• BMB Reports
• /
• v.35 no.2
• /
• pp.178-185
• /
• 2002

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine $Cys^{50}$, $Cys^{83}$, and $Cys^{230}$, and $lys^{81}$ residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the $Cys^{50}$ and $Cys^{230}$ mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the $Cys^{50}$ and $Cys^{230}$ mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the $Cys^{50}$ and $Cys^{230}$ residues are structurally important.

• BMB Reports
• /
• v.41 no.8
• /
• pp.593-596
• /
• 2008

ADPKD (Autosomal Dominant Polycystic Kidney Disease) is characterized by the progressive expansion of multiple cystic lesions in the kidneys. ADPKD is caused by mutations in Ed-pl. consider PKD1 and PKD2. Recently a relation between c-myc and the pathogenesis of ADPKD was reported. In addition, c-Myc is a downstream effector of PKD1. To identify the gene regulated by PKD2 and c-Myc, we performed gene expression profiling in PKD2 and c-Myc overexpressing cells using a human 8K cDNA microarray. NCAM (neuronal cell adhesion molecule) levels were significantly reduced in PKD2 overexpressing systems in vitro and in vivo. These results suggest that NCAM is an important molecule in the cystogenesis induced by PKD2 overexpession.

• BMB Reports
• /
• v.51 no.3
• /
• pp.119-125
• /
• 2018

The Hippo pathway plays prominent and widespread roles in various forms of human carcinogenesis. Specifically, the Yes-associated protein (YAP), a downstream effector of the Hippo pathway, can lead to excessive cell proliferation and the inhibition of apoptosis, resulting in tumorigenesis. It was reported that the YAP is strongly elevated in multiple types of human malignancies such as breast, lung, small intestine, colon, and liver cancers. Recent work indicates that, surprisingly, Hippo signaling components' (SAV1, MST1/2, Lats1/2) mutations are virtually absent in human cancer, rendering this signaling an unlikely candidate to explain the vigorous activation of the YAP in most, if not all human tumors and an activated YAP promotes the resistance to RAF-, MAPK/ERK Kinase (MEK)-, and Epidermal growth factor receptor (EGFR)-targeted inhibitor therapy. The analysis of YAP expressions can facilitate the identification of patients who respond better to an anti-cancer drug treatment comprising RAF-, MEK-, and EGFR-targeted inhibitors. The prominence of YAP for those aspects of cancer biology denotes that these factors are ideal targets for the development of anti-cancer medications. Therefore, our report strongly indicates that the YAP is of potential prognostic utility and druggability in various human cancers.

• BMB Reports
• /
• v.51 no.1
• /
• pp.27-32
• /
• 2018

Non-small-cell lung cancer (NSCLC) is commonly caused by a mutation in the epidermal growth factor receptor (EGFR) and subsequent aberrant EGFR signaling with uncontrolled kinase activity. A deletion mutation in EGFR exon 19 is frequently observed in EGFR gene mutations. We designed a DNAzyme to suppress the expression of mutant EGFR by cleaving the mutant EGFR mRNA. The DNAzyme (named Ex19del Dz) specifically cleaved target RNA and decreased cancer cell viability when transfected into gefitinib-resistant lung cancer cells harboring EGFR exon 19 deletions. The DNAzyme decreased EGFR expression and inhibited its downstream signaling pathway. In addition to EGFR downregulation, Ex19del Dz containing CpG sites activated Toll-like receptor 9 (TLR9) and its downstream signaling pathway via p38 kinase, causing an immunostimulatory effect on EGFR-mutated NSCLC cells. Thus, dual effects of this DNAzyme harboring the CpG site, such as TLR9 activation and EGFR downregulation, leads to apoptosis of EGFR-mutated NSCLC cells.

• BMB Reports
• /
• v.52 no.12
• /
• pp.718-727
• /
• 2019

Severe combined immunodeficiency (SCID) is a group of inherited disorders characterized by compromised T lymphocyte differentiation related to abnormal development of other lymphocytes [i.e., B and/or natural killer (NK) cells], leading to death early in life unless treated immediately with hematopoietic stem cell transplant. Functional NK cells may impact engraftment success of life-saving procedures such as bone marrow transplantation in human SCID patients. Therefore, in animal models, a T cell-/B cell-/NK cell+ environment provides a valuable tool for understanding the function of the innate immune system and for developing targeted NK therapies against human immune diseases. In this review, we focus on underlying mechanisms of human SCID, recent progress in the development of SCID animal models, and utilization of SCID pig model in biomedical sciences. Numerous physiologies in pig are comparable to those in human such as immune system, X-linked heritability, typical T-B+NK- cellular phenotype, and anatomy. Due to analogous features of pig to those of human, studies have found that immunodeficient pig is the most appropriate model for human SCID.

• Animal cells and systems
• /
• v.10 no.3
• /
• pp.115-120
• /
• 2006

Parkinson's disease (PD) is a neurodegenerative disease caused by selective degeneration of dopaminergic neurons in the substantia nigra. Mutations in ${\alpha}$-synuclein have been causally linked to the pathogenesis of hereditary PD. In addition, it is a major component of Lewy body found in the brains of sporadic cases as well. In the present study, we examined whether overexpression of wild type or PD-related mutant ${\alpha}$-synuclein induces unfolded protein response (UPR) and triggers the known signaling pathway of the resulting endoplasmic reticulum (ER) stress in SH-SY5Y cells. Overexpression of wild type, A30P, and A53T ${\alpha}$-synuclein all induced XBP-1 mRNA splicing, one of the late stage UPR events. However, activation of ER membrane kinases and upregulation of ER or cytoplsmic chaperones were not detected when ${\alpha}$-synuclein was overexpressed. However, basal level of cytoplsmic calcium was elevated in ${\alpha}$-synuclein-expressing cells. Our observation suggests that overexpression of ${\alpha}$-synuclein induces UPR independent of the known ER membrane kinase-mediated signaling pathway and induces ER stress by disturbing calcium homeostasis.

• Molecules and Cells
• /
• v.37 no.5
• /
• pp.357-364
• /
• 2014

Medulloblastoma, the most common malignant brain tumor in children, is a disease whose mechanisms are now beginning to be uncovered by high-throughput studies of somatic mutations, mRNA expression patterns, and epigenetic profiles of patient tumors. One emerging theme from studies that sequenced the tumor genomes of large cohorts of medulloblastoma patients is frequent mutation of RNA binding proteins. Proteins which bind multiple RNA targets can act as master regulators of gene expression at the post-transcriptional level to co-ordinate cellular processes and alter the phenotype of the cell. Identification of the target genes of RNA binding proteins may highlight essential pathways of medulloblastomagenesis that cannot be detected by study of transcriptomics alone. Furthermore, a subset of RNA binding proteins are attractive drug targets. For example, compounds that are under development as anti-viral targets due to their ability to inhibit RNA helicases could also be tested in novel approaches to medulloblastoma therapy by targeting key RNA binding proteins. In this review, we discuss a number of RNA binding proteins, including Musashi1 (MSI1), DEAD (Asp-Glu-Ala-Asp) box helicase 3 X-linked (DDX3X), DDX31, and cell division cycle and apoptosis regulator 1 (CCAR1), which play potentially critical roles in the growth and/or maintenance of medulloblastoma.

• Molecules and Cells
• /
• v.37 no.4
• /
• pp.302-306
• /
• 2014

Myostatin represses muscle growth by negatively regulating the number and size of muscle fibers. Myostatin loss-of-function can result in the double-muscling phenotype and increased muscle mass. Thus, knockout of myostatin gene could improve the quality of meat from mammals. In the present study, zinc finger nucleases, a useful tool for generating gene knockout animals, were designed to target exon 1 of the myostatin gene. The designed ZFNs were introduced into porcine primary fibroblasts and early implantation embryos via electroporation and microinjection, respectively. Mutations around the ZFNs target site were detected in both primary fibroblasts and blastocysts. The proportion of mutant fibroblast cells and blastocyst was 4.81% and 5.31%, respectively. Thus, ZFNs can be used to knockout myostatin in porcine primary fibroblasts and early implantation embryos.

• Molecules and Cells
• /
• v.38 no.1
• /
• pp.33-39
• /
• 2015

The correction of disease-causing mutations by single-strand oligonucleotide-templated DNA repair (ssOR) is an attractive approach to gene therapy, but major improvements in ssOR efficiency and consistency are needed. The mechanism of ssOR is poorly understood but may involve annealing of oligonucleotides to transiently exposed single-stranded regions in the target duplex. In bacteria and yeast it has been shown that ssOR is promoted by expression of $Red{\beta}$, a single-strand DNA annealing protein from bacteriophage lambda. Here we show that $Red{\beta}$ expression is well tolerated in a human cell line where it consistently promotes ssOR. By use of short interfering RNA, we also show that ssOR is stimulated by the transient depletion of the endogenous DNA mismatch repair protein MSH2. Furthermore, we find that the effects of $Red{\beta}$ expression and MSH2 depletion on ssOR can be combined with a degree of cooperativity. These results suggest that oligonucleotide annealing and mismatch recognition are distinct but interdependent events in ssOR that can be usefully modulated in gene correction strategies.

• Journal of Radiation Industry
• /
• v.9 no.4
• /
• pp.181-185
• /
• 2015

Today the biosafety evaluation, a common problem of vital importance, is based on internationally proved test-systems, standards and techniques. The paradigm of biosafety includes multidisciplinary approach, a combination of physical, chemical and biological tests to monitor the environmental level of pollutants and needs to be improved by modern approaches. The genetic risk of environmental pollutions has long been studied by many researchers. In this study, used was the known sensitive plant test-system, clones of plant Tradescantia (spiderwort) able to detect gene mutations (frequency of mutational events and formation of micronuclei) in combination with chemical and, in some instances, with radiological measurements. In addition, male gametophyte generation of fruit trees was applied as bioindicators of genotoxicity. The obtained results did not show any significant increase along with wind direction. As for the male gametophyte assay, the fertility of the investigated fruit-trees near to NPP did not significantly differ from that of the control point. The influence of the NPP on the male generative system of the investigated taxa of fruit trees for the investigated year was not revealed. The system described needs to be expanded by species of interest (human) as there is a difficulty to transfer the revealed dose correlations to humans. The development of this idea includes various levels: population (epidemiological studies), individual, cellular, molecular (DNA), etc.