Endan Li;Jiwoo Choi;Hye-Ri Sim;Jiyeon Kim;Jae Hyun Jun;Jangbeen Kyung;Nina Ha;Semi Kim;Keun Ho Ryu;Seung Soo Chung;Hyun Sook Kim;Sungsu Lee;Wongi Seol;Jihwan Song
BMB Reports
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v.56
no.3
/
pp.178-183
/
2023
Huntington's disease (HD) is a neurodegenerative disorder, of which pathogenesis is caused by a polyglutamine expansion in the amino-terminus of huntingtin gene that resulted in the aggregation of mutant HTT proteins. HD is characterized by progressive motor dysfunction, cognitive impairment and neuropsychiatric disturbances. Histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase, has been shown to induce transport- and release-defect phenotypes in HD models, whilst treatment with HDAC6 inhibitors ameliorates the phenotypic effects of HD by increasing the levels of α-tubulin acetylation, as well as decreasing the accumulation of mutant huntingtin (mHTT) aggregates, suggesting HDAC6 inhibitor as a HD therapeutics. In this study, we employed in vitro neural stem cell (NSC) model and in vivo YAC128 transgenic (TG) mouse model of HD to test the effect of a novel HDAC6 selective inhibitor, CKD-504, developed by Chong Kun Dang (CKD Pharmaceutical Corp., Korea). We found that treatment of CKD-504 increased tubulin acetylation, microtubule stabilization, axonal transport, and the decrease of mutant huntingtin protein in vitro. From in vivo study, we observed CKD-504 improved the pathology of Huntington's disease: alleviated behavioral deficits, increased axonal transport and number of neurons, restored synaptic function in corticostriatal (CS) circuit, reduced mHTT accumulation, inflammation and tau hyperphosphorylation in YAC128 TG mouse model. These novel results highlight CKD-504 as a potential therapeutic strategy in HD.
Jin Young Kim;Seon-Young Han;Kiho Sung;Jeong Yeon Seo;Cheol Hwan Myung;Chan Song Jo;Jee Hoe Yoon;Ji Yun Park;Jae Sung Hwang
Biomolecules & Therapeutics
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v.31
no.4
/
pp.466-472
/
2023
Exon skipping is an efficient technique to inhibit specific gene expression induced by a short-sequence peptide nucleic acid (PNA). To date, there has been no study on the effects of PNA on skin pigmentation. In melanocytes, the tripartite complex is responsible for the transport of mature melanosomes from the nucleus to the dendrites. The tripartite complex is composed of Rab27a, Mlph (Melanophilin), and Myosin Va. Defects in the protein Mlph, a melanosome transport-related protein, are known to cause hypopigmentation. Our study shows that Olipass peptide nucleic acid (OPNA), a cell membrane-permeable PNA, targets exon skipping in the Mlph SHD domain, which is involved in Rab27a binding. Our findings demonstrate that OPNA induced exon skipping in melan-a cells, resulting in shortened Mlph mRNA, reduced Mlph protein levels, and melanosome aggregation, as observed by microscopy. Therefore, OPNA inhibits the expression of Mlph by inducing exon skipping within the gene. These results suggest that OPNA, which targets Mlph, may be a potential new whitening agent to inhibit melanosome movement.
Araceli Hernandez-Sanchez;Edgar D. Paez-Perez;Elvia Alfaro-Saldana;Vanesa Olivares-Illana;J. Viridiana Garcia-Meza
Journal of Microbiology and Biotechnology
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v.34
no.3
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pp.525-537
/
2024
Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.
Familial Amyotrophic lateral sclerosis (FALS) is a progressive neurodegenetative disorder induced by mutations of the SOD1 gene. Heat shock protein 27 (HSP27) is well-defined as a stress-inducible protein, however the its role in ALS protection has not yet been established. To investigate the role HSP27 may have in SOD1 mutant-mediated apoptosis, human SOD1 or HSP27 genes were fused with a PEP-1 peptide in a bacterial expression vector to produce a genetic in-frame fusion protein, which was then transduced into cells. We found the purified PEP-1-HSP27 fusion proteins can be transduced efficiently into neuronal cells and protect against cell death by enhancing mutant SOD1 activity. Moreover, transduced PEP-1-HSP27 efficiently prevents protein aggregation produced by oxidative stress. These results suggest that transduced HSP27 fusion protein may be explored as a potential therapeutic agent for FALS patients.
Hwang, Hae-Gwang;Kim, Dae-Hwan;Lee, Jeongmin;Mo, Youngwon;Lee, Se-Hoon;Lee, Yongjin;Hyeon, Jae Wook;Lee, Sol Moe;Cheon, Yong-Pil;Choi, Eun-Kyoung;Kim, Su Yeon;Lee, Yeong Seon;Son, Young-Jin;Ryou, Chongsuk
Journal of Microbiology and Biotechnology
/
v.28
no.10
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pp.1749-1759
/
2018
Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with >95% purity. The purified recPrPs predominantly adopted an ${\alpha}$-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.
Myxococcus xanthus is a Gram negative, rod-shaped, soil bacterium that displays a social behaviors, and multicellular development upon nutrient deprivation. The csgA gene encoding a cell surface protein is essential for developmental behaviors including rippling, aggregation, fruiting body formation and sporulation. csgA mutants show normal vegetative growth, but lack all these developmental phenotypes. Expression of the CsgA (C-signal) specific genes are eliminated or dramatically reduced in csgA mutants. In order to identify components of C-signal transduction pathway, second site mutations were introduced into csgA mutants and were identified which can fully or partially restore development of csgA mutants (Rhie, H. G. et. al. 1989. J. Bacteriol. 171, 3268-3276). One of such csgA suppressor mutations, socD500 restores only sporulation to csgA mutants at 15$^{\circ}C$. The socD500 mutaion however eliminates the three basic developmental requirements, starvation, high cell density and a solid surface. Only sporulation, not accompanied with fruiting body formation is induced simply by shifting the temperature of vegetatively growing cells from $32^{\circ}C$ to $15^{\circ}C$. Spores induced by socD500 mutation is not as thick as that of wild-type fruiting body. In socD500 genetic background, two of ten C-signal dependent genes, $\Omega$DK4506 and $\Omega$DK4406 are more highly expressed in growing cells at $15^{\circ}C$. These results indicate that the socD500 mutation may be partly involved in the regulation of expression of two C-signal dependent genes and genes for sporulation in this transduction pathway.
The aim of this study was to assess the effect of the frequency of the L$N_2$ infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of $H_2O$$_2$ was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7% vs. 34.6% respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7%, 76.7% vs. 44.0% for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5$\pm$12.9, 71.6$\pm$8.0, and 62.5$\pm$4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of $H_2O$$_2$ was higher in group 2 than other groups (15.3$\pm$3.0, 16.6$\pm$1.6 vs. 23.4$\pm$1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2$\pm$3.8, 17.4$\pm$1.3 vs. 13.2$\pm$2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8%, 36.0% vs. 65.6%, p<0.05). The frequency of the L$N_2$ infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.
Objective: It has been recently reported that very small stem cells with pluripotency are detected in murine and human. The purposes of this study are to confirm whether very small putative stem cells (VSPSCs), which have the proper characteristics of stem cells as well as the expression of stem cell markers, are detected in human endometrium. Methods: The endometrial cells of 5 women, which were obtained by endometrial biopsy, were cultured for 2 weeks and were confirmed for the expressions of alkaline phosphatase, OCT-4 and CXCR4 by immunochemistry. Subsequently VSPSCs were separated by percoll density gradient method and were cultured. Also VSPSCs and their derived cells were confirmed for the expressions of OCT-4 and CXCR4. Results: The colonies, which is composed with VSPSCs less than 3 ${\mu}m$ and the 5~15 ${\mu}m$ sized hyperchromatic round cells, were detected in the endometrium of all of women and showed the strong expressions of alkaline phosphatase, OCT-4 and CXCR4. In culture after the separation of VSPSCs by percoll, these cells showed the morphological and functional characteristics of stem cells; self-renewal, colony formation, embryoid body-like formation and differential plasticity. VSPSCs formed gradually the 5~15 ${\mu}m$ sized hyperchromatic round cells and the 10~20 ${\mu}m$ sized sphere-shaped cells by cell-to-cell aggregation or cell fusion. Then these cells differentiated the various cells including fibroblast-like cells, nerve-like cells and endothelium-like cells. VSPSCs and their derived cells often showed the strong expressions of OCT-4 and CXCR4. Conclusion: VSPSCs less than 3 ${\mu}m$ and their derived cells are detected in human endometrium and these cells have the proper characteristics of stem cells and the expressions of stem cell markers as alkaline phosphatase, OCT-4 and CXCR4.
Acanthopanax sessiliflorus (A. sessiliflorus) has been known as a traditional medicine having anti-stress, antioxidative and platelet aggregation inhibitory effects. This study was undertaken to investigate the functional properties of water extracts from four parts of A. sessiliflorus. Root, stem, leaf and fruit extracts from A. sessiliflorus were prepared with hot water ($80^{\circ}C$). The contents of functional substances, eleutheroside B and E, polyphenol, antioxidative activity, nitrite scavenging ability and anti-cancer activity of the extracts were determined. The contents of eleutheroside E in stem, root and fruit extracts were 542.50 ${\mu}$g/g, 343.35 ${\mu}$g/g and 30.78 ${\mu}$g/g, respectively. A large part of eleutheroside B was found in fruit (372.01 ${\mu}$g/g) and root (289.33 ${\mu}$g/g) extracts. Root and stem extracts contained 227.21 mg/100g and 131.22 mg/100g of polyphenols, respectively. Antioxidative activities (electron donating ability) of stem and root extracts were 79.87% and 77.27%, respectively. It appears that the antioxidative activities were related to polyphenol contents of the extracts. Most extracts showed 76-81.5% of nitrite scavenging ability at pH 1.2. It reveals that water extract from parts of A. sessiliflorus can inhibit formation of nitrosoamine in food. Effects of the extracts on the growth of normal and cancer cell lines were investigated. Extracts showed no cytotoxicity to normal dendritic cell line (DC2.4). Especially, the root extract promoted the growth of normal cell line. Root and stem extracts had 20-23% of inhibitory effect against stomach cancer cell line (SNU-719) and liver cancer cell line (Hep3B). These result indicated that the extracts from A. sessiliflorus can be used as functional food materials with antioxidative activity and nitrite scavenging ability to eliminate nitrosoamine in food.
Baek Seung In;Lim Hyun Suk;Shin Weon Hye;Ko Cheol Woo;Koo Ja Hoon;Kwak Jung Sik
Childhood Kidney Diseases
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v.2
no.2
/
pp.138-144
/
1998
Purpose : The use of cyclosporine and mitomycin in various immunologic or neoplastic disorders has been known to cause wide-ranged nephrotoxic effects including thrombotic microangiopathy. However, the mechanism of nephrotoxicity of these drugs has not been studied adequately, so that present experimental study has been undertaken to find out whether these drugs can cause direct damage to the kidney and to clarify the pathogenetic mechanism of nephrotoxic effect of these drugs. Materials and methods : Sprague-Dawley rats weighing 250-300 gm were used for experimental animals and unilateral renal perfusion technique, modified from the method described by Hoyer et al was used. Isolation of left kidney from systemic circulation was made by clamping aorta and left renal vein and a hole was punctured in the anterior wall of the left renal vein. Cyclosporine (2.5 mg in 4 ml solution) and mitomycin (1.6 mg in 4ml solution) were infused through left renal artery and normal saline was used in control rats. Forty-eight hours after infusion of the drugs, animals were sacrificed and left kidney removed and processed for histologic examination. Total ischemic time of left kidney was less than 15 minutes: Results : Cyclosporine-perfused group showed severe swelling of glomerular endothelial ceil along with swelling of glomerular epithelial cell and interstitial vascular endothelial cell. Mitomycin-perfused group also showed severe swelling of glomerular endothelial and epithelial cells. And in addition to these findings, they demonstrated platelets aggregation, swelling and degranulation of platelets and fibrin accumulation in some of the capillaries, indicating occurrance of thrombotic microangiopathy. Conclusion : present experiment indicates that cyclosporine and mitomycin can cause direct toxic injury to renal endothelial cell. And this direct toxic damage to endothelial cell seems to be an important initiating event for the development of thrombotic microangiopathy.
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