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Kwang-Hoon Lee

Publications and source records attributed to Kwang-Hoon Lee.

10 recordsLinked to original sources
PubMed2006

Identification of alpha-enolase as an autoantigen associated with severe asthma.

BACKGROUND: Approximately 5% to 10% of patients with asthma have severe disease that is not effectively controlled by typical therapies. The existence of an autoantigen associated with severe asthma has been previously reported. OBJECTIVE: We attempted to identify the autoantigen. METHODS: Severe asthma was defined as patients having at least 1 severe asthmatic exacerbation requiring an emergency department visit or admission in the last year despite continuous typical therapies. Autoantibodies to airway epithelial cells (A549) were examined in sera from patients with severe asthma by immunoblot analysis. RESULTS: IgG autoantibodies to the 52-kd airway epithelial cell antigen were detected in sera from 32 of 78 patients with severe asthma (41%), 9 of 83 patients with mild-to-moderate asthma (11%), and 2 of 58 healthy controls (3%; P < .001). The 52-kd autoantigen was identified as alpha-enolase by mass spectrometry analysis and confirmed by using recombinant human alpha-enolase protein. The detection of IgG autoantibodies to alpha-enolase was the most significant indicator for distinguishing severe asthma from mild-to-moderate asthma, even after adjusting for the effects of other clinical variables, including age, sex, atopy, and FEV(1) (adjusted odds ratio, 5.2; 95% CI, 2.1-12.9; P < .001). CONCLUSION: The alpha-enolase was identified as an autoantigen associated with severe asthma. Further studies are needed to determine the significance of this autoantigen in severe asthma. CLINICAL IMPLICATIONS: IgG autoantibodies to alpha-enolase could be a biological marker for severe asthma.

Adult↗
PubMed2005

Highly efficient protein expression and purification using bacterial hemoglobin fusion vector.

Recently developed bacterial hemoglobin (VHb) fusion expression vector has been widely used for the production of many target proteins due to its distinctive properties of expressing fusion protein with red color which facilitates visualization of the steps in purification, and increasing solubility of the target proteins. However, after intensive use of the vector, several defects have been found. In this report, we present a modified VHb fusion vector (pPosKJ) with higher efficiency, in which most of the defects were eliminated. First, it was found that thrombin protease often digests target protein as well as inserted thrombin cleavage site, so it was replaced by a TEV cleavage site for more specific cleavage of VHb from target protein. Second, a glycine-rich linker sequence was inserted between 6x his-tag and VHb to improve the affinity of 6x his-tag to Ni-NTA resin, resulting in higher purity of eluted fusion protein. Third, EcoRI and XhoI restriction sites located elsewhere in the vector were removed to make these restriction sites available for the cloning of target protein coding genes. A pPosKJ vector was fully examined with an anti-apoptotic BCL-2 family member of Caenorhabditis elegans, CED-9. A C-terminal VHb fusion expression vector (pPosKJC) was also constructed for stable expression of target proteins that may be difficult to express with an N-terminal fusion. Vaccinia-related kinase 1 (VRK1) was also successfully expressed and purified using the vector with high yield. Taken together, we suggest that the VHb fusion vector may be well suited for high-throughput protein expression and purification.

Amino Acid Sequence↗
PubMed2005

Biochemical and crystallographic studies reveal a specific interaction between TRAPP subunits Trs33p and Bet3p.

Transport protein particle (TRAPP) comprises a family of two highly related multiprotein complexes, with seven common subunits, that serve to target different classes of transport vesicles to their appropriate compartments. Defining the architecture of the complexes will advance our understanding of the functional differences between these highly related molecular machines. Genetic analyses in yeast suggested a specific interaction between the TRAPP subunits Bet3p and Trs33p. A mammalian bet3-trs33 complex was crystallized, and the structure was solved to 2.2 angstroms resolution. Intriguingly, the overall fold of the bet3 and trs33 monomers was similar, although the proteins had little overall sequence identity. In vitro experiments using yeast TRAPP subunits indicated that Bet3p binding to Trs33p facilitates the interaction between Bet3p and another TRAPP subunit, Bet5p. Mutational analysis suggests that yeast Trs33p facilitates other Bet3p protein-protein interactions. Furthermore, we show that Trs33p can increase the Golgi-localized pool of a mutated Bet3 protein normally found in the cytosol. We propose that one of the roles of Trs33p is to facilitate the incorporation of the Bet3p subunit into assembling TRAPP complexes.

Amino Acid Sequence↗
PubMed2004

Two-promoter vector is highly efficient for overproduction of protein complexes.

The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes. The major problem of this method is the much lower expression of the second gene compared with that of the first gene next to the promoter. We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem. Compared with bicistronic vectors, corresponding two-promoter vectors yielded four to nine times larger amounts of the complexes between BCL-2 family proteins, BCL-X(L):BAD, BCL-X(L):BIM-S, and CED-9:EGL-1 in bacterial cells as a result of significantly increased expression of the second genes in a manner independent of the order of the target genes. With the two-promoter system, we produced two other complexes in large quantity suitable for extensive crystallization trial. The method does not accompany any technical disadvantages, and represents a significant improvement from the conventional method, which should enjoy wide application for the coexpression of binary or higher order protein complexes by extension.

Circular Dichroism↗
PubMed2003

Biological characterization of EDC-crosslinked collagen-hyaluronic acid matrix in dermal tissue restoration.

Porous collagen matrices crosslinked with various amounts of hyaluronic acid (HA) by 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) were developed as scaffolds for dermal tissue regeneration. The effect of HA on cells in accordance with HA concentrations in the collagenous matrices was investigated using cultures of fetal human dermal fibroblasts, and the effect of EDC-crosslinked collagen-HA matrix on wound size reduction was also evaluated in vivo. Scanning electron microscopic views of the matrices demonstrated that all of the collagen-HA matrices had interconnected pores with mean diameters of 150-250 microm. An HA matrix retention test showed that the concentration of HA decreased slowly after an initial rapid decrease over 24h. Fetal human dermal fibroblasts adhered well to all of the collagen-based matrices as compared with the Porous polyurethane matrix used as a control. An 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide based proliferation test and the hematoxylin and eosin staining of a 2 week cultured matrix showed that the proliferation of fibroblasts was enhanced on a 9.6% HA contained collagen matrix. No significant difference was in terms of fibroblast migration into the various types of scaffolds as HA content was increased. In vivo testing showed that dermis treated with collagen or collagen-HA matrix was thicker than the control, and epithelial regeneration was accelerated, and collagen synthesis increased. However, no significant effect of HA on wound size reduction was found.

Animals↗
PubMed2003

Clinical features of patients with Behçet's disease and epididymitis.

PURPOSE: Epididymitis is a rare manifestation of Behçet's disease but its clinical significance is still not fully understood. We evaluated the clinical significance of epididymitis in patients with Behçet's disease. MATERIALS AND METHODS: Of 780 male patients attending our clinics between 1985 and 2002 who were diagnosed with Behçet's disease by international criteria or who had the complete or incomplete type of Behçet's disease by Japanese criteria 36 were identified with epididymitis. Clinical data on these patients were reviewed. RESULTS: The 36 patients were categorized into the complete (13 or 36.1%) and incomplete (23 or 63.9%) types of Behçet's disease with an average age at onset of 27.4 years. The frequency of individual symptoms were oral ulcers and cutaneous involvement in all 36 cases (100%), genital ulcers in 32 (88.9%), arthritis in 18 (50.0%), ocular involvement in 17 (47.2%), central nervous system involvement in 2 (5.6%), gastrointestinal ulcer in 1 (2.8%) and a positive pathergy test in 4 (11.1%). A significantly higher number of patients with epididymitis had genital ulcers (p <0.05), cutaneous involvement (p <0.001), arthritis (p <0.05), central nervous system involvement (p <0.05) and a positive pathergy test (p <0.05) compared with the other 744 with Behçet's disease without epididymitis who served as controls. CONCLUSIONS: To our knowledge there has been no controlled study of the clinical significance of epididymitis in Behçet's disease. Our results suggest a tendency toward severe Behçet's disease manifestations in patients with epididymitis, prompting physicians to evaluate closely and meticulously treat such patients.

Adolescent↗
PubMed2003

Influence of sex on patients with Behçet's disease in Korea.

Behçet's disease (BD) is a multisystemic inflammatory disorder known as having a histopathological findings of vasculitis. The influence of sexual difference on BD is a well-known fact and there are several reports suggesting a more severe course of the disease among young males. The purpose of our study was to determine the effects of gender on the severity and clinical features of BD patients in Korea. The study included 1,901 patients with BD who fulfilled the criteria of International Study Group for Behçet's Disease or corresponded to the complete or incomplete type for the revised criteria of Behçet's Disease Research Committee of Japan. BD in Korea showed a female predominance (M:F=0.61:1). The skin lesions were observed in 79.9% of patients, of which 77.6% had erythema nodosum-like lesion, which was more frequent in females. The ocular lesions were more common in males showing a higher frequency of uveitis. Ocular and vascular symptoms as clinical features with severe complications or mortality were more frequent in males than in females. The mean age at the onset of patients with the worst prognosis such as ocular, gastrointestinal, neurologic, and vascular involvements was significantly younger in male than in female patients (p<0.05). In conclusion, this study elucidated the influences of sexual difference on BD in Korea.

Adolescent↗