constant at around 33-45%.

• Journal of the Korean Magnetic Resonance Society
• /
• v.2 no.2
• /
• pp.85-105
• /
• 1998

Prions cause neurodegenerative diseases in animals and humans. The scrapie prion protein (PrPSc) is the major-possibly only-component of the infectious prion and is generated from the cellular isoform (PrPC) by a conformational change. Limited proteolysis of PrPSc produces an polypeptide comprised primarily of residues 90 to 231, which retains infectivity. The three-dimensional structure of rPrP(90-231), a recombinant protein resembling PrPC with the Syrian hamster (SHa) sequence, was solved using multidimensional NMR. Low-resolution structures of rPrP(90-231), synthetic peptides up to 56 residues, a longer (29-231, full-length) protein with SHa sequence, and a short here further structure refinement of rPrP(90-231) and dynamic features of the protein. Consideration of these features in the context of published data suggests regions of conformational heterogeneity, structural elements involved in the PrPC\longrightarrowPrPSc transformation, and possible structural features related to a species barrier to transmission of prion diseases.

• IMMUNE NETWORK
• /
• v.12 no.6
• /
• pp.240-246
• /
• 2012

Natural killer (NK) cells play a pivotal role in early surveillance against virus infection and cellular transformation, and are also implicated in the control of inflammatory response through their effector functions of direct lysis of target cells and cytokine secretion. NK cell activation toward target cell is determined by the net balance of signals transmitted from diverse activating and inhibitory receptors. A distinct feature of NK cell activation is that stimulation of resting NK cells with single activating receptor on its own cannot mount natural cytotoxicity. Instead, specific pairs of co-activation receptors are required to unleash NK cell activation via synergy- dependent mechanism. Because each co-activation receptor uses distinct signaling modules, NK cell synergy relies on the integration of such disparate signals. This explains why the study of the mechanism underlying NK cell synergy is important and necessary. Recent studies revealed that NK cell synergy depends on the integration of complementary signals converged at a critical checkpoint element but not on simple amplification of the individual signaling to overcome intrinsic activation threshold. This review focuses on the signaling events during NK cells activation and recent advances in the study of NK cell synergy.

• BMB Reports
• /
• v.40 no.6
• /
• pp.966-972
• /
• 2007

Pituitary tumor transforming gene (PTTG1) is widely detected in many tumors. Increasing evidence reveals that PTTG1 is associated with cell proliferation, cellular transformation and apoptosis. However, the functions of PTTG1, especially its role in DNA damage-induced apoptosis, remain largely unclear. In this report, we used UV irradiation to induce apoptosis in HeLa cells to examine the role of PTTG1 in UV-induced apoptosis by RNAi-mediated knockdown and overexpression of PTTG1. RNAi-mediated knockdown of PTTG1 expression increased and overexpression of PTTG1 decreased the UV-induced apoptosis. Furthermore, UV irradiation decreased PTTG1 mRNA and protein expression. These effects were found to be mediated by JNK pathway. Therefore, PTTG1 had an important anti-apoptotic role in UV-induced apoptosis and this role was mediated by JNK pathway. These results may provide important information for understanding the exact role and the regulation mechanism of PTTG1 in UV-induced apoptosis.

• The Plant Pathology Journal
• /
• v.35 no.6
• /
• pp.692-697
• /
• 2019

Agrobacterium rhizogenes-mediated transformation of sugar beet hairy roots expressing single-chain variable fragment (scFv) was exploited to evaluate the efficacy of four antibody-based constructs for interfering with the Beet necrotic yellow vein virus infection. The scFv specific to a major coat protein of virus, p21, was targeted to various cellular compartments including the cytosol (pIC and pICC constructs), apoplast (pIA), and mitochondrion (pIM). After mechanical virus inoculation, most of the hairy root clones expressing scFv in the cytosol displayed low virus titers while the majority of transgenic hairy root clones accumulated antibody in outer membrane of mitochondria or apoplast were infected. This hairy root system provided an efficient and rapid approach to initially investigating root disease resistance like rhizomania prior to transform whole recalcitrant plants such as sugar beet.