Remodeling of N-glycosylation pathway of the methylotrophic yeast Hansenula polymoprha toward human complex-type glycans

Seon Ah Cheon,Jeong-Nam Park,Doo-Byoung Oh,Ohsuk Kwon,Hyun Ah Kang 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1

We have previously shown that the glycoengineered methylotrophic yeast Hansenula polymorpha strains, the Δoch1 and Δoch1Δalg3 double deletion strains, with the targeted expression of Aspergillus saitoi α-1,2-mannosidase in the ER, were able to produce human mannose-type N-glycans (Man5GlcNAc2 or core Man3GlcNAc2)1, 2. Here we report the further modification of yeast glycosylation pathway to synthesize the complex-type N-glycans with a terminal N-acetyl glucosamine in both the glycoengineered ΔHpoch1 and ΔHpoch1 ΔHpalg3 strains, respectively. First, several combinatorial synthetic leaders were constructed to localize efficiently active human β -1,2 N-acetyl glucosaminyl transferase I (GnT I) in the Golgi apparatus of yeast. The short, medium, and long N-terminal leader sequences with the various length of stem region of yeast type II membrane proteins (ScMnn9, HpOch1, and HpOcr1), located in the early Golgi compartment, were fused in-frame to the catalytic domain of human GnT I lacking its own N-terminal leader sequence. The GnT I constructs combined with various yeast N-terminal leader sequences were introduced into the H. polymorpha och1 and och1 alg3 strains carrying the ER-targeted α-1,2 mannosidase, and the obtained transformants were screened for proper localization of GnT1 into the Golgi compartment by the size fractional centrifugation and Western blotting. Subsequently, the production of the complex-type glycans with monoantennary N-acetyl glucosamine was analyzed by a capillary electrophoresis of ATPS-labeled cell wall glycans. Our data strongly suggested that the ΔHpoch1 single deletion strain would be a better host for the production of human complex-type N-glycans than the ΔHpoch1 ΔHpalg3 double deletion strain in the respect of the glycosylation site occupancy and the byproduct Hex6GlcNAcs formation.

The unfolded protein response (UPR) pathway in Cryptococcus

Cheon, Seon Ah,Jung, Kwang-Woo,Bahn, Yong-Sun,Kang, Hyun Ah unknown 2014 Virulence Vol.5 No.2

New selectable host–marker systems for multiple genetic manipulations based on TRP1, MET2 and ADE2 in the methylotrophic yeast Hansenula polymorpha

Cheon, Seon Ah,Choo, Jinho,Ubiyvovk, Vera M.,Park, Jeong-Nam,Kim, Moo Woong,Oh, Doo-Byoung,Kwon, Ohsuk,Sibirny, Andriy A.,Kim, Jeong-Yoon,Kang, Hyun Ah John Wiley Sons, Ltd. 2009 Yeast Vol.26 No.9

<P>Interest has been increasing in the thermotolerant methylotrophic yeast Hansenula polymorpha as a useful system for fundamental research and applied purposes. Only a few genetic marker genes and auxotrophic hosts are yet available for this yeast. Here we isolated and developed H. polymorpha TRP1, MET2 and ADE2 genes as selectable markers for multiple genetic manipulations. The H. polymorpha TRP1 (HpTRP1), MET2 (HpMET2) and ADE2 (HpADE2) genes were sequentially disrupted, using an HpURA3 pop-out cassette in H. polymorpha to generate a series of new multiple auxotrophic strains, including up to a quintuple auxotrophic strain. Unexpectedly, the HpTRP1 deletion mutants required additional tryptophan supplementation for their full growth, even on complex media such as YPD. Despite the clearly increased resistance to 5-fluoroanthranilic acid of the HpTRP1 deletion mutants, the HpTRP1 blaster cassette does not appear to be usable as a counter-selection marker in H. polymorpha. Expression vectors carrying HpADE2, HpTRP1 or HpMET2 with their own promoters and terminators as selectable markers were constructed and used to co-transform the quintuple auxotrophic strain for the targeted expression of a heterologous gene, Aspergillus saitoi MsdS, at the ER, the Golgi and the cell surface, respectively. The nucleotide sequences presented here were submitted to GenBank under Accession Nos AY795576 (HpTRP1), FJ226453 (HpMET2) and FJ493241 (HpADE2), respectively. Copyright © 2009 John Wiley & Sons, Ltd.</P>

Unique Evolution of the UPR Pathway with a Novel bZIP Transcription Factor, Hxl1, for Controlling Pathogenicity of <i>Cryptococcus neoformans</i>

Cheon, Seon Ah,Jung, Kwang-Woo,Chen, Ying-Lien,Heitman, Joseph,Bahn, Yong-Sun,Kang, Hyun Ah Public Library of Science 2011 PLoS pathogens Vol.7 No.8

<▼1><P>In eukaryotic cells, the unfolded protein response (UPR) pathway plays a crucial role in cellular homeostasis of the endoplasmic reticulum (ER) during exposure to diverse environmental conditions that cause ER stress. Here we report that the human fungal pathogen <I>Cryptococcus neoformans</I> has evolved a unique UPR pathway composed of an evolutionarily conserved Ire1 protein kinase and a novel bZIP transcription factor encoded by <I>HXL1</I> (<I><U>H</U>AC1</I> and <I><U>X</U>BP1</I>-<I><U>L</U></I>ike gene <I>1</I>). <I>C. neoformans HXL1</I> encodes a protein lacking sequence homology to any known fungal or mammalian Hac1/Xbp1 protein yet undergoes the UPR-induced unconventional splicing in an Ire1-dependent manner upon exposure to various stresses. The structural organization of <I>HXL1</I> and its unconventional splicing is widely conserved in <I>C. neoformans</I> strains of divergent serotypes. Notably, both <I>C. neoformans ire1</I> and <I>hxl1</I> mutants exhibited extreme growth defects at 37°C and hypersensitivity to ER stress and cell wall destabilization. All of the growth defects of the <I>ire1</I> mutant were suppressed by the spliced active form of Hxl1, supporting that <I>HXL1</I> mRNA is a downstream target of Ire1. Interestingly, however, the <I>ire1</I> and <I>hxl1</I> mutants showed differences in thermosensitivity, expression patterns for a subset of genes, and capsule synthesis, indicating that Ire1 has both Hxl1-dependent and -independent functions in <I>C. neoformans</I>. Finally, Ire1 and Hxl1 were shown to be critical for virulence of <I>C. neoformans</I>, suggesting UPR signaling as a novel antifungal therapeutic target.</P></▼1><▼2><P><B>Author Summary</B></P><P>The unfolded protein response (UPR) is a widely conserved signaling pathway in eukaryotic cells and protects cells from the ER stress causing accumulation of toxic unfolded or misfolded proteins. Nevertheless, the UPR pathway has been poorly exploited as a therapeutic target for treatment of eukaryotic fungal pathogens, mainly due to its evolutionarily conserved features. The present study reports unique evolution of the UPR pathway in the basidiomycetous human fungal pathogen, <I>Cryptococcus neoformans</I>, which causes life-threatening meningoencephalitis in both immunocompromised and immunocompetent individuals. Here, for the first time we identified and characterized the <I>C. neoformans</I> UPR pathway, which is composed of an evolutionarily conserved and a distinct signaling component, an ER stress sensor Ire1 and its downstream bZIP transcription factor Hxl1, respectively. Intriguingly, <I>Cryptococcus</I> Hxl1 is very divergent from yeast Hac1 and human Xbp1, but subject to Ire1-mediated unconventional splicing. The Ire1-Hxl1-dependent UPR pathway functions not only in the major response to ER stress, but also plays critical roles in controlling cell wall integrity, growth at host physiological temperature, antifungal drug susceptibility, and virulence of <I>C. neoformans</I>. Therefore we propose Hxl1 is an ideal target for antifungal drug development, based on its marked divergence from the host Xbp1 transcription factor.</P></▼2>

Distinct roles of two ceramide synthases, CaLag1p and CaLac1p, in the morphogenesis of <i>Candida albicans</i>

Cheon, Seon Ah,Bal, Jyotiranjan,Song, Yunkyoung,Hwang, Hai‐,min,Kim, Ah Ruem,Kang, Woo Kyu,Kang, Hyun Ah,Hannibal‐,Bach, Hans K.,Knudsen, Jens,Ejsing, Christer S.,Kim, Jeong‐,Yoon Blackwell Publishing Ltd 2012 Molecular microbiology Vol.83 No.4

<P><B>Summary</B></P><P>Lag1p and Lac1p catalyse ceramide synthesis in <I>Saccharomyces cerevisiae.</I> This study shows that Lag1 family proteins are generally required for polarized growth in hemiascomycetous yeast. However, in contrast to <I>S. cerevisiae</I> where these proteins are functionally redundant, <I>C. albicans</I> Lag1p (CaLag1p) and Lac1p (CaLac1p) are functionally distinct. Lack of CaLag1p, but not CaLac1p, caused severe defects in the growth and hyphal morphogenesis of <I>C. albicans</I>. Deletion of <I>CaLAG1</I> decreased expression of the hypha‐specific <I>HWP1</I> and <I>ECE1</I> genes. Moreover, overexpression of <I>CaLAG1</I> induced pseudohyphal growth in this organism under non‐hypha‐inducing conditions, suggesting that CaLag1p is necessary for relaying signals to induce hypha‐specific gene expression. Analysis of ceramide and sphingolipid composition revealed that CaLag1p predominantly synthesizes ceramides with C24:0/C26:0 fatty acid moieties, which are involved in generating inositol‐containing sphingolipids, whereas CaLac1p produces ceramides with C18:0 fatty acid moieties, which are precursors for glucosylsphingolipids. Thus, our study demonstrates that CaLag1p and CaLac1p have distinct substrate specificities and physiological roles in <I>C. albicans</I>.</P>

백두옹의 소염진통작용

천선아(Seon Ah Cheon),최병기(Byung Kee Choi),김성연(Seung Yeun Kim),이은방(Eun Bang Lee) 한국응용약물학회 2000 Biomolecules & Therapeutics(구 응용약물학회지) Vol.8 No.3

The root extract of pulsatilla koreana has been used as antibacterial, antiparasitic and anti-inflammatory analgesic agents in Traditional Medicine in Korea. Thus anti-inflammatory and analgesic actions of the methanol and water extracts of the root were investigated by administration in oral and intravenous route. From the results, it is concluded that the extracts exhibited the potent anti-inflammatory and analgesic actions in intravenous administration, but did not show the actions in oral administration in animals.

재조합 인간 상피세포 성장인자(DWP 401)의 일반약리작용

천선아(Seon Ah Cheon),김상미(Sang Mee Kim),이은방(Eun Bang Lee),임승욱(Seung Wook Lim),유영효(Young Hyo Yu),박명환(Myung Hwan Park) 대한약학회 1995 약학회지 Vol.39 No.5

The recombinant human epidermal growth factor(DWP 401) was investigated on the pharmacological actions. DWP 401 had no effects on the hexobarbital-induced sleeping time, locomotor activity, rotarod test, body temperature, analgesic action and anticonvulsant action in mice. It also had no influences on the isolated tracheal muscle and ileum of guinea-pig, isolated uterus and fundus strip of rats. Slight hypotensive action with effect on respiration was revealed at a dose of 8g/kg i.v. of DWP 401 in rabbits. DWP 401 exhibited a weak inhibitory action of glucose tolerance in normal rats, significantly lowered the blood glucose contents in adrenalectomized rats at a concentration of 160g/kg, and produced a significant inhibitory effect on leucocyte migration in CMC-pouch of rats at a concentration of 32g/rat. Furthermore, DWP 401 showed a significant decrease on gastric juice volume and acidity. However, DWP 401 had no intestinal propulsion rate and influence on urine excretion.

The outcome of endoscopic management of bile leakage after hepatobiliary surgery

( Seon Ung Yun ),( Young Koog Cheon ),( Chan Sup Shim ),( Tae Yoon Lee ),( Hyung Min Yu ),( Hyun Ah Chung ),( Se Woong Kwon ),( Taek Gun Jeong ),( Sang Hee An ),( Gyung Won Jeong ),( Ji Wan Kim ) 대한내과학회 2017 The Korean Journal of Internal Medicine Vol.32 No.1

Background/Aims: Despite improvements in surgical techniques and postoperative patient care, bile leakage can occur after hepatobiliary surgery and may lead to serious complications. The aim of this retrospective study was to evaluate the efficacy of endoscopic treatment of bile leakage after hepatobiliary surgery. Methods: The medical records of 20 patients who underwent endoscopic retrograde cholangiopancreatography because of bile leakage after hepatobiliary surgery from August 2009 to September 2014 were reviewed retrospectively. Endoscopic treatment included insertion of an endoscopic retrograde biliary drainage stent after endoscopic sphincterotomy. Results: Most cases of bile leakage presented as percutaneous bile drainage through a Jackson-Pratt bag (75%), followed by abdominal pain (20%). The sites of bile leaks were the cystic duct stump in 10 patients, intrahepatic ducts in five, liver beds in three, common hepatic duct in one, and common bile duct in one. Of the three cases of bile leakage combined with bile duct stricture, one patient had severe bile duct obstruction, and the others had mild strictures. Five cases of bile leakage also exhibited common bile duct stones. Concerning endoscopic modalities, endoscopic therapy for bile leakage was successful in 19 patients (95%). One patient experienced endoscopic failure because of an operation-induced bile duct deformity. One patient developed guidewire-induced microperforation during cannulation, which recovered with conservative treatment. One patient developed recurrent bile leakage, which required additional biliary stenting with sphincter-otomy. Conclusions: The endoscopic approach should be considered a first-line modality for the diagnosis and treatment of bile leakage after hepatobiliary surgery.

근거리에서 공포탄에 의한 두부손상

박현선,백광제,김준식,전영진,한승백,신동운,김아진 대한외상학회 1999 大韓外傷學會誌 Vol.12 No.2

Blank cartridge guns are generally regarded as being harmless and are not considered to be firearms in most countries. We experienced a case of brain injury causedby an accidental blank cartridge shot at close range. The patient suffered from open compound comminuted skull fracture at th frontal area, including the orbital roof. The initial blood pressure was uncheckable, and the patient showed Cheyne-Stokes respiration. The mental status was a coma state. Bleeding was noticed continuously at the open frontal area wound, and bleeding rhinorrhea and otorrhea were noticed. After fluid resuscitation, the vital signs were stabilized, and brain computerized tomogram (CT) scan was performed. It revealed open compound comminuted skull fracture at the frontal area with intracranial hemorrhage (ICH), subdural hemorrhage (SDH), and subarachnoid hemorrhage (SAH). After resuscitation, an open craniectomy and removal of the ICH were performed. However, the patient died 6days after the operation due to severe lethal brain swelling.

요양보호사의 입소노인에 대한 구강관리 인식에 관한 연구

정진아(Jin-Ah Jung),천혜원(Hye-Won Cheon),김경선(Kyoung-Seon Kim),강정희(Jung-Hee Kang) 한국보건복지학회 2021 보건과 복지 Vol.23 No.1

본 연구는 노인요양시설 입소노인의 구강관리 시 필요한 임상실무지침서와 구강관리 교육 프로그램 개발에 필요한 근거를 마련하기 위해 수행하였다. 사회적 기업 소속의 일부 치과위생사가 방문하여 입소노인의 구강관리를 수행하고 있는 J도에 소재한 5개 노인요양시설 요양보호사 236명의 자료를 수집하였으며, SPSS 19.0을 이용하여 분석하였다. 연구결과, 요양보호사의 구강관리 지식수준은 치주병의 치료, 치주병과 노화, 경관영양시행과 타액분비, 생리식염액과 구강건조증에 대한 지식부분이 부족함을 알 수 있었으며, 구강관리에 대한 지식(p <.01)과 구강관리에 대한 교육이 필요하다고 느낄수록(p <.001) 구강관리에 대한 인식이 높게 나타났다. 따라서 요양보호사가 인지하고 있는 구강관리에 대한 지식과 인식을 통해 입소노인의 구강관리에 중요한 영향을 미칠 것으로 사료되므로 요양보호사에게 실질적인 구강보건교육이 될 수 있는 임상실무지침서와 교육 프로그램 개발이 필요하며 구강보건증진을 위해 예방과 교육에 대해 체계적으로 훈련받은 구강보건전문인력의 배치가 필요하기에 국가적으로 보건의료정책적인 제도의 지원이 이루어져야 할 것이다. This study was conducted to provide a clinical practice guideline necessary for the oral management for elderlies in care facilities and a basis for the development of the oral management education program. It collected the data from 236 care workers in 5 elderly care facilities in J Province where dental hygienists of Social Enterprise had performed the oral management for elderlies and performed it by using SPSS 19.0. As a result, it showed that the care workers’ level of knowledge of the oral management for the elderlies was lacking in periodontal disease’s treatment, periodontal disease and aging, intubation feeding and saliva production, and the physiological salt solution and xerostomia, and the more they felt the knowledges(p <.01) and educations(p <.001) of the oral management were necessary, the higher awareness of the oral management. Therefore, it is thought that the knowledge and the awareness about the oral management, which care workers are recognizing, have important role for the oral management of their caring elderlies, so the clinical practice guideline and the developing an educational program, which can be practical oral health educations to the care workers, are needed, and as it is necessary to arrange oral health specialists systematically trained about the prevention and the education for improving the oral health, support for health care policy systems should be provided nationally.