• Plant Biotechnology Reports
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• v.2 no.1
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• pp.75-85
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• 2008

Prune dwarf virus (PDV) is an Ilarvirus systemically infecting almond trees and other Prunus species and spreading through pollen, among other means. We have studied strategies based on coat protein (cp) gene to block PDV replication in host plant cells. A Portuguese isolate of PDV was obtained from infected almond leaves and used to produce the cDNA of the cp gene. Various constructs were prepared based on this sequence, aiming for the transgenic expression of the original or modified PDV coat protein (cpPDVSense and cpPDVMutated) or for the expression of cpPDV RNA (cpPDVAntisense and cpPDVwithout start codon). All constructs were tested in a PDV host model, Nicotiana benthamiana, and extensive molecular characterization and controlled infections were performed on transformants and their progenies. Transgenic plants expressing the coat protein RNA were able to block the proliferation of a PDV isolate sharing only 91% homology with the isolate used for cpPDV cloning, as evaluated by DAS-ELISA on newly developed leaves. With cp expression, the blockage of PDV proliferation in newly developed leaves was only achieved with the construct cpPDV Mutated, where the coat protein has a substitution in the 14th aa residue, with arginine replaced by alanine. This result points to a possible role of the mutated amino acid in the virus ability to replicate and proliferate. This work reveals the possibility of achieving protection against PDV through either coat protein RNA or mutated cp sequence.

• Development and Reproduction
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• v.24 no.4
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• pp.249-261
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• 2020

Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.1
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• pp.37-44
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• 2017

Regulation of vascular smooth muscle cell (VSMC) phenotype plays an essential role in many cardiovascular diseases. In the present study, we provide evidence that $kr{\ddot{u}}ppel$-like factor 8 (KLF8) is essential for tumor necrosis factor ${\alpha}$ ($TNF{\alpha}$)-induced phenotypic conversion of VSMC obtained from thoracic aorta from 4-week-old SD rats. Stimulation of the contractile phenotype of VSMCs with $TNF{\alpha}$ significantly reduced the VSMC marker gene expression and KLF8. The gene expression of KLF8 was blocked by $TNF{\alpha}$ stimulation in an ERK-dependent manner. The promoter region of KLF8 contained putative Sp1, KLF4, and $NF{\kappa}B$ binding sites. Myocardin significantly enhanced the promoter activity of KLF4 and KLF8. The ectopic expression of KLF4 strongly enhanced the promoter activity of KLF8. Moreover, silencing of Akt1 significantly attenuated the promoter activity of KLF8; conversely, the overexpression of Akt1 significantly enhanced the promoter activity of KLF8. The promoter activity of SMA, $SM22{\alpha}$, and KLF8 was significantly elevated in the contractile phenotype of VSMCs. The ectopic expression of KLF8 markedly enhanced the expression of SMA and $SM22{\alpha}$ concomitant with morphological changes. The overexpression of KLF8 stimulated the promoter activity of SMA. Stimulation of VSMCs with $TNF{\alpha}$ enhanced the expression of KLF5, and the promoter activity of KLF5 was markedly suppressed by KLF8 ectopic expression. Finally, the overexpression of KLF5 suppressed the promoter activity of SMA and $SM22{\alpha}$, thereby reduced the contractility in response to the stimulation of angiotensin II. These results suggest that cross-regulation of KLF family of transcription factors plays an essential role in the VSMC phenotype.

• Korean Journal of Poultry Science
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• v.39 no.3
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• pp.241-244
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• 2012

This study was performed to analyze the generational transmission and the expression of the foreign gene in the GFP transgenic chickens. The transmission rate and the expression of the GFP gene was investigated from the GFP transgenic rooster (G2) as the first founder to the ninth (G8). Analysis of GFP expression in hatched chickens was used the UV lamp. When GFP was expressed in the wings, bill and legs of a chick, the bird only was selected as a transgenic chick. The average transmission rate of the overall transgenic was 38~58%. These results showed that the transmission of the GFP gene in the transgenic chickens in accordance with the laws of Mendel's continues to the next generation without gene silencing.

• BMB Reports
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• v.53 no.6
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• pp.323-328
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• 2020

Matrix metalloproteinase 1 (MMP-1), a calcium-dependent zinccontaining collagenase, is involved in the initial degradation of native fibrillar collagen. Tissue necrosis factor-alpha (TNFα) is a pro-inflammatory cytokine that is rapidly produced by dermal fibroblasts, monocytes/macrophages, and keratinocytes and regulates inflammation and damaged-tissue remodeling. MMP-1 is induced by TNFα and plays a critical role in tissue remodeling and skin aging processes. However, the regulation of the MMP1 gene by TNFα is not fully understood. We aimed to find additional cis-acting elements involved in the regulation of TNFα-induced MMP1 gene transcription in addition to the nuclear factor-kappa B (NF-κB) and activator protein 1 (AP1) sites. Assessments of the 5'-regulatory region of the MMP1 gene, using a series of deletion constructs, revealed the requirement of the early growth response protein 1 (EGR-1)-binding sequence (EBS) in the proximal region for proper transcription by TNFα. Ectopic expression of EGR-1, a zinc-finger transcription factor that binds to G-C rich sequences, stimulated MMP1 promoter activity. The silencing of EGR-1 by RNA interference reduced TNFα-induced MMP-1 expression. EGR-1 directly binds to the proximal region and transactivates the MMP1 gene promoter. Mutation of the EBS within the MMP1 promoter abolished EGR-1-mediated MMP-1 promoter activation. These data suggest that EGR-1 is required for TNFα-induced MMP1 transcriptional activation. In addition, we found that all three MAPKs, ERK1/2, JNK, and p38 kinase, mediate TNFα-induced MMP-1 expression via EGR-1 upregulation. These results suggest that EGR-1 may represent a good target for the development of pharmaceutical agents to reduce inflammation-induced MMP-1 expression.

• BMB Reports
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• v.49 no.6
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• pp.343-348
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• 2016

GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts.

• Molecules and Cells
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• v.39 no.5
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• pp.375-381
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• 2016

MicroRNAs (miRNAs) are small non-coding RNAs (~22 nucleotides) regulating gene expression at the post-transcriptional level. By directing the RNA-induced silencing complex (RISC) to bind specific target mRNAs, miRNA can repress target genes and affect various biological phenotypes. Functional miRNA target recognition is known to majorly attribute specificity to consecutive pairing with seed region (position 2-8) of miRNA. Recent advances in a transcriptome-wide method of mapping miRNA binding sites (Ago HITS-CLIP) elucidated that a large portion of miRNA-target interactions in vivo are mediated not only through the canonical "seed sites" but also via non-canonical sites (~15-80%), setting the stage to expand and determine their properties. Here we focus on recent findings from transcriptome-wide non-canonical miRNA-target interactions, specifically regarding "nucleation bulges" and "seed-like motifs". We also discuss insights from Ago HITS-CLIP data alongside structural and biochemical studies, which highlight putative mechanisms of miRNA target recognition, and the biological significance of these non-canonical sites mediating marginal repression.

• BMB Reports
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• v.49 no.3
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• pp.137-138
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• 2016

Satiety cues a feeding animal to cease further ingestion of food, thus protecting it from excessive energy gain. Impaired control of satiety is often associated with feeding-related disorders such as obesity. In our recent study, we reported the identification of a neural pathway that expresses the myoinhibitory peptide (MIP), critical for satiety responses in Drosophila. Targeted silencing of MIP neuron activity strikingly increased the body weight (BW) through elevated food intake. Similarly, genetic disruption of the gene encoding MIP also elevated feeding and BW. Suppressing the MIP pathway behaviorally transformed the satiated flies to feed similar to the starved ones, with augmented sensitivity to food. Conversely, temporal activation of MIP neuron markedly reduced the food intake and BW, and blunted the sensitivity of the starved flies to food as if they have been satiated. Shortly after termination of MIP neuron activation, the reduced BW reverted to the normal level along with a strong feeding rebound. Together our results reveal the switch-like role of the MIP pathway in feeding regulation by controlling satiety.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.15
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• pp.6753-6759
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• 2015

Background: Loss of function of the p53 gene is implicated in defective apoptotic responses of tumors to chemotherapy. Although the pro-apoptotic roles of eugenol and capsaicin have been amply reported, their dependence on p53 for apoptosis induction in gastric cancer cells is not well elucidated. The aim of the study was to elucidate the role of p53 in the induction of apoptosis by eugenol and capsaicin in a human gastric cancer cell line, AGS. Materials and Methods: AGS cells were incubated with or without various concentrations of capsaicin and eugenol for 12 hrs, in the presence and absence of p53 siRNA. Cell cycling, annexin V and expression of apoptosis related proteins Bax, Bcl-2 ratio, p21, cyt c-caspase-9 association, caspase-3 and caspase-8 were studied. Results: In the presence of p53, capsaicin was a more potent pro-apoptotic agent than eugenol. However, silencing of p53 significantly abrogated apoptosis induced by capsaicin but not that by eugenol. Western blot analysis of pro-apoptotic markers revealed that as opposed to capsaicin, eugenol could induce caspase-8 and caspase-3 even in the absence of p53. Conclusions: Unlike capsaicin, eugenol could induce apoptosis both in presence and absence of functional p53. Agents which can induce apoptosis irrespective of the cellular p53 status have immense scope for development as potential anticancer agents.

• The Plant Pathology Journal
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• v.29 no.4
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• pp.454-459
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• 2013

The Potexvirus Alternanthera mosaic virus (AltMV) has multifunctional triple gene block (TGB) proteins, among which our studies have focused on the properties of the TGB1 protein. The TGB1 of AltMV has functions including RNA binding, RNA silencing suppression, and cell-to-cell movement, and is known to form homologous interactions. The helicase domains of AltMV TGB1 were separately mutated to identify which regions are involved in homologous TGB1 interactions. The yeast two hybrid system and Bimolecular Fluorescence Complementation (BiFC) in planta were utilized to examine homologous interactions of the mutants. Helicase motif I of AltMV TGB1 was found to be critical to maintain homologous interactions. Mutations in the remaining helicase motifs did not inhibit TGB1 homologous interactions. In the absence of homologous interaction of TGB1, subcellular localization of helicase domain I mutants showed distinctively different patterns from that of WT TGB1. These results provide important information to study viral movement and replication of AltMV.