Liquid Ordered State of Detergent Resistant Lipid Rafts is Maintained at Physiological Condition

김기범 School of Life Science & Biotechnology Korea Unive 2004 국내박사

Lipid Raft란 세포막에 존재하는 cholesterol, sphingolipid-rich micro domain으로서, 다양한 신호전달 물질이 모이게 되는 signaling platform의 역할을 하는 것으로 알려져 있다. 이러한 특이구조의 존재는 insulin signaling, cholesterol-homeostasis 등의 다양한 signaling event 들이 세포 내에서 보다 효율적으로 매개, 조절되는 것을 가능하게 하며, 이를 뒷받침하는 다양한 연구결과들이 보고되어 있다. 반면, 연구를 위한 이들의 분리정제 방법에 있어서 여러가지 논란이 야기되고 있는데, 그 중 가장 큰 논란이 되는 점은 이들의 분리가 세포막의 구성분간의 결합양상을 변질 시킬 수 있는 4C˚ 에서 이루어진다는 것이다. 또한 분리에 사용되는 Detergent에 따라서 raft내의 단백질 조성이 다르게 나타나는 사실 또한 관련 연구진행의 난점으로 지적되고 있다. 상기 주제에 대한 연구를 위해, HepG2 cell line내에 존재하는 raft 의 분리 정제를 다양한 방법을 통해 진행하였으며, 각 방법을 통해 추출한 raft들로부터 내부의 단백질만을 분리한 뒤, 이차원 전기영동을 통해 각각의 분리 방법에 따른 단백질 profile들의 차이점을 비교 분석하였다. 그 결과, 현재 이용되고 있는 TX-100를 사용한 raft의 저온 (4C˚)분리 법에 비해, brij35를 이용한 분리방법이 보다 많은 raft protein들을 보존시키고 있음을 확인하였다. 또한 2차원 전기영동 결과를 토대로 raft에 존재하는 lipid modified 단백질들인 Ras, Src, Fyn들의 western blot을 수행한 바, 기존의 보고와는 달리 저온의 분리조건에 비해 생리활성 조건인 37C˚ 에서 정성적으로, 정량적으로 더 많은 raft protein들이 안정화 되어있다는 사실을 알 수 있었다. 이와 같은 방법으로 분리한 raft 단백질들은, raft내의 cholesterol 을 제거 시킴에 따라 대부분 raft로부터 분리되는 양상을 보였으며, 이를 통해 Brij 35을 이용한 생리활성조건에서의 분리결과가 외부 단백질의 contamination에 의해 야기된 것이 아님을 증명할 수 있었다. 반면 detergent를 이용하지 않고, 세포막의 pH변화를 야기시켜 raft를 분리시키는 Detergent Free 방법의 경우, 다수의 protein들이 cholesterol 제거 시 별다른 영향을 받지 않은 체, raft에 그대로 상존하였는데, 이러한 결과는 상기의 분리법이 raft의 연구에 적절치 않을 수도 있음을 암시하는 것으로 볼 수 있을 것이다. 이들 연구결과를 종합해 볼 때, raft의 고유한 liquid-ordered structure는 생리 활성조건에서도 유지가 될 수 있으며, Brij 35를 이용한 raft의 분리방법이 기존의 방법에 비해 보다 정확한 raft의 연구를 가능케 한다는 결론을 내릴 수 있었다. Different proteins were found in lipid rafts depending on their isolation methods. For example, more insulin receptor was found in raft fractions prepared from HepG2 cells with Brij35 than with Triton X-100. In order to assess the effect of detergent type and temperature on raft isolation, raft proteins from HepG2 cells were analyzed by two-dimensional electrophoresis. More raft protein spots appeared when rafts were isolated by Brij35 at room temperature or 37℃ than by Triton X-100 at 0℃, indicating that lipid rafts are not disrupted in the presence of detergent at physiological temperature (37℃). Indeed, lipid-modified proteins such as Ras, Src, and Fyn were found in raft fractions although detergent-resistant rafts were isolated at room or physiological temperature. The 2-D gel profile of raft proteins isolated by detergent-free (high pH/carbonate) method was considerably similar to that of detergent-resistant raft proteins but showed more raft proteins. Whereas many detergent-resistant raft proteins disappeared upon cellular exposure with methyl-β-cyclodextrin, high pH/carbonate-resistant raft proteins did not, suggesting that many proteins isolated by high pH/carbonate could be contaminants. Taken together all these data, we conclude that liquid-ordered state of detergent-resistant lipid rafts are not destroyed at physiological temperature.

Establishment of an alternative in vitro potency assay for performing quality control testing on the Japanese encephalitis virus vaccine

김도근 School of Life Sciences & Biotechnology, Korea Uni 2012 국내박사

Hepatitis B Virus X Protein : Molecular Basis of Intracellular Instability

김정환 Graduate School of Life Sciences & Biotechnology K 2003 국내박사

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in viral replication and in the development of hepatocellular carcinoma. HBx has the capability to transactivate the expression of all HBV proteins including the viral core protein HBc. Consistent with its regulatory role HBx is relatively unstable and is present at low levels in the cell. I report here that the level of HBx was significantly reduced by the co-expression of HBc in cultured human hepatoma cells, whereas the level of HBx mRNA was unaffected. The repression of HBx by HBc was relieved by treating cells with the proteasome inhibitor MG132, indicating that HBc acts by stimulating the proteasome-mediated degradation of HBx. The inhibitory effect of HBc was specific to HBx and did not affect other proteins, including p53, a known target of proteasome. Although no direct physical interaction between HBc and HBx could be demonstrated in this study, mutational analysis indicated that the C-terminal half of the HBc is responsible for its inhibitory effect. These results suggest that HBc functions as a novel regulator of the HBV life cycle and of hepatocellular carcinogenesis through the control of HBx level via an inhibitory feedback-type of mechanism. Furthermore, it was shown that HBx contains bipartite recognition signals for proteasome-mediated degradation in its middle and C-terminal parts. In addition, lysine residues that serve as ubiquitination target site were characterized. Through site-directed mutagenesis, it was shown that ubiquitination was not restricted to any of the 6 lysine residues and that all the 6 lysines can be ubiquitinated with diverse susceptibility. Among the 6 lysines, the 140th lysine residue was ubiquitinated with highest efficiency. This result indicates that the 140th lysine may act as the main ubiquitin acceptor in physiological condition. Interestingly, one mutant in which all the 6 lysines were substituted by alanine still served a proteasome substrate, although it was free from being ubiquitination. It is thought, therefore, that the degradation of HBx is achieved through both ubiquitin-dependent as well as ubiquitin-independent pathway.

Antioxidant and Anticancer Activities of Platycodon Grandiflorum A. De Candolle Root

이지영 Graduate School of Life Sciences & Biotechnology K 2004 국내박사

한국산 도라지는 오랫동안 전통 한약재로 사용되거나 식용으로 널리 사용되어 왔다. 본 논문에서는 한국산 도라지(4 년근)의 생리활성 검색을 위해 도라지를 석유에텔로 추출하여 얻은 분획을 가지고 항산화 및 항암 활성을 측정한 후, 각 활성물질을 분리, 정제 및 동정하여 분석하기까지의 과정을 기술하였다. 먼저 도라지의 석유에텔 추출물을 실리카겔 컬럼크로마토그래피를 통해 석유에텔과 에틸에텔을 9:1-5:5 (v/v) 비율로 분리하여 총 5 개의 분획 (fraction I-V)을 얻었다. 분획의 항산화 활성 측정은 크게 과산화지질 형성억제능력과 자유 라디칼 소거능력 측정으로 나누었는데, 먼저 과산화지질 형성억제효과를 측정하기위해 Ferric thiocyanate test 와 Thiobarbituric acid test를 행한 결과, 다른 분획보다 FII(8:2)분획에서 높은 과산화지질 형성억제효과를 보였고, 기존의 항산화제와 비교하였을 때 BHA 보다는 못하지만 α-tocopheol 보다 활성정도가 높게 나타났다. 또한 자유라디칼 소거효과(free radical scavenging)를 측정한 결과에서도 FII(8:2) 분획이 다른 분획에 비해 높은 활성을 보였으며 농도에 따라 효과가 증가하는 경향을 나타내었다. 많은 식물에서 밝혀진 상당수의 항산화 물질이 페놀성 물질로 밝혀졌기 때문에 각 분획에서 페놀성 물질 함량을 조사하였으며, 그 결과, FII(8:2) 분획에서 4.80 ±0.26mg/g 으로 다른 분획보다 높게 나타났다. 이를 보다 더 명확하게 분리하기 위해 preparative TLC (thin layer chromatography)를 통해 FII 를 FII-1∼5 분획들로 분리하여 각각의 자유 라디칼 소거능을 측정한 결과, 높은 활성을 보이는 분획을 얻을 수 있었다. 최초 분획들의 항암활성 측정에서는 인체결장암세포인 HT-29, 직장암세포인 HRT-18 과 간암세포인 HepG2 를 대상으로 MTT assay 방법을 통해 각각 분획들의 세포증식억제 효과를 실험한 결과 다른 분획에 비해 FIII(7:3) 분획이 가장 효과적인 활성을 나타내었다. 이것을 역시 Preparative TLC 를 통해 FIII-1∼6 의 분획들로 분리하여 활성을 조사하였고, 특히 FIII-2 에서 가장 높은 활성과 농도 의존적인 세포사멸효과를 관찰하였다. 이 실험을 통해 도라지의 석유에텔 추출물에서 기존의 항산화제와 비교해 높은 활성을 가진 항산화 물질과, 암세포에 따라 약간의 차이가 있으나 항암효과를 나타내는 물질의 존재를 확인하고, 이것은 각각 계속되는 실험을 통해 활성물질의 분리, 정제 후, 동정 및 분석이 이루어졌다. 항산화 물질 연구에서는 앞에서 가장 높은 항산화 효과를 보였던 FII(8:2) 분획을 실리카겔 컬럼 크로마토그래피를 통해 재정제하였고, 그 중 가장 높은 활성을 보이는 FII-3 분획을 HPLC 를 사용하여 높은 항산화 효과를 보이고 서로 비슷한 구조를 가지는 것으로 추측되는 2 개의 물질을 분리, 정제하였다. 이들을 UV spectra, HRFAB-MS, FT-IR 그리고 ^(13)C, ^(1)H-NMR 을 통해 구조를 분석한 결과, 두개의 물질은 페놀성 물질과 지방산이 에스테르 결합으로 연결된 wax esters 임이 밝혀졌다. 두개의 물질 모두 페놀성 부분으로 coniferyl alcohol 을 가지고 있으며 연결된 지방산으로서 compound 1 (분자량 418)이 palmitic acid, compound 2 (분자량 444)가 oleic acid 의 esters 형태로 밝혀졌다. 분리, 정제된 compound 1, 2를 가지고 DPPH 자유 라디칼 소거능을 측정한 실험에서, compound 2 가 1 보다 좀 더 높은 활성을 나타내었고 이는 합성 항산화제인 BHA 보다도 높은 것이었다. 또한 superoxide 와 nitric oxide 자유라디칼 소거능 실험에서도 compound 1, 2 모두 높은 항산화 활성을 보여, BHA 와 BHT 보다 효과적임을 확인하였다. 이러한 페놀성 wax ester 는 보통 식물의 cuticle 이나 suberin 층에서 발견되었으나, coniferyl alcohol 을 페놀부분으로 가지는 wax ester는 지금까지 거의 보고된 바가 없고, 또한 그것의 식물체내 역할 및 생리활성에 대해서도 거의 알려진 바가 없다. 따라서 이 실험을 통해 처음으로 도라지에서 wax ester 물질이 분리되었고 이것의 항산화 활성이 확인되었다. 항암효과를 가지고 있는 물질에 대한 연구에서는, 처음에 분리한 분획 중 항암활성이 좋았던 FIII(7:3)분획을 가지고 preparative-TLC 및 HPLC 를 통해 1개의 활성물질을 분리 및 정제하였다. 이 물질은 추가적인 구조분석이 이루어지지 않았으나, 분리과정에 있어서 UV 흡수 스펙트럼을 통해 전형적인 polyacetylene 구조를 가지고 있음을 확인하였다. 이 물질을 가지고 결장암세포인 HCT-116 세포를 대상으로 실험한 결과, 뚜렷한 세포성장억제의 효과를 보였으며, 20, 40 그리고 60 μg/mL 의 농도와 24, 48, 72 시간 배양한 결과, 시간과 농도 의존적인 활성을 가지고 있음을 확인하였다. 특히 60 μg/mL 의 농도로 처리한 군에서 24시간 이내에 이미 50% 이상의 세포사멸율을 나타내었고, 72 시간 배양 후에는 약 90 %에 가까운 효과를 나타내었다. 이러한 세포성장억제 또는 사멸에 관련되는 세포의 사멸 메커니즘(apoptosis)을 살펴 보기 위해, 인체 결장암세포인 HCT-116을 분리된 활성물질과 함께 배양하였을 때 apoptosis 시 발생하는 세포 형태 및 DNA 의 특징적인 변화여부를 관찰하였다. 세포 모양 및 성장 관찰실험에서 HCT-116 에 활성물질을 처리 후 시간에 따라 세포가 수축되고 세포수도 감소되는 것을 관찰하였고, acridine orange / ethidium bromide 염색 후 세포핵의 변화관찰에서도 세포핵의 분획화와 apoptotic body 로 형성되는 것을 관찰하였는데, 이들은 모두 세포의 apoptosis 중 일어나는 현상으로 잘 알려져 있다. 또한 apoptosis 중 DNA 의 fragmentation 은 DNA ladder 를 통해 확인되었으며, 세포주기의 분포 변화 관찰실험을 통해 이러한 apoptosis 가 세포의 G0/G1 기에서 arrest 함으로써 일어나는 것을 확인할 수 있었다. 결론적으로, 본 실험을 통해 한국산 도라지에서 항산화와 항암효과를 가지는 생리활성물질이 각각 검색, 분리되었는데, 분리된 페놀성 wax ester 는 도라지에서 처음으로 발견됨과 동시에 그 항산화 활성이 처음으로 보고되었고, 도라지에서 분리된 polyacetylene 은 여러 종류의 암세포에서 성장을 억제시키는데, 특히 인체 결장암세포인 HCT-116 에서는 apoptosis를 유도하는 것을 확인하였다. In this study, the petroleum ether extracts from the root of Platycodon grandiflorum A. De Candolle (balloonflower), a plant used as traditional oriental medicine and also as a food material in Korea, were investigated on their antioxidant and anticancer activities. Crude petroleum ether extracts from dried P. grandiflorum root were fractionated into FI ∼ FV by a silica gel column chromatography with gradient solvents (petroleum ether : ethyl ether, 9:1∼5:5, v/v). First, the antioxidant activity of the fractions was evaluated based on lipid peroxidation inhibition and free radical scavenging activity. Among the fractions, FII (8:2) showed the highest antioxidant activity, and its antioxidant activity was closely related with the phenolic content in the fraction. The FII was further separated into 5 sub-fractions, FII-A∼E, by a preparative thin layer chromatography (TLC), and then the sub-fractions were screened for the antioxidant activity, which was comparable to that of commercial antioxidants such as α-tocopherol and BHA. The anticancer activity of FI ∼ FV was evaluated by MTT assay using human cancer cell lines (HT-29, HRT-18 and HepG2), and FIII(7:3) showed the highest growth inhibition effect on all cancer cell lines tested. The FIII was also sub-fractionated into FIII-A∼D by preparative TLC, and among the sub-fractions, FIII-B exhibited the highest and concentration-dependent cytotoxicity. From these results of activity screening, the potential as a source of highly active antioxidant and anticancer agents was confirmed in the petroleum ether extracts of P. grandiflorum root, and accordingly, the extract was further investigated for separation and evaluation of active compounds. For more specialized study on antioxidant, FII (8:2) that had the highest antioxidant activity was subjected to 2nd silica gel column chromatography using more detailed solvent gradients (petroleum ether : ethyl ether, 10:0∼8:2), and six sub-fractions FII-1∼6 were obtained. Then, the most active sub-fraction, FII-3, was separated again by HPLC to obtain two pure antioxidant compounds, Compounds 1 and 2. From the analysis by UV spectra, FAB-MS, IR, 13C and 1H NMR, both compounds were identified as wax esters that had the similar structures consisting of phenolic alcohol and fatty acid. Both had coniferyl alcohol as phenolic alcohol, but Compound 1 (M.W. 418) contained palmtic acid as fatty acid, whereas Compound 2 (M.W. 444) had oleic acid. The antioxidant activity, based on DPPH radical scavenging activity, was higher in Compound 2 than in Compound 1, which was also higher than that of BHA. In superoxide and nitric oxide radical scavenging tests, both compounds also exhibited higher activity than BHA and BHT. These wax ester compounds mostly have been identified in cuticle or suberin of vascular plants, but their function or activity, especially related with antioxidant activity, have been rarely studied. In this study, two wax ester compounds were newly identified in P. grandiflorum, and their antioxidant activity was also firstly reported. For the study of anticancer activity of P. grandiflorum extracts, FIII (7:3) showing the highest activity was also separated again into six sub-fractions FIII- 1∼6 using preparative TLC and subsequent four fractions FIII-2a∼d from FIII-2 using HPLC, and one active compound was obtained from active FIII-2c fraction. Although additional structural analysis was not performed, its basic structure was identified as a polyacetylene compound based on its typical UV absorbance spectrum. By MTT assay, this compound showed a significant growth inhibiting effect on human colon carcinoma cell, HCT-116, in time and concentration dependent manners at conditions tested. Especially at a concentration of 60 μg/mL, the compound showed over 50 % of cytotoxicity on cancer cells within 24 h, and after 72 h, almost 90 % of cancer cells were induced to cell death. For the study of this cytotoxicity in relation to apoptosis, a specific form of programmed cell death, the characteristic changes on cell morphology and DNA that commonly occur during apoptosis were investigated. Cell shrinkage and reduced cell number were induced by incubating HCT-116 with the compound, and cell nuclear fragmentation and apoptotic body formation were also observed after acridine orange / ethidium bromide staining, which are all typical morphological changes during apoptosis. DNA fragmentation during apoptosis was also confirmed by observing DNA ladder using electrophoresis. From all those evidences, it is concluded that this compound induced apoptosis in HCT-116 human colon carcinoma cell, and from the flow cytometry study, the apoptosis occurred by the arrest on the G0/G1 phase of cell cycle. Conclusively through this study, two wax ester compounds exhibiting highly antioxidant activity were firstly separated from the petroleum ether extracts of P. grandiflorum root. Also one polyacetylene compound showing cytotoxicity in human colon carcinoma cell by inducing apoptosis was obtained from those P. grandiflorum extracts.

Red wine oxidition and its polyphenolic effect on cholesterol homeostasis in vitro and in vivo

이동현 School of Life Sciences and Biotechnology, Korea U 2014 국내박사

The present studies aim to investigate the oxidation effect on flavor changes in Cabernet Sauvignon wine during secondary shelf life and its polyphenolic effect on cholesterol homeostasis in vitro and in vivo. In oxidation effect on flavor of the red wine, headspace solid-phase microextraction coupled to gas chromatography-quadrupole mass spectrometry and sensory tests, with the support of multivariate statistical analyses such as orthogonal projection to latent structure discriminant analysis (OPLS-DA) loading plot and PCA score plot, were employed to identify several flavors related to oxidation. Isoamyl acetate, ethyl decanoate, nonanoic acid, n-decanoic acid, undecanoic acid, 2-furancarboxylic acid, dodecanoic acid, and phenylacetaldehyde were determined to be associated with the oxidation of the wine. In cholesterol homeostasis by the red wine polyphenols, the alcohol-free red wine concentrates was found to be more effective for the reduction of total cholesterol than lovastatin via in vitro test. For in vivo model, despite a high cholesterol diet, a significant reduction in both total cholesterol and LDL-cholesterol was observed when supplemented with alcohol-free red wine concentrates (AFRWC), but the increase of HDL-cholesterol was not observed.

Modified atmosphere (MA) packages using SPI/MMT coating film for prolonging the shelf-life of sprouts

이정은 School of life sciences and biotechnology Korea un 2009 국내석사

Shelf-life extension of Luffa cylindrica with various packaging films : quality properties of Luffa cylindrica in modified atmosphere

나종희 School of Life Sciences and Biotechnology, Korea U 2010 국내석사

Structural changes in mitochondria of the Drosophila melanogaster flight muscle after spaceflight

정덕화 School of Life Sciences and Biotechnology, Korea U 2010 국내석사

The purpose of this work was the analysis of some ultra structural changes in mitochondria of the Drosophila melanogaster (Drosophila). These results show that spaceflight affects mitochondria structure. The aerospace provides many infinite areas of scientific research. For exploring out of the earth, space science is indispensable. Drosophila is an excellent material for space study. This study has utilized Drosophila to test the effects of spaceflight of a Soyuz-type space vehicle for 12 days. Under space based experimental condition, mitochondria clustering and morphological alterations were observed in wild type Drosophila. After 12 days spaceflight, a larger area of mitochondria was observed in wild type flight muscle by transmission electron microscopy (TEM). Changes in the area and the number of mitochondria were analyzed by ImageJ (National Institutes of Health). The results suggest that the area of mitochondria was increased in the cells exposed to the altered gravity.

Effects of Tau on the activity of triose phosphate isomerase (TPI) in brain cells

박승아 School of Life Sciences and Biotechnology, Korea U 2010 국내박사

Tau is a main component of the aberrant paired helical filaments (PHF) found in Alzheimer’s disease (AD). It has also been reported to enhance oxidative stress, which is a major factor in the pathogenesis of neurodegenerative diseases. However, protective functions of Tau have recently been reported, including antagonizing apoptosis, in addition to its role in stabilizing microtubules. In this study, the interaction between Tau and triose phosphate isomerase (TPI) in a normal, control state as well as in a neurodegeneration state was examined and demonstrated for the first time. More importantly, it also showed that Tau protects TPI against oxidative damage. An oxidative stress-induced decrease in the activity of TPI was attenuated in Tau-overexpressing cells, indicating that Tau protects TPI against oxidative damage. By contrast, the activity of TPI was decreased in Tau-transgenic (Tg) mice compared to non-Tg (NTg) mice even though protein levels were not changed in both groups. Some TPIs were found on the PHF in Tg mice, which explains the decrease in the activity of TPI. Taken together, it is concluded that while Tau binds and protects TPI in normal cells, and conversely, the formation of PHF induced by Tau phosphorylation trap some TPI and trigger the functional loss of TPI in the development of neurodegenerative diseases. These results provide new insights into understanding the in-depth involvement of Tau in the development of neurodegenerative disorders.

Functional study of ASC1 in adhesion and virulence in Candida albicans

김세웅 School of Life Sciences and Biotechnology, Korea U 2010 국내석사

Candida albicans, the common human fungal pathogen, can switch morphology from yeast to puseudohyphal or hyphal form by various environmental cues. It is well known that the ability of morphological conversion as well as biofilm formation and adhesion activity, renders C. albicans virulent. It is noteworthy that every factor involved in the morphogenesis is known to be important for the virulence in this pathogen. According to our previous two-dimensional differential gel electrophoresis data, Asc1p is decreased by a factor of 6 during the morphological conversion. To examine a functional relevance of this protein in morphogenesis and virulence, asc1 homozygous null mutant was generated by PCR based gene disruption method. Although a normal morphological transition of the asc1 deletion strain in liquid media was found, it did not change its morphology on solid media. Moreover it was also found that the adhesion activity and the hyphal specific gene expressions were defective by ASC1 deletion. Finally, it was revealed that asc1 null mutant is avirulent in a mouse model. These results provided that Asc1 plays a pivotal role for cellular adhesion and virulence through regulation of specific gene expression in C. albicans. 캔디다증을 유발하는 Candida albicans는 잘 알려진 human fungal pathogen이다. Candida albicans는 yeast growth form, pseudohyphal growth from, hyphal growth form으로 다양한 환경에 의해 자신의 형태를 변화시킴으로써 변화된 환경에 적응하면 살아가는 fungi다. 형태 변화나, 바이오필름 형성, 숙주세포의 표면에 부착 정도 등은 Candida albicans가 병원성을 띄게 하는 중요한 병원성 인자들이다. 이러한 표현형을 띄기 위해서 세포내에서 진행되는 기작들 또한 병원성에 중요하다고 할 수 있다. hyphal growth form과 yeast growth form 상황에서의 2 DIGE data를 비교해 봄으로써 ASC1의 단백질 수준에서의 발현이 hyphal growth를 할 때 감소하는 것을 확인 할 수 있었다. CaASC1은 40S 라이보솜 소단위체로서 단백질 합성, 여러 가지 signal들의 transducer로써 역할을 한다고 알려져 있다. Candida albicans에서 ASC1의 병원성과 관련된 기능을 알아보기 위하여 PCR-based gene disruption method를 통해 asc1 null strain을 제작 하였고 이 strain에서 병원성 인자로 알려진 adhesion activity, invasion activity의 감소를 확인 할 수 있었다. 또한, asc1 null strain에서 특정하게 전사과정에서 발현이 감소하는 adhesion gene (ECE1, HWP1, ALS3)을 찾았고, wild type 과 asc1 null mutant를 mouse 모델에 injection 함으로써 병원성이 감소하는 것을 확인 할 수 있었다. 그러므로 C. albicans에서 Asc1이 병원성 인자로 알려진 adhesion을 조절하는 regulator로 작용하는 것을 알 수 있었다.