Publications by authors named "Kyuboem Han"

14 Publications

  • Page 1 of 1

Effect of cysteine-free human fibroblast growth factor-5s mutant (FGF5sC93S) on hair growth.

Dermatol Ther 2020 11 18;33(6):e14530. Epub 2020 Nov 18.

Paean Biotechnology, Inc., Daejeon, South Korea.

Treatment for hair loss is largely limited, and any beneficial effects are often transient. Based on the critical role of the FGF5 isoform, FGF5s, in the hair growth cycle, it may be a good therapeutic candidate for the prevention of hair loss, as well as the promotion of hair growth. To investigate its potential use for hair growth, a mutant form of the FGF5s protein (FGF5sC93S) was generated, expressed, and purified. The FGF5sC93S mutant was able to antagonize FGF5-induced mitogenic activity, which normally triggers the conversion of hair follicles from the anagen phase to the catagen phase. In addition, the FGF5sC93S mutant efficiently suppressed gene expression induced by FGF5 both human outer root sheath (hORS) and human dermal papilla (hDP) cells. Administration of FGF5sC93S proteins onto the scalps of human subjects significantly increased the total number of hairs at 24 weeks. Together, our data demonstrate that a mutant form of the FGF5s protein could be used as a potential hair promoting agent.
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http://dx.doi.org/10.1111/dth.14530DOI Listing
November 2020

Nanoinjection system for precise direct delivery of biomolecules into single cells.

Lab Chip 2019 02;19(4):580-588

Department of Biotechnology, CHA University, 335 Pankyoro, Bundang-gu, Seongnam, Gyeonggi-do 13488, Republic of Korea.

Intracellular delivery of functional molecules such as proteins, transcription factors and DNA is effective and promising in cell biology. However, existing transfection methods are often unsuitable to deliver big molecules into cells or require carriers such as viruses and peptides specific to the target molecules. In addition, the nature of bulk processing does not generally provide accurate dose control of individual cells. The concept of single-cell-based material injection based on electrokinetic pumping through nanocapillaries could overcome these problems, yet the fabrication and operation of nanoscale 3-dimensional structures have remained unsolved. In this research, a hybrid (PDMS/glass) microfluidic chip with a true 3-dimensional nanoinjection structure (called "nanoinjection system") is presented. The nanoinjection structure was fabricated by femtosecond-laser (fs-laser) ablation in a single solid glass, which showed very successful delivery of red fluorescent protein (RFP) and expression of plasmid DNA in several different types of cells. This system is promising in that the amount of molecules to be delivered is controllable and the processed cells are systematically separated into a harvesting chamber, which can radically improve the purity of the processed cells. In addition, it was confirmed that the cells were healthy even after the molecule injection for a few seconds, indicating that the injection time can be significantly elongated, further improving the delivery efficiency of biomolecules without affecting the cell viability. We envision that the nanoinjection system having the major features of being carrier-free and dose-controllable, having an unlimited injection period, and ease of harvesting will greatly contribute to the next-generation research studies in the fields of cell biology and cell therapeutics.
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http://dx.doi.org/10.1039/c8lc00709hDOI Listing
February 2019

Enhanced delivery of protein fused to cell penetrating peptides to mammalian cells.

BMB Rep 2019 May;52(5):324-329

Paean Biotechnology, Inc., Daejeon 34028, Korea.

Recent progress in cellular reprogramming technology and lineage-specific cell differentiation has provided great opportunities for translational research. Because virus-based gene delivery is not a practical reprogramming protocol, protein-based reprogramming has been receiving attention as a safe way to generate reprogrammed cells. However, the poor efficiency of the cellular uptake of reprogramming proteins is still a major obstacle. Here, we reported key factors which improve the cellular uptake of these proteins. Purified red fluorescent proteins fused with 9xLysine (dsRED-9K) as a cell penetrating peptide were efficiently delivered into the diverse primary cells. Protein delivery was improved by the addition of amodiaquine. Furthermore, purified dsRED-9K was able to penetrate all cell lineages derived from mouse embryonic stem cells efficiently. Our data may provide important insights into the design of protein-based reprogramming or differentiation protocols [BMB Reports 2019; 52(5): 324-329].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549919PMC
May 2019

Corrigendum: The Effect of Micro-Spicule Containing Epidermal Growth Factor on Periocular Wrinkles.

Ann Dermatol 2017 Dec 30;29(6):828. Epub 2017 Oct 30.

Department of Dermatology, Chungnam National University School of Medicine, Daejeon, Korea.

[This corrects the article on p. 187 in vol. 29, PMID: 28392646.].
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http://dx.doi.org/10.5021/ad.2017.29.6.828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705381PMC
December 2017

The Effect of Micro-Spicule Containing Epidermal Growth Factor on Periocular Wrinkles.

Ann Dermatol 2017 Apr 24;29(2):187-193. Epub 2017 Mar 24.

Department of Dermatology, Chungnam National University School of Medicine, Daejeon, Korea.

Background: Micro-needle patches have been recently used to increase skin permeability, which improves drug delivery, and for cosmetic purposes. However, these patches may often have limited efficacy due to insufficient skin penetration and reduced compliance caused by discomfort.

Objective: We evaluated the efficacy and the safety of soluble micro-spicule containing epidermal growth factor (MS-EGF) for the treatment of periocular wrinkles.

Methods: Twenty healthy volunteers aged 33 to 54 years were enrolled in a randomized, controlled, split-face study. For 4 weeks, a periocular wrinkle was treated daily with either a soluble MS-EGF cream or a cream containing EGF alone. All subjects underwent 8 weeks of follow-up. Efficacy was assessed using an ultrasonic measurement of dermal depth and density, digital skin image analysis, 5-point photonumeric scale for periocular wrinkles and subjective satisfaction.

Results: MS-EGF group showed statistically significant increase of dermal depth and density compared to EGF alone group after 4 and 8 weeks. In addition, there was a marked improvement shown in clinical and 3-dimensional skin image in MS-EGF group. The treatments were well-tolerated; no significant side-effect was noted.

Conclusion: The MS-EGF formulation may represent an effective and biocompatible advance in the treatment of periocular wrinkles.
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http://dx.doi.org/10.5021/ad.2017.29.2.187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383744PMC
April 2017

Effect of rice cell-derived human granulocyte-macrophage colony-stimulating factor on 5-fluorouracil-induced mucositis in hamsters.

Biol Pharm Bull 2013 ;36(3):425-31

Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, 221 Huksuk-dong, Dongjak-gu, Seoul 156–756, South Korea.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important regulator of the maturation and function of cells in the granulocyte and macrophage lineages, and also plays a significant role in wound healing. In a previous study, we expressed human GM-CSF in rice cells (rice cell-derived human GM-CSF; rhGM-CSF). The purpose of the present study was to evaluate its effect on wound healing in oral mucositis. Oral mucositis was induced in Syrian hamster cheek pouches by 5-fluorouracil treatment and mechanical scratching. Ulcerated areas were treated from days 3 to 14 with an application of 200 µL saline, or of the same volume of a solution containing 0.04, 0.2, or 1 µg/mL rhGM-CSF. Treatment of hamsters with rhGM-CSF reduced the ulcerated areas of the oral mucosa, compared with the control. Early in the healing process, the mucositis tissue layer of the rhGM-CSF-treated group showed significantly decreased myeloperoxidase activity and increased numbers of proliferating cell nuclear antigen (PCNA)-positive cells. Treatment with rhGM-CSF also affected expression of inflammatory cytokines in the ulcerative mucosal tissue. These results demonstrate the efficacy of plant-produced rhGM-CSF in wound healing and have significant implications for the development of rhGM-CSF as a therapeutic agent for ulcerative oral mucositis.
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http://dx.doi.org/10.1248/bpb.b12-00869DOI Listing
August 2013

Identification of a cyclosporine-specific P450 hydroxylase gene through targeted cytochrome P450 complement (CYPome) disruption in Sebekia benihana.

Appl Environ Microbiol 2013 Apr 25;79(7):2253-62. Epub 2013 Jan 25.

Department of Biological Engineering, Inha University, Incheon, South Korea.

It was previously proposed that regio-specific hydroxylation of an immunosuppressive cyclosporine (CsA) at the 4th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycete Sebekia benihana. This modification is thought to be the reason for the hair growth-promoting side effect without the immunosuppressive activity of CsA. Through S. benihana genome sequencing and in silico analysis, we identified the complete cytochrome P450 complement (CYPome) of S. benihana, including 21 CYPs and their electron transfer partners, consisting of 7 ferredoxins (FDs) and 4 ferredoxin reductases (FDRs). Using Escherichia coli conjugation-based S. benihana CYPome-targeted disruption, all of the identified CYP, FD, and FDR genes in S. benihana were individually inactivated. Among the 32 S. benihana exconjugant mutants tested, only a single S. benihana CYP mutant, ΔCYP-sb21, failed to exhibit CsA hydroxylation activity. The hydroxylation was restored by CYP-sb21 gene complementation. Since all S. benihana FD and FDR disruption mutants maintained CsA hydroxylation activity, it can be concluded that CYP-sb21, a new member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific CsA hydroxylation process in S. benihana. Moreover, expression of an extra copy of the CYP-sb21 gene increased CsA hydroxylation in wild-type S. benihana and an NADPH-enriched Streptomyces coelicolor mutant, by 2-fold and 1.5-fold, respectively. These results show for the first time that regio-specific hydroxylation of CsA is carried out by a specific P450 hydroxylase present in S. benihana, and they set the stage for the biotechnological application of regio-specific CsA hydroxylation through heterologous CYP-sb21 expression.
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http://dx.doi.org/10.1128/AEM.03722-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623247PMC
April 2013

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica.

Appl Microbiol Biotechnol 2012 Jul 1;95(1):157-68. Epub 2012 Mar 1.

Department of Biological Engineering, Inha University, Incheon 402-751, South Korea.

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.
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http://dx.doi.org/10.1007/s00253-012-3955-xDOI Listing
July 2012

Targeted gene disruption and functional complementation of cytochrome P450 Hydroyxlase involved in cyclosporin A hydroxylation in Sebekia benihana.

J Microbiol Biotechnol 2011 Jan;21(1):14-9

Department of Biological Engineering, Inha University, Incheon 402-751, Korea.

A cyclic undecapeptide-family natural product, cyclosporin A (CyA), which is one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex in a filamentous fungal strain named Tolypocladium niveum. Previously, structural modifications of cyclosporins such as a regionspecific hydroxylation at the 4th N-methyl leucine in a rare actinomycetes called Sebekia benihana were reported to lead to dramatic changes in their bioactive spectra. However, the reason behind this change could not be determined since a system to genetically manipulate S. benihana has not yet been developed. To address this limitation, in this study, we utilized the most commonly practiced gene manipulation techniques including conjugation-based foreign gene transfer-and-expression as well as targeted gene disruption to genetically manipulate S. benihana. Using these optimized genetic manipulation systems, a putative cytochrome P450 hydroxylase (CYP) gene named CYP506, which is involved in CyA hydroxylation in S. benihana, was specifically disrupted and genetically complemented. The S. benihana deltaCYP506 exhibited a significantly reduced CyA hydroxylation yield as well as considerable yield restoration by functional complementation of the S. benihana CYP506 gene, suggesting that the genetically manipulated S. benihana CYP mutant strains may serve as a more efficient bioconversion host for various valuable metabolites including CyA.
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http://dx.doi.org/10.4014/jmb.1009.09026DOI Listing
January 2011

Analysis and functional expression of NPP pathway-specific regulatory genes in Pseudonocardia autotrophica.

J Ind Microbiol Biotechnol 2011 Apr 23;38(4):573-9. Epub 2011 Jan 23.

Department of Biological Engineering, Inha University, Incheon 402-751, Korea.

Using the genomics-guided polyene screening method, a rare actinomycetes called Pseudonocardia autotrophica was previously identified to contain functionally clustered nystatin-like biosynthetic genes and to produce a presumably novel polyene compound named nystatin-like Pseudonocardia polyene (NPP) (Kim et al., J Ind Microbiol Biotechnol 36:1425-1434, 2009). Since very low NPP productivity was observed in most P. autotrophica culture conditions, its biosynthetic pathway was proposed to be tightly regulated. Herein we report in silico analysis of six putative NPP pathway-specific regulatory genes present in its biosynthetic gene cluster, followed by functional overexpression of these regulatory genes in P. autotrophica. Three pathway-specific regulatory genes (nppRI, RIII, and RV) were predicted to belong to a typical LAL-type transcriptional family. Each regulatory gene was cloned under the strong constitutive ermE* promoter in a Streptomyces integrative pSET152 plasmid, followed by direct intergeneric conjugation from a plasmid-containing E. coli donor cell to P. autotrophica. While all the P. autotrophica exconjugants exhibited improved NPP productivity, the one containing nppRIII showed the highest NPP productivity improvement. In addition, culture-time-dependent analysis revealed that the nppRIII-stimulated NPP biosynthesis was more significant in the late exponential growth stage than in the stationary stage. Moreover, the P. autotrophica nppRIII-disruption mutant failed to produce NPP, with significantly reduced transcription levels of most npp biosynthetic genes. The results described suggest that identification and overexpression of key pathway-specific regulatory gene, followed by optimum harvest timing, should be critical factors to maximize the productivity of an intrinsically low-level metabolite such as NPP produced by rare actinomycetes species.
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http://dx.doi.org/10.1007/s10295-011-0939-2DOI Listing
April 2011

Cloning-independent expression and analysis of omega-transaminases by use of a cell-free protein synthesis system.

Appl Environ Microbiol 2010 Sep 23;76(18):6295-8. Epub 2010 Jul 23.

Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, South Korea.

Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative omega-transaminase (omega-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative omega-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.
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http://dx.doi.org/10.1128/AEM.00029-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937503PMC
September 2010

Combination strategy to increase cyclosporin A productivity by Tolypocladium niveum using random mutagenesis and protoplast transformation.

J Microbiol Biotechnol 2009 Sep;19(9):869-72

Department of Biological Engineering, Inha University, Incheon 402-751, Korea.

The cyclic undecapeptide cyclosporin A (CyA), one of the most valuable immunosuppressive drugs, is produced nonribosomally by a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. To increase CyA productivity by wild-type T. niveum (ATCC 34921), random mutagenesis was first performed using an antifungal agar-plug colony assay (APCA) selection approach. This generated a mutant strain producing more than 9-fold greater CyA than the wild-type strain. Additionally, a foreign bacterial gene, Vitreoscilla hemoglobin gene (VHb), was transformed via protoplast regeneration and its transcription was confirmed by RT-PCR in the UV-irradiated mutant cell. This led to an additional 33.5% increase of CyA production. Although most protoplastregenerated T. niveum transformants tend to lose CyA productivity, the optimized combination of random mutagenesis and protoplast transformation described here should be an efficient strategy to generate a commercially valuable, yet metabolite low-producing, fungal species, such as CyA producing T. niveum.
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http://dx.doi.org/10.4014/jmb.0811.629DOI Listing
September 2009

Subcutaneous Four-Week Repeated Dose Toxicity Studies of Rice Cell-Derived Recombinant Human Granulocyte-Macrophage Colony Stimulating Factor in Rats.

Toxicol Res 2008 Dec 1;24(4):315-320. Epub 2008 Dec 1.

110Department of Environmental & Health Chemistry, Physical Pharmacy Laboratory, College of Pharmacy, Chung-Ang University, Seoul, 156-756 Korea.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a glycoprotein and hematopoietic growth factors that regulates the proliferation of myeloid precursor cells and activates mature granulocytes and macrophages. In a previous study, we reported that hGM-CSF could be produced in transgenic rice cell suspension culture, termed rhGM-CSF. In the present study, we examined the repeated dose toxicity of rhGM-CSF in SD rats. The repeated dose toxicity study was performed at each dose of 50 and 200 µg/kg subcutaneous administration of rhGM-CSF everyday for 28-days period. The results did not show any changes in food and water intake. There were also no significant changes in both body and organ weights between the control and the tested groups. The hematological and blood biochemical parameters were statistically not different in all groups. These results suggest that rhGM-CSF may show no repeated dose toxicity in SD rats under the conditions.
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http://dx.doi.org/10.5487/TR.2008.24.4.315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006265PMC
December 2008

Isolation and partial characterization of a cryptic polyene gene cluster in Pseudonocardia autotrophica.

J Ind Microbiol Biotechnol 2006 Feb 12;33(2):84-7. Epub 2005 Aug 12.

Department of Biotechnology, Inha University, 402-751, Incheon, Korea.

The polyene antibiotics, a category that includes nystatin, pimaricin, amphotericin, and candicidin, comprise a family of very promising antifungal polyketide compounds and are typically produced by soil actinomycetes. The biosynthetic gene clusters for these polyenes have been previously investigated, revealing the presence of highly similar cytochrome P450 hydroxylase (CYP) genes. Using polyene CYP-specific PCR screening with several actinomycete genomic DNAs, Pseudonocardia autotrophica was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using the polyene-specific CYP gene probe identified a positive cosmid clone, which contained a DNA fragment of approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete and two incomplete open reading frames, which were found to be highly similar, but still unique, when compared to previously known polyene biosynthetic genes. These results suggest that the polyene-specific screening approach may constitute an efficient method for the isolation of potentially valuable cryptic polyene biosynthetic gene clusters from various rare actinomycetes.
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http://dx.doi.org/10.1007/s10295-005-0018-7DOI Listing
February 2006