• Experimental and Molecular Medicine
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• v.50 no.12
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• pp.6.1-6.10
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• 2018

Bmal1 is one of the key molecules that controls the mammalian molecular clock. In humans, two isoforms of Bmal1 are generated by alternative RNA splicing. Unlike the extensively studied hBmal1b, the canonical form of Bmal1 in most species, the expression and/or function of another human-specific isoform, hBmal1a, are poorly understood. Due to the lack of the N-terminal nuclear localization signal (NLS), hBMAL1a does not enter the nucleus as hBMAL1b does. However, despite the lack of the NLS, hBMAL1a still dimerizes with either hCLOCK or hBMAL1b and thereby promotes cytoplasmic retention or protein degradation, respectively. Consequently, hBMAL1a interferes with hCLOCK:hBMAL1b-induced transcriptional activation and the circadian oscillation of Period2. Moreover, when the expression of endogenous hBmal1a is aborted by CRISPR/Cas9-mediated knockout, the rhythmic expression of hPer2 and hBmal1b is restored in cultured HeLa cells. Together, these results suggest a role for hBMAL1a as a negative regulator of the mammalian molecular clock.

• Development and Reproduction
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• v.23 no.3
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• pp.285-295
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• 2019

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonic development. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

• Mycobiology
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• v.47 no.4
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• pp.473-482
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• 2019

Rice blast disease, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most important diseases in rice production. PAS (period circadian protein, aryl hydrocarbon receptor nuclear translocator protein, single-minded protein) domains are known to be involved in signal transduction pathways, but their functional roles have not been well studied in fungi. In this study, targeted gene deletion was carried out to investigate the functional roles of the PAS-containing gene MoPAS1 (MGG_02665) in M. oryzae. The deletion mutant ΔMopas1 exhibited easily wettable mycelia, reduced conidiation, and defects in appressorium formation and disease development compared to the wild type and complemented transformant. Exogenous cAMP restored appressorium formation in ΔMopas1, but the shape of the restored appressorium was irregular, indicating that MoPAS1 is involved in sensing the hydrophobic surface. To examine the expression and localization of MoPAS1 in M. oryzae during appressorium development and plant infection, we constructed a MoPAS1:GFP fusion construct. MoPAS1:GFP was observed in conidia and germ tubes at 0 and 2 h post-infection (hpi) on hydrophobic cover slips. By 8 hpi, most of the GFP signal was observed in the appressoria. During invasive growth in host cells, MoPAS1:GFP was found to be fully expressed in not only the appressoria but also invasive hyphae, suggesting that MoPAS may contribute to disease development in host cells. These results expand our knowledge of the roles of PAS-containing regulatory genes in the plant-pathogenic fungus M. oryzae.

• Bulletin of the Korean Chemical Society
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• v.34 no.12
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• pp.3665-3670
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• 2013

The R3V6 peptide includes a hydrophilic arginine stretch and a hydrophobic valine stretch. In previous studies, the R3V6 peptide was evaluated as a gene carrier and was found to have low cytotoxicity. However, the transfection efficiency of R3V6 was lower than that of poly-L-lysine (PLL) in N2A neuroblastoma cells. In this study, the transfection efficiency of R3V6 was improved in combination with high mobility group box 1A domain (HMGA). HMGA is originated from the nuclear protein and has many positively-charged amino acids. Therefore, HMGA binds to DNA via charge interaction. In addition, HMGA has a nuclear localization signal peptide and may increase the delivery efficiency of DNA into the nucleus. The ternary complex with HMGA, R3V6, and DNA was prepared and evaluated as a gene carrier. First, the HMGA/DNA complex was prepared with a negative surface charge. Then, R3V6 was added to the complex to coat the negative charges of the HMGA/DNA complex, forming the ternary complex of HMGA, R3V6, and DNA. A physical characterization study showed that the ternary complex was more stable than the PLL/DNA complex. The HMGA/R3V6/DNA complex had a higher transfection efficiency than the PLL/DNA, HMGA/DNA, or R3V6/DNA complexes in N2A cells. Furthermore, the HMGA/R3V6/DNA complex was not toxic to cells. Therefore, the HMGA/R3V6/DNA complex may be a useful gene delivery carrier.

• Investigative Magnetic Resonance Imaging
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• v.8 no.2
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• pp.94-99
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• 2004

Purpose : To investigate the signal enhancement ratio by NOE effect on in vivo $^{31}P$ MRS in human heart muscle and liver. we also evaluated the enhancement ratios of different phosphorus metabolites, which are important in 31P MRS for each organ. Materials and Methods : Ten normal subjects (M:F = 8:2, age range = 24-32 yrs) were included for in vivo $^{31}P$ MRS measurements on a 1.5 T whole-body MRI/MRS system using $^1H-^{31}P$ dual tuned surface coil. Two-dimensional Chemical Shift Imaging (2D CSI) pulse sequence for $^{31}P$ MRS was employed in all $^{31}P$ MRS measurements. First, $^{31}P$ MRS performed without NOE effect and then the same 2D CSI data acquisitions were repeated with NOE effect. After postprocessing the MRS raw data in the time domain, the signal enhancements in percent were estimated from the major metabolites. Results : The calculated NOE enhancement for liver $^{31}P$ MRS were $\alpha-ATP\;(7\%),\;\beta-ATP\;(9\%),\;\gamma-ATP\;(17\%),\;Pi\;(1\%),\;PDE\;(19\%)$ and $PME\;(31\%)$. Because there is no creatine kinase activity in liver, PCr signal is absent. For cardiac $^{31}P$ MRS, whole body coil gave better scout images and thus better localization than surface coil. In $^{31}P$cardiac multi-voxel spectra, DPG signal increased from left to right according to the amount of blood included. The calculated enhancement for cardiac $^{31}P$ MRS were : $\alpha-ATP\;(12\%),\;\beta-ATP\;(19\%),\;\gamma-ATP\;(30\%),\;PCr\;(34\%),\;Pi\;(20\%),\;(PDE)\;(51\%),\;and\;DPG\;(72\%)$. Conclusion : Our results revealed that the NOE effect was more pronounced in heart muscle than in liver with different coupling to 1H spin system and thus different heteronuclear cross-relaxation.

• The Korean Journal of Nuclear Medicine
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• v.33 no.3
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• pp.289-297
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• 1999

Purpose: Misonidazole is a radiosensitizer that binds in hypoxic cells. The purpose of this study was to find out the feasibility of I-131-Iodomisonidazole (IMISO) for imaging of tumor hypoxia. Materials and Methods: Tosyl precursor was dissolved in acetonitrile and I-131-NaI was added to synthesize IMISO. Balb/c mice inoculated with CT-26 adenocarcinoma were injected with IMISO. Mice were sacrificed at 1, 2, 4, 24 hr and % of injected dose per gram of tissue (%ID/g) was determined. For scintigraphy and MRI, mouse bearing CT-26 adenocarcinoma was administered with IMISO and imaging was performed 4 hr after. Then, mouse body was fixed and microtomized slice was placed on radiographic film for autoradiography Results: %ID/g of tumor was 1.64 (1h), 0.98 (2h), 0.85 (4h) and 0.20 (24h), respectively. At 24h, %ID/g of tumor was higher than that of all other tissues except thyroid. Tumor to muscle ratio increased with time and tumor to blood ratio also increased with time and reached 1.53 at 24 hr. On autoradiogram, tumor was well visualized as an increased activity in central hypoxic area of the tumor which corresponds to the area of high signal intensity on T2-weighted MR image. On scintigraphy, tumor uptake was visualized. Conclusion: This results suggest that IMISO may have a potential for tumor hypoxia imaging in mouse model. However, further study is needed to improve it's localization in tumor tissue and to achieve acceptable images of tumor hypoxia.

• Journal of Microbiology and Biotechnology
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• v.30 no.1
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• pp.109-117
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• 2020

Cre recombinase is widely used to manipulate DNA sequences for both in vitro and in vivo research. Attachment of a trans-activator of transcription (TAT) sequence to Cre allows TAT-Cre to penetrate the cell membrane, and the addition of a nuclear localization signal (NLS) helps the enzyme to translocate into the nucleus. Since the yield of recombinant TAT-Cre is limited by formation of inclusion bodies, we hypothesized that the positively charged arginine-rich TAT sequence causes the inclusion body formation, whereas its neutralization by the addition of a negatively charged sequence improves solubility of the protein. To prove this, we neutralized the positively charged TAT sequence by proximally attaching a negatively charged poly-glutamate (E12) sequence. We found that the E12 tag improved the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) compared with those of TAT-NLS-Cre (TAT-Cre) when expressed in E. coli. Furthermore, the growth of cells expressing E12-TAT-Cre was increased compared with that of the cells expressing TAT-Cre. Efficacy of the purified TAT-Cre was confirmed by a recombination test on a floxed plasmid in a cell-free system and 293 FT cells. Taken together, our results suggest that attachment of the E12 sequence to TAT-Cre improves its solubility during expression in E. coli (possibly by neutralizing the ionic-charge effects of the TAT sequence) and consequently increases the yield. This method can be applied to the production of transducible proteins for research and therapeutic purposes.

• Journal of Veterinary Clinics
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• v.21 no.3
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• pp.253-258
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• 2004

DNA double-strand break (DSB) is a serious treat for the cells including mutations, chromosome rearrangements, and even cell death if not repaired or misrepaired. Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) bound to double strand DNA breaks are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and the interaction is essential for DNA-dependent protein kinase (DNA-PK) activity. The Ku80 mutants were designed to bind Ku70 but not DNA end binding activity and the peptides were treated in breast cancer cells for co-therapy strategy to see whether the targeted inhibition of DNA-dependent protein kinase (DNA-PK) activity sensitized breast cancer cells to ionizing irradiation or chemotherapy drug to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. We designed domains of Ku80 mutants, 26 residues of amino acids (HN-26) as a control peptide or 38 (HNI-38) residues of amino acids which contain domains of the membrane-translocation hydrophobic signal sequence and the nuclear localization sequence, but HNI-38 has additional twelve residues of peptide inhibitor region. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, resulting in inactivation of DNA-PK complex activity in breast cancer cells (MDA-465 and MDA-468). Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to irradiation or chemotherapy drugs. The growth of breast cancer cells was also inhibited. These results demonstrate the possibility of synthetic peptide to apply breast cancer therapy to induce apoptosis of cancer cells.

• Journal of Crop Science and Biotechnology
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• v.11 no.4
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• pp.237-242
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• 2008

A liguleless mutant, which showed complete loss of lamina joint region at the junction between leaf blade and leaf sheath, was isolated from a Ds insertional mutants derived from the source cultivar, Dongjin. This mutant could not affect other developmental patterns like phyllotaxis. Southern blot analysis, using GUS as a probe, revealed that the liguleless mutant contained three Ds copies transposed in the rice genome. Among the four genomic sequences flanking the Ds, one was mapped in the intergenic region (31661640 - 31661759), and the other two predicted a protein kinase domain (12098980 - 12098667) as an original insertion site within a starter line used for massive production of Ds insertional mutant lines. Another predicted and inserted in first exon of liguleless 1 protein (OsLG1) that was mapped in coding region (LOC_Os04g56170) of chromosome 4. RT-PCR revealed that the OsLG1 gene was not expressed liguleless mutants. Structure analysis of OsLG1 protein revealed that it predicted transcription factor with a highly conserved SBP domain consisting of 79 amino acids that overlapped a nuclear localization signal (NLS). RT-PCR revealed that OsLG1 is mainly expressed in vegetative organs.

• Journal of Plant Biotechnology
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• v.46 no.2
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• pp.97-105
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• 2019

Dehydration Responsive Element Binding (DREB) gene is one of the essential transcription factors plants use for responding to stress conditions including salinity, drought, and cold stress. The purpose of this study was to isolate the full length and characterize the DREB gene from three different genotypes of sugarcane, wild, commercial cultivar, and interspecific hybrid sugarcane. The length of the gene, designated ScDREB was 789 bp, and coding for a putative polypeptide of 262 amino acid residues. Sequences of the gene were submitted to the GenBank database with accession numbers of KX280722.1, KX280721.1, and KX280719.1 for wild sugarcane, commercial cultivar (KPS94-13), and interspecific hybrid (Biotec2), respectively. In silico characterization indicated that the deduced polypeptide contains a putative nuclear localization signal (NLS) sequence, and a conserved AP2/ERF domain of the DREB family, at 82-140 amino residues. Based on multiple sequence alignment, sequences of the gene from the three sugarcane genotypes were classified in the DREB2 group. Gene expression analysis indicated, that ScDREB2 expression pattern in tested sugarcane was up-regulated by salt stress. When the plants were under 100 mM NaCl stress, relative expressions of the gene in leaves was higher than those in roots. In contrast, under 200 mM NaCl stress, relative expressions of the gene in roots was higher than those in leaves. This is the first report on cloning the full length and characterization, of ScDREB2 gene of sugarcane. Results indicate that ScDREB2 is highly responsive to salt stress.