Expression of N-terminal truncated desmoglein 3 (Delta NDg3) in epidermis and its role in keratinocyte differentiation

During a search for keratinocyte differentiation-related genes, we obtained a cDNA fragment from the 5'-untranslated region of a previously identified splicing variant of desmoglein 3 (Dg3). This transcript encodes a protein of 282 amino acids, which corresponds to the N-terminal truncated intracellular domain of Dg3 (Delta NDg3). Northern blot analysis detected a 4.6-kb transcript matching the predicted size of Delta NDg3 mRNA, and Western blot analysis with an antibody raised against the Dg3 C-terminus (H-145) detected a 31-kDa protein. Increased Delta NDg3 expression was observed in differentiating keratinocytes by RT-PCR and Western blot analysis, suggesting that Delta NDg3 is indeed a differentiation-related gene product. In immunohistochemical studies of normal and pathologic tissues, H-145 antibody detected the protein in the cytoplasm of suprabasal layer cells, whereas an antibody directed against the N-terminal region of Dg3 (AF1720) reacted with a membrane protein in the basal layer. In addition, Delta NDg3 transcript and protein were upregulated in psoriatic epidermis, and protein expression appeared to increase in epidermal tumors including Bowen's disease and squamous cell carcinoma. Moreover, overexpression of Delta NDg3 led to increased migration and weakening of cell adhesion. These results suggest that Delta NDg3 have a role in keratinocyte differentiation, and that may be related with tumorigenesis of epithelial origin.

Serological Characterization of Soochong and Muju Virus as New Serotype of Hantavirus

Apodemus agrarius, which accounts for three-fourths of the wild rodents, mainly inhabits in cultivated fields of Korea. Apodemus peninsulae and Eothenomys regulus are the second and third dominant species, respectively. Soochong virus (SOOV) from A. peninsulae and Puumala-related Muju virus (MUJV) from E. regulus were isolated in 1997 and 1998 in Korea, respectively. But serological characterizations of SOOV and MUJV were not identified clearly. Thus, in order to determine the serotypic classification, simultaneous cross-indirect immunofluorescent antibody (IFA) assay and cross-plaque reduction neutralization (PRN) test against four different hantaviruses were conducted with sera from 17 A. agrarius, 19 A. peninsulae, and 8 E. regulus strains. IFA titers of sera from A. agrarius and A. peninsulae were the highest to Hantaan virus (HTNV) and SOOV, respectively. However, most sera showed similar IFA titers to Seoul virus (SEOV). Therefore it was difficult to do serotyping using the sera from A. agrarius and A. peninsulae by IFA. In case of sera of E. regulus, IFA titers to Puumala virus (PUUV) were higher than HTNV, SOOV and SEOV. Cross-PRN result of A. agrarius to HTNV, SOOV, SEOV and PUUV was 6,890, 5,120, 110 and 30, respectively. In case of A. peninsulae, the mean PRN titer was the highest to SOOV (1:6,820) and those to HTNV, SEOV and PUUV were 1,580, 100 and 30, respectively. The mean PRN titers of E. regulus to HTNV, SOOV, SEOV and PUUV were 70, 10, 80 and 640. SOOV and MUJV could be distinguished from HTNV and SEOV by cross-PRNT. These results demonstrate that SOOV and MUJV could be classified as new serotype of hantavirus.

Production and Characterization of Monoclonal Antibodies to Mumps Virus Isolated in Korea

Classical mumps patients develop bilateral or less commonly unilateral parotitis. Mumps virus belongs to the genus Rubulavirus in the family Paramyxoviridae. It contains single stranded RNA genome with negative polarity. To characterize the antigenicity of mumps virus isolated in Korea, nineteen hybridoma cell lines producing monoclonal antibodies to mumps virus were established by fusion of Sp2/0-Ag14 mouse myeloma cells with spleen cells of mice immunized with mumps virus strain 98-40. The specificity of these monoclonal antibodies was established by immunofluorescence microscopy and immunoblotting analysis. Fifteen out of nineteen hybridoma cell lines secreted IgG monoclonal antibodies (MAbs) against mumps virus, and the remaining four secreted IgM. The isotypes of thirteen clones of 19 MAbs were IgG1, two were IgG2a, and four were IgM. Eight MAbs reacted with a 68 kDa nucleocapsid protein, six MAbs reacted with a 46 kDa phosphoprotein, and five MAbs reacted with a 42 kDa matrix protein.

Seroprevalence and Phylogenetic Analysis of Orientia tsutsugamushi from Apodemus peninsulae in Korea

To investigate the seroprevalence of the Orientia tsutsugamushi infection of Apodemus peninsulae and genomic variations in O. tsutsugamushi isolates, 246 A. peninsulae were trapped in 14 mountainous areas approximately 500 meter above sea level in Korea during the period of 1997 and 2000. Seropositive rate of O. tsutsugamushi among A. peninsulae was 31.8% in Kyunggi, 8.2% in Chunbuk and 7.1% in Kangwon provinces by microimmunofluorescent test. The 56 kDa protein gene was amplified by PCR in the spleens of seropositive A. peninsulae. Two amplicons from seropositive A. peninsulae were sequenced and their phylogeny was analysed on the basis of sequence homology. The 56 kDa genes of A. peninsulae 98-12 strain and A. peninsulae 98-16 strain showed 98.7% nucleotide homology and 96.6% amino acid similarity. A. peninsulae 98-12 and A. peninsulae 98-16 strain were related to Kuroki, Boryong and Karp strains showing 93.3~92.2%, and 87.1~84.6% homologies in nucleotide and amino acids levels, respectively. In the phylogenetic analysis, A. peninsulae 98-12 and A. peninsulae 98-16 strain formed a distinct group with Boryong, Kuroki and Nishino strains and were clearly distinguished from other genetic groups. The results suggest that A. peninsulae might be an important reservoir of O. tsutsugamushi in Korea.

Seroepidemiologic Analysis of Acute Febrile Illness in Korea during 1997~1998

Hemorrhagic fever with renal syndrome (HFRS), scrub typhus, murine typhus and leptospirosis have been the principal acute febrile diseases in Korea for many years. To evaluate the seroepidemiologic patterns of the acute febrile illness, sera collected from 4,503 patients in 1997~1998 were examined for antibodies against Hantaan virus, Orientia tsutsugamushi and Rickettsia typhi by indirect immunofluorescent antibody technique (IFA) and macroscopic agglutination test for Leptospira interogans. Seropositive cases for Orientia tsutsugamushi, Rickettsia typhi, Leptospira interogans and Hantaan virus were 261 (12.4%), 242 (11.5%), 11 (0.5%), and 250 (11.9%) in 1997, and 415 (17.3%), 273 (11.4%), 16 (0.7%), and 357 (14.9%) in 1998, respectively. Male was affected more frequently by HFRS and leptospirosis while scrub typhus was more prevalent in female. Old age group was more susceptible to the acute febrile diseases. Most positive cases were occurred during October and November for scrub typhus, and during November and December for HFRS. These results showed similar patterns with previous epidemiological data obtained during recent several years, except the single scrub typhus epidemic in 1998, and implied that no significant changes occurred in ecologic system for acute febrile diseases in Korea.

Prevalence of Leptospira interrogans in Wild Rodents in Korea

Leptospirosis has been known as endemic disease in Korea since 1984. Wild rodent, mostly Apodemus agrarius, has been known as an important source of leptospiral infection especially in rainy circumstances in harvest reason of rural area. The infection rates of Leptospira interrogans in field rodents, Apodemus agrarius, was investigated by culture and PCR detection of leptospiral DNA, and compared with previous data. Furthermore, the serogroup and serovar were investigated. Two hundred twenty two Apodemus agrarius were captured during October to December 1996. Spirohaetes were isolated from 22 (9.9%) and leptospiral DNA was detected in an additional six rodents (12.6%). Subsequent cross-agglutinin absorption test, monoclonal antibody reactivity classified 21 cultures among 22 isolates as Leptospira interrogans serogroup Icterohemorrhagiae serovar lai. The above data did not differ from previous survey in 1984 to 1987. There was no significant change of Leptospira interrogans infection in field rodents in Korea.

Serovar Identification and Genetic Characterization of Leptospira Isolates by Arbitrarily Primed PCR and Ribotyping

Serovars of 22 leptospiral field isolates from rats trapped in Korea were identified by cross-agglutinin absorption test (CAAT). Genomic characteristics of 7 selected isolates and 6 antigenically closely related reference serovars of lai, yeonchon, birkini, gem, mwogolo, and canicola were differentiated by arbitrarily primed PCR (AP-PCR) and southern blot hybridization using 16S rRNA gene probe from Borrelia burgdorferi. Among the 22 isolates, 21 strains were identified as serovar lai by CAAT, while the serological reactivity of NR13 did not accord with that of serovar lai. Results of AP-PCR using primers RSP, KF and PB-1 were in general agreement with those obtained by serological identification, and all 7 isolates including NR13 showed the same profile with serovar lai or yeonchon. In the southern blot hybridization with 16S rRNA gene probe, the isolates were divided into two ribotype groups when HindIII and BamHI digests were employed: isolates NR4, NR13, and serovar lai showed the same profile, and isolates JR34, JR57, KR48, JR77, and JR82 were classified as the another ribotype group. Isolate NR13 and serovar yeonchon, which were isolated in Korea and showed serological differences with serovar lai, were indistinguishable from serovar lai in this DNA study using AP-PCR and ribotyping. These results demonstrate that Korean leptospiral isolates were closely related in DNA level, and ribotyping would be useful for subgrouping of field isolates.

Production and Characterization of Monoclonal Antibodies Against Seoul Virus

Hantaviruses are members of the family Bunyaviridae, the etiologic agents of Hemorrhagic fever with renal syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). They are negative-sense, single-stranded RNA viruses possessing a large (L), medium (M) and small (S) genomic segment which encodes a viral polymerase, envelope glycoproteins (G1 and G2) and a nucleocapsid (N) protein, respectively. Seoul (SEO) virus, the causative agent of clinically mild HFRS in worldwide, was isolated from lung tissues of urban rat (Rattus norvegicus) captured in Seoul, Korea, 1982. To clarify the antigenic characteristics and the differentiation of serotypes of hantavirus, 8 hybridoma cell lines secreting monoclonal antibodies (MAbs) against SEOV 80-39 strain were produced by fusion of SP2/0-Ag14 mouse myeloma cells with spleen cells of BALB/c mice, immunized with SEOV. Reactivities of these MAbs were examined by the indirect immunofluorescence assay (IFA), enzyme linked immunosorbent assay (ELISA), immunoblot, high density particle agglutination (HDPA) and plaque reduction neutralization test (PRNT). Eight out of 235 hybridomas secreted MAbs of Seoul virus 80-39 continuously, and these eight MAbs were all IgG. The isotypes of these 8 MAbs are; one clone (F3-3C) was IgG1, six (F1-1B9B, F1-3B, F1-3D, F4-1E, F4-3F, F4- 6C) were IgG2a and one (F1-1B9F) was IgG2b. Seven MAbs (F1-1B9B, F1-1B9F, F1-3B, F1- 3D, F3-3C, F4-1E, F4-3F) reacted with nucleocapsid protein (M.W. 50K) of SEOV by immunoblot. All eight MAbs were cross-reacted with Hantaan (HTN) virus, one (F4-3F) was cross-reacted with Puumala (PUU) virus and two (F1-1B9B, F1-3B) were cross-reacted with Prospect Hill (PH) virus by IFAT. None of these 8 MAbs had neutralizing activity.

Expression of Human Papillomavirus Type 16, Prototype and Natural Variant E7 Proteins using Baculovirus Expression System

Human papillomavirus (HPV) 16, E7 proteins derived from the prototype (Bac73) and natural variant (Bac101) E7 open reading frame were produced in Sf9 insect cells. The variant E7 gene occurred naturally by substitution mutation at the position of 88 nucleotide, resulting serine instead of asparagine. Using E7 specific monoclonal antibody (VD6), both E7 proteins were identified in recombinant baculovirus infected SF9 cells. Radiolabelling and immunoprecipitation analysis revealed that both E7 proteins were phosphoproteins. Immunostaining result showed that E7 proteins were mainly localized in the cytoplasm. Nuclear form of E7 proteins was also detected after a sequential fractionation procedure for removing chromatin structure. Considering that the VD6 recognition site in E7 protein is located within 10 amino acid at the N-terminus, this region appears to be blocked by the nuclear component. Western blot analysis revealed that nuclear form was more abundant than cytoplasmic E7 proteins. Time course immunostaining showed that the primary location of E7 protein was the nucleus and exported to the cytoplasm as proteins were accumulated. These events occurred similarly in both Bac73 and Bac101 infected Sf9 cells, suggesting that these two proteins may have similar biological functions.

Change of the Antigenecity of Human Papillomavirus Type 16 E7 Oncoprotein according to Phosphorylation / 대한암학회지

PURPOSE: It was suggested that immunogenic region of E7 proteins of human papillo- mavirus (HPV) type 16 encompass casein kinase (CK) II phosphorylation site and the resulting negative charge may affect the various biologic function of E7 protein. This study was undertaken to analyze the change of antigenic characteristics of HPV type 16, E7 oncoprotein according to phosphorylation. MATERIALS AND METHODS: We produced two monoclonal antibodies (VD6 and IB10) which showed different reactivities to E7 proteins expressed from bacteria or extracted from CaSki cell. These reaction were analyzed by Western blotting. Also the antigenic sites estimation of these antibodies using nested deletion sets was done. On the basis of above experiments, we performed in vitro phosphorylation assay using CK II and its specific inhibitor, DRB (5, 6-dichloro-l-beta-D-ribofuranosylbenzimidazole), to analyze the IB10 reactivity to E7 oncoproteins according to phosphorylation. RESULTS: In Westem blot analysis, VD6 and IB10 antibodies reacted strongly to bacterially expressed E7 protein. But using E7 extracted from CaSki cell, VD6 reacted to 2.0 kDa E7 protein whereas IB10 showed weak reactivity. The antigenic sites estimation of these antibodies showed that antigenic site of VD6 was located in amino terminal region and that of IB10 in the middle portion in the range of approximate amino acid 25-45. The antigenic site of IB10 might contain the possible phosphorylation sites (Ser-31, 32) in E7. Considering this, the different reactivities of IB10 to E7 proteins expressed in bacteria and extracted from CaSki cell might be due to phosphorylation. In in vitro phosphorylation assay using CK II, the phosphorylation of E7 increased according to reaction time. And this phosphorylation reduced the reactivity of IB10 to E7 protein whereas the reactivity of VD6 did not change. Also the reactivity of IB10 to E7 protein increased in a dose dependent manner with CK II specific inhibitor, DRB treated CaSki cell extracts. CONCLUSION: These result showed the antigenecity is affected by the degree of phosphorylation of E7 protein.

Repair of Cartilage Defect by Transplantation of Cultured Chondrocyte in the Rabbit: Difference related to Size of Defect / 대한정형외과연구학회지

Chondrocytes were harvested from knee joints of immature rabbits and cultured as monolayer until confluence, then split, and grown in collgen gel. The cells were transplanted into osteochondral defect of two different sizes (Group I : 16.1mm2, Group II : 7.1mm2) made on the patellar groove of distal femur in 21 mature rabbits. Left legs were used as experimental group and right leg as control. After 7 weeks, 1 out of 5 in experimental side and none out of 5 in the control side showed cartilaginous repair in Group I and 3 out of 8 in experimental side and 2 out of 8 in control side showed cartilaginous repair in Group II. After 14 weeks, 1 out of 4 in experimental side and none out of 4 in control side showed cartilaginous repair in Group I and 2 out of 4 in experimental side and 2 out of 4 in control side showed cartilaginous repair in Group II.

Identification of Serotypes of Orientia Tsutsugamushi Using Nested PCR / 감염

BACKGROUND: For the early diagnosis of tsutsugamushi disease, polymerase chain reaction (PCR) using skin specimen might be a precise and useful diagnostic tool. The purpose of this study is to detect and identify the serotype of the Orientia tsutsugamushi from the skin lesions of the patients with tsutsugamushi disease by nested PCR. METHODS: Nested PCR was used to diagnose tsutsugamushi disease and identify the serotype of the O. tsutsugamushi; Karp, Kato, Gilliam or Boryong/Kuroki. The primer sets were derived from serotype-specific DNA sequences encoding the 56kDa antigen of O. tsutsugamushi. The PCR products were analyzed by using direct cyclic sequencing. RESULTS: The serotype-specific DNA bands with 1% agarose gel electrophoresis of amplified DNAs by nested PCR were observed in all 11 skin biopsy specimens from 8 patients with tsutsugamushi disease. Among 8 patients, 7 were proved to be infected with Boryong/Kuroki strains and one with Karp. One Karp strain showed one base mutation with amino acid sequence variation, but all the Boryong/ Kuroki strains showed no mutation. CONCLUSION: We suggest that serotype-specific nested PCR is a simple, rapid and precise diagnostic method, and useful for early diagnosis of tsutsugamushi disease. Furthermore, we might detect the sequence variations of serotype-specific DNA sequences encoding 56-kDa antigen among strains.

Enzyme-Linked Immunosorbent Assay Using Fusion Protein for Detection of Serum Antibodies to Human Papillomavirus Type 16 E7 in Patients with Cervical Cancer / 대한산부인과학회잡지

BACKGROUND: Human papillomavirus(HPV) 16 is the most frequently found oncogenic HPV type in cervical cancer and the early protein E7 is considered to be one of the two major proteins involved in malignant transformation and maintenance of the transformed phenotype of the cells. It is suggested that serologic detection of anti-HPV antibody in serum can be used as a marker for HPV-associated cervical cancer. We evaluated the efficacy of enzyme-linked immunosorbent assay(ELISA) method for detecting antibodies circulating in human sera against E7 proteins of HPV 16 in patients with cervical cancer/cervical intraepithelial neoplasia(CIN) and in normal controls. MATERIALS AND METHODS: We have developed a ELISA method using fusion proteins of HPV 16 E7, expressed in E. coli, as antigen to detect the anti-E7 antibody in human sera. Sera from 276 women(90 patients with invasive cervical cancers, 8 patients with CIN III and 178 healthy women) were tested for the presence of antibodies to E7 proteins. The results were compared with that of polymerase chain reaction(PCR) method. RESULTS: Fifteen(16.7 %) of the 90 cervical cancer sera, one(12.5 %) of the 8 CIN sera and twelve (6.7 %) of the 178 normal sera were reactive with E7 proteins(cut-off value : absorbance (A) = 0.079, x + 3 SD). The detection rate of HPV 16 DNA by PCR were 45.5 %(41/90) in cervical cancer patients, 37.5 %(3/8) in CIN III patients, and 11.8 %(21/178) in normal controls Twelve(29.3 %) of 41 cervical cancer patients harboring HPV 16 DNA showed positive for the anti-E7 antobody. Although the positivity with ELISA was rather lower that of PCR. A statistically significant trend of increasing seropositivity was obtained( 2=6.48 ; df=1 ; p=0.011). The concordance rate between the results of ELISA and PCR was 64.4 %. CONCLUSION: The increasing seropositivity for HPV 16 E7 antibodies in association with malignant progression suggest that these antibodies may be a useful marker for HPV 16-associated cervical cancer. But the facts that in only about one-fifth portion of patients with cervical cancer showed the positive results in ELISA restricts the direct clinical applications. A search for more sensitive and specific serologic method for the detection of antibodies to HPV 16 E7 is needed.

Infection Rate and Serotype Distribution of Orientia tsutsugamushi arnong Field Rodents in Chollanamdo using Nested Polymerase Chain Reaction

The 86 strains of field rodents captured in Chollanamdo were analyzed its infection rates of Orientia tsutsugamushi by nested PCR. The detection rate of O. tsutsugamushi was 14.3 % in A. agrarius whereas O. tsutsugamushi could not be detected in C. lasiura. In locality, the rodents captured in the mountainous area showed higher detection rate than suburban area. In the case of detection rate by organs, the spleen was most appropriate specimen, but in two cases, O. tsutsugamushi could be detected in only kidney specimens. The major serotype of detected O. tsutsugamushi was serotype Karp, but four cases were serotype Boryong. These serotypes were confirmed by nucleotide sequence determination of amplified PCR products. In conclusion, the serotype Karp was the major O. tsutsugamushi in Chollanamdo.

Generation of Monoclonal Antibodies Against Human Papillomavirus Type16 E7 Protein : Usefulness for Various E7 Detection Systems

The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.

Genetic Heterogeneity in 56 kDa gene of Orientia tsutsugamushi Genotype Karp

The type-specific PCR and the sequence analysis of 56 kDa gene of Orientia tsutsugamushi infected in field rodents specimens have shown intratypic genetic heterogeneity in genotype Karp. In sequence comparison, this genetic heterogeneity was mainly due to insertion or deletion of a repeated unit in variable domain I (VDI) region. These results suggested that genetic duplication or deletion of the specific sequence rnight be involved in intratypic genetic heterogeneity of Orientia tsutsugamushi.

Retrospective survey of antibody response against borrelia burgdorferi in febrile pateints in 1990

No abstract available.

Seroepidemiological survey of scrub typhus in Korea, 1991

No abstract available.

Significance of IgG and IgM antibodies in the diagnosis of scrub typhus and evaluation of rickettsia tsutsugamushi strain Boryong as a diagnostic antigen

No abstract available.

Significance of IgG and IgM antibodies in the diagnosis of scrub typhus and evaluation of rickettsia tsutsugamushi strain Boryong as a diagnostic antigen

No abstract available.