• Molecules and Cells
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• v.37 no.4
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• pp.322-329
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• 2014

N-acetylglucosamine kinase (GlcNAc kinase or NAGK; EC 2.7.1.59) is highly expressed and plays a critical role in the development of dendrites in brain neurons. In this study, the authors conducted structure-function analysis to verify the previously proposed 3D model structure of GlcNAc/ATP-bound NAGK. Three point NAGK mutants with different substrate binding capacities and reaction velocities were produced. Wild-type (WT) NAGK showed strong substrate preference for GlcNAc. Conversion of Cys143, which does not make direct hydrogen bonds with GlcNAc, to Ser (i.e., C143S) had the least affect on the enzymatic activity of NAGK. Conversion of Asn36, which plays a role in domain closure by making a hydrogen bond with GlcNAc, to Ala (i.e., N36A) mildly reduced NAGK enzyme activity. Conversion of Asp107, which makes hydrogen bonds with GlcNAc and would act as a proton acceptor during nucleophilic attack on the ${\gamma}$-phosphate of ATP, to Ala (i.e., D107A), caused a total loss in enzyme activity. The overexpression of EGFP-tagged WT or any of the mutant NAGKs in rat hippocampal neurons (DIV 5-9) increased dendritic architectural complexity. Finally, the overexpression of the small, but not of the large, domain of NAGK resulted in dendrite degeneration. Our data show the effect of structure on the functional aspects of NAGK, and in particular, that the small domain of NAGK, and not its NAGK kinase activity, plays a critical role in the upregulation of dendritogenesis.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1371-1379
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• 2015

The Ca_v1.2 Ca²⁺ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca_v1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca_v1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca_v1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca_v1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca_v1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca_v1.2/WT. Tunicamycin treatment of oocytes expressing Ca_v1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Ca_v1.2.

• Journal of Microbiology
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• v.43 no.spc1
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• pp.110-117
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• 2005

Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. A critical virulence determinant of Salmonella is the ability to invade mammalian cells. The expression of genes required for invasion is tightly regulated by environmental conditions and a variety of regulatory genes. The hilA regulator encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. Work from several laboratories has highlighted that regulation of hilA expression is a key point for controlling expression of the invasive phenotype. A number of positive regulators of hilA expression have been identified including csrAB, sirA/barA, pstS, hilC/sirC/sprA, fis, and hilD. HilD, an AraC/XylS type transcriptional regulator, is of particular importance as a mutation in hilD results in a 14-fold decrease in chromosomal hilA::Tn5lacZY-080 expression and a 53-fold decrease in invasion of HEp-2 cells. It is believed that HilD directly regulates hilA expression as it has been shown to bind to hilA promoter sequences. In addition, our research group, and others, have identified genes (hilE, hha, pag, and lon) that negatively affect hilA transcription. HilE appears to be an important Salmonella-specific regulator that plays a critical role in inactivating hilA expression. Recent work in our lab has been directed at understanding how environmental signals that affect hilA expression may be processed through a hilE pathway to modulate expression of hilA and the invasive phenotype. The current understanding of this complex regulatory system is reviewed.

• Biomolecules & Therapeutics
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• v.23 no.4
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• pp.339-344
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• 2015

Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. However, more detailed mechanism by which NAR exerts anti-cancer properties still remains unanswered. Thus, in this study, we have shown that NAR down-regulates the level of cyclin D1 in human colorectal cancer cell lines, HCT116 and SW480. NAR inhibited the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. Inhibition of proteasomal degradation by MG132 blocked NAR-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with NAR. In addition, NAR increased the phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine blocked cyclin D1 downregulation by NAR. p38 inactivation attenuated cyclin D1 downregulation by NAR. From these results, we suggest that NAR-mediated cyclin D1 downregulation may result from proteasomal degradation through p38 activation. The current study provides new mechanistic link between NAR, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

• Molecules and Cells
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• v.38 no.8
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• pp.723-728
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• 2015

Smurf2, a member of the HECT domain E3 ligase family, is well known for its role as a negative regulator of TGF-${\beta}$ signaling by targeting Smads and TGF-${\beta}$ receptor. However, the regulatory mechanism of Smurf2 has not been elucidated. Arginine methylation is a type of post-translational modification that produces monomethylated or dimethylated arginine residues. In this report, we demonstrated methylation of Smurf2 by PRMT1. In vitro methylation assay showed that Smurf2, not Smurf1, was methylated by PRMT1. Among the type I PRMT family, only PRMT1 showed activity for Smurf2. Transiently expressed Smurf2 was methylated by PRMT1, indicating Smurf2 is a novel substrate of PRMT1. Using deletion constructs, methylation sites were shown to be located within amino acid region 224-298 of Smurf2. In vitro methylation assay following point mutation of putative methylation sites confirmed the presence of Arg232, Arg234, Arg237, and Arg239. Knockdown of PRMT1 resulted in increased Smurf2 expression as well as inhibition of TGF-${\beta}$-mediated reporter activity. Although it is unclear whether or not increased Smurf2 expression can be directly attributed to lack of methylation of arginine residues, our results suggest that methylation by PRMT1 may regulate Smurf2 stability and control TGF-${\beta}$ signaling.

• Molecules and Cells
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• v.43 no.9
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• pp.784-792
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• 2020

Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2014.05a
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• pp.513-515
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• 2014

Ultra High Definition(hereafter UHD), Next-generation video technology, was quickening in the request of many years of the recipient and the authors who have been towards the higher sense of reality. The change of the medium has resulted in a mutation in the detailed content and the point of view overlooking the world as evidenced through the history. Thus the emergence of UHD will also be expected to require to manufacture unique technology. Therefore, this paper, especially in the documentary among the video content, tries to study the changes in the manufacturing process by UHD video technology. Moreover UHD video screen technology such as high resolution tries to analyse for the fitness for purpose of the documentary that transmission of the accuracy and the change from a documentary video production. In addition, I try to explore the improvement in the choice of shooting material expected to be a problem, even for the advance of the work on the second half and backup according to the enormous data. We derive the developmental aspects that UHD video technology in documentary production is brought about by the research plan described above. It is intended to contribute to the development of content production with the development of technology.

• Clinical and Experimental Pediatrics
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• v.62 no.5
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• pp.193-197
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• 2019

Alport syndrome (ATS) is an inherited glomerular disease caused by mutations in one of the type IV collagen novel chains (${\alpha}3$, ${\alpha}4$, and ${\alpha}5$). ATS is characterized by persistent microscopic hematuria that starts during infancy, eventually leading to either progressive nephritis or end-stage renal disease. There are 3 known genetic forms of ATS, namely X-linked ATS, autosomal recessive ATS, and autosomal dominant ATS. About 80% of patients with ATS have X-linked ATS, which is caused by mutations in the type IV collagen ${\alpha}5$ chain gene, COL4A5. Although an 80% mutation detection rate is observed in men with X-linked ATS, some difficulties do exist in the genetic diagnosis of ATS. Most mutations are point mutations without hotspots in the COL4A3, COL4A4, and COL4A5 genes. Further, there are insufficient data on the detection of COL4A3 and COL4A4 mutations for their comparison between patients with autosomal recessive or dominant ATS. Therefore, diagnosis of ATS in female patients with no apparent family history can be challenging. Therefore, in this study, we used whole-exome sequencing (WES) to identify mutations in type IV collagen in 2 girls with glomerular basement membrane structural changes suspected to be associated with ATS; these patients had no relevant family history. Our results revealed de novo c.4688G>A (p.Arg1563Gln) and c.2714G>A (p.Gly905Asp) mutations in COL4A5. Therefore, we suggest that WES is an effective approach to obtain genetic information in ATS, particularly in female patients without a relevant family history, to detect unexpected DNA variations.

• Infection and chemotherapy
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• v.50 no.4
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• pp.357-361
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• 2018

While carbapenems are the drug of choice to treat extended-spectrum-${\beta}$-lactamase (ESBL)-producing strains, some alternative carbapenem-sparing regimens are suggested for antibiotic stewardship. We experienced a case of ciprofloxacin treatment failure for acute pyelonephritis caused by an apparently susceptible Escherichia coli. A 71-year-old woman presented the emergency department with fever for 7 days and bilateral flank pain for 2 days. The laboratory results and abdominopelvic computed tomography finding were compatible with acute pyelonephritis. During 3-day ciprofloxacin therapy, the patient remained febrile with persistent bacteremia. After the change in antibiotics to ertapenem, the patient's clinical course started to improve. ESBL-producing E. coli isolates were identified in all three consecutive blood samples. Pulsed-field gel electrophoresis (PFGE) patterns, serotypes, and sequence types showed the three isolates were derived from the identical strain. The isolates produced CTX-M-14 type ESBL belonging to the ST69 clonal group. Despite in vitro susceptibility, the failure was attributed to a gyrA point mutation encoding Ser83Leu within quinolone resistance-determining regions. This case suggests that ciprofloxacin should be used cautiously in the treatment of serious infections caused by ciprofloxacin-susceptible, ESBL-producing E. coli, even in acute pyelonephritis because in-vitro susceptibility tests could fail to detect certain genetic mutations.

• Molecules and Cells
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• v.45 no.7
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• pp.479-494
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• 2022

Human mesenchymal stem cells (MSCs) are multipotent stem cells that have been intensively studied as therapeutic tools for a variety of disorders. To enhance the efficacy of MSCs, therapeutic genes are introduced using retroviral and lentiviral vectors. However, serious adverse events (SAEs) such as tumorigenesis can be induced by insertional mutagenesis. We generated lentiviral vectors encoding the wild-type herpes simplex virus thymidine kinase (HSV-TK) gene and a gene containing a point mutation that results in an alanine to histidine substitution at residue 168 (TK(A168H)) and transduced expression in MSCs (MSC-TK and MSC-TK(A168H)). Transduction of lentiviral vectors encoding the TK(A168H) mutant did not alter the proliferation capacity, mesodermal differentiation potential, or surface antigenicity of MSCs. The MSC-TK(A168H) cells were genetically stable, as shown by karyotyping. MSC-TK(A168H) responded to ganciclovir (GCV) with an half maximal inhibitory concentration (IC₅₀) value 10-fold less than that of MSC-TK. Because MSC-TK(A168H) cells were found to be non-tumorigenic, a U87-TK(A168H) subcutaneous tumor was used as a SAE-like condition and we evaluated the effect of valganciclovir (vGCV), an oral prodrug for GCV. U87-TK(A168H) tumors were more efficiently ablated by 200 mg/kg vGCV than U87-TK tumors. These results indicate that MSC-TK(A168H) cells appear to be pre-clinically safe for therapeutic use. We propose that genetic modification with HSV-TK(A168H) makes allogeneic MSC-based ex vivo therapy safer by eliminating transplanted cells during SAEs such as uncontrolled cell proliferation.