• BMB Reports
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• v.36 no.6
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• The Journal of Korean Society of Virology
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• v.30 no.2
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• pp.101-112
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• 2000

Rotaviruses are the most common cause of severe vomiting and diarrhea in children worldwide and classified as a genus in the family Reoviridae. Rotavirus has eleven segmented dsRNAs and the virion consists of three shells. Outer capsid VP7 and VP4 induce neutralizing antibodies and are classified into G types (glycoprotein VP7) and P types (protease-sensitive VP4). Characterization of VP7 gene of Korean isolates of human rotavirus was performed using multiplex PCR and nucleotide sequence analysis. After RT-PCR amplification of full length (1,062 bp) of VP7 genes, the amplified PCR products were G typed by multiplex PCR and the nucleotide sequences were compared with those of reference rotavirus from GenBank. The G type analysis revealed that 25% (2/8) belong to G1, whereas 37.5% (3/8) benong to G2 and G4, respectively. The Korean isolates within the same serotypes showed high homology of nucleotide sequences and could be discriminated from foreign isolates exception with two strains (CAU009 and CAU022). But Korean isolates CAU009 and CAU022 were close related into japanease isolates 417 (99.2%) and indian isolates (97.6%) than Korean isolatese. Our results showed that these two strains were supposed to be originated from abroad. As a results, The G typing and nucleotide sequence analysis of VP7 gene of rotavirus isolated from infants in Korea could be used for identification, serotying and determination of novel or unusual strains of rotaviruses.

• Parasites, Hosts and Diseases
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• v.50 no.4
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• pp.287-293
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• 2012

Knowledge of the prevalence of human Toxoplasma gondii infection is required in the Republic of Korea. In this study, we surveyed the seroprevalence of T. gondii infection and analyzed the risk factors associated with seropositivity among residents in 2 administrative districts; Seoul and the island of Jeju-do, which have contrasting epidemiologic characteristics. Sera and blood collected from 2,150 residents (1,114 in Seoul and 1,036 in Jeju-do) were checked for IgG antibody titers using ELISA and for the T. gondii B1 gene using PCR. In addition, participants completed a questionnaire that solicited information on gender, age, occupation, eating habits, history of contact with animals, and travel abroad. The T. gondii B1 gene was not detected in all residents examined. However, ELISA showed 8.0% (89 of 1,114 sera) positive for IgG antibodies against T. gondii in Seoul and 11.3% (117 of 1,036 sera) in Jeju-do. In both districts, the positive rates were higher in males than in females, and those 40-79 years of age showed higher rates than other ages. In Seoul, residents older than 70 years of age showed the highest positive rate, 14.9%, whereas in Jeju-do the highest prevalence, 15.6%, was in those in their sixties. The higher seropositive rate in Jeju-do than in Seoul may be related to eating habits and occupations. The present results and a review of related literature are indicative of an increased seroprevalence of T. gondii in Korea in recent years.

• Parasites, Hosts and Diseases
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• v.51 no.3
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• pp.363-367
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• 2013

The prevalence of Toxoplasma gondii infection in birds has epidemiological significance because birds are indeed considered as a good indicator of environmental contamination by T. gondii oocysts. In this study, the prevalence of T. gondii in 313 house sparrows in Lanzhou, northwestern China was assayed by the modified agglutination test (MAT). Antibodies to T. gondii were positive in 39 (12.46%) of 313 samples (MAT titer ${\geq}$ 1:5). Tissues of heart, brain, and lung from the 39 seropositive house sparrows were tested for T. gondii DNA, 11 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 9 genetic markers, including 8 nuclear loci, i.e., SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8 and an apicoplast locus Apico. Of them, 4 isolates were genotyped with complete data for all loci, and 2 genotypes (Type II variants; ToxoDB #3 and a new genotype) were identified. These results showed that there is a potential risk for human infection with T. gondii in this region. To our knowledge, this is the first report of T. gondii seroprevalence in house sparrows in China.

• Korean Journal of Veterinary Research
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• v.39 no.2
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• pp.247-256
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• 1999

A disease of young Holstein calves characterized by recurrent pneumonia, ulcerative and granulomatous stomatitis, enteritis with bacterial overgrowth, periodontitis, delayed wound healing, persistent neutrophilia and death at an early age had been originally described in 1983 and again in 1987. Most of these calves had stunted growth and a persistent, progressive neutrophilia (often exceeding 100,000/ml). By investigation of pedigrees, all of the affected calves have now been traced to a common sire and confirmed by polymerase chain reaction (PCR) diagnostic DNA testing to be homozygous carriers of a defective allele for bovine CD18. Neutrophils from these calves have several functional deficits and, most importantly, fail to adhere in a ${\beta}_2$-integrin dependent manner. The ${\beta}_2$-integrins represent a family of glycoproteins which participate in various leukocyte adhesion reactions during host defense. The presence or absence of ${\beta}_2$-integrin molecules can be demonstrated on the surface of neutrophils, monocytes and lymphocytes from normal or affected calves using specific monoclonal antibodies and flow cytometry, or by colloidal gold immunolabeling and scanning electron microscopy in backscatter mode. Deficiency of the ${\beta}_2$-integrins on all leukocyte types in Holstein calves is analogous to leukocyte adhesion deficiency (LAD) seen in humans. Neutrophils in bovine (BLAD) and human LAD patients are unable to adhere to the endothelial lining of the cardiovascular system thus interrupting egression of neutrophils into infected tissues. Other leukocytes, while still deficient in expression of the ${\beta}_2$-integrins, are still able to efficiently egress from the blood stream due to interactions of other adhesion molecules that are not as highly expressed on neutrophils. Both BLAD cattle and LAD children (who do not receive bone marrow transplants) often die at an early age as a result of the failure of neutrophils to extravasate into infected tissues. In 1991, Shuster, et $al^{27}$, identified two point mutations within the alleles encoding bovine CD18 in a Holstein calf afflicted with leukocyte adhesion deficiency. One mutation causes an aspartic acid to glycine substitution at amino acid 128 (D128G) in an extracellular region of this adhesion glycoprotein that is highly conserved (> 95% identity) between humans, cattle and mice. The other mutation is silent. Numerous calves with clinical symptoms of leukocyte adhesion deficiency have since been tested and all have been found homozygous for the D128G allele. In addition, calves homozygous far the D128G allele have been identified during widespread DNA testing in the United States. All cattle with the mutant allele are related to one bull, who through artificial insemination (A.I.), sired many calves in the 1950's and 1960's. The carrier frequency of the D128G CD18 allele among U.S. Holstein cattle had reached approximately 15% among active A.I. bulls and 8% among cows. By 1993, the organization of the dairy industry and the diagnostic test developed to genotype cattle, enabled virtually complete eradication of bovine leukocyte adhesion deficiency among current and future A.I. bulls.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.3
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• pp.105-111
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• 2015

In late December 2013, the Ebola virus emerged from West Africa. The outbreak started in Guinea and rapidly spread to Liberia and Sierra Leone. Initially, the virus is spread to the human population after contact with infected wildlife and then spread person-to-person through direct contact with body fluids such as blood, sweat, urine, semen, and breast milk. The Ebola virus infects endothelial cells, mononuclear phagocytes and hepatocytes. It causes massive damage to internal tissues and organs, such as blood vessels and the liver, and ultimately death. Most tests for the virus RNA rely on a technology called reverse-transcriptase polymerase chain reaction (RT-PCR). While this method is highly sensitive, it is also expensive, requiring skilled scientists, and delicate power supplies. The strip analytical technique (enzyme-linked immunosorbent assay or ELISA) detects antigens or antibodies to the Ebola virus. This test is cheap and does not require electricity or refrigeration. Despite ongoing efforts directed at experimental treatments and vaccine development, current medical work on the Ebola viral disease is largely limited to supportive therapy. Thus, rapid and reliable diagnoses of the Ebola virus are critically important for patient management, infections, prevention, and control measures.

• Journal of Life Science
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• v.21 no.11
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• pp.1565-1572
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• 2011

Eye-specific lactate dehydrogenase (EC 1.1.1.27, LDH) $C_4$ isozyme in the eyes of greenlings (Hexagrammos otakii) was successfully purified by affinity chromatography and continuous-elution electrophoresis. The molecular weight of the purified eye-specific LDH $C_4$ isozyme was 154.8 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal pH for enzymatic reaction of the eye-specific LDH $C_4$ isozyme was pH 8.5. $K^{PYR}_m$ value of the purified eye-specific LDH $C_4$ isozyme was $1.88{\times}10^{-5}$ M using pyruvate as a substrate. These results indicate that we must consider pH when measuring eye-specific LDH $C_4$ isozyme activity. The eye-specific LDH $C_4$ isozyme had a higher binding affinity for the substrate as a pyruvate than LDH A4 isozyme. Antibodies produced against the purified eye-specific LDH $C_4$ isozyme may be used in the diagnosis of several human diseases and in comparative physiological studies of fishes.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.1
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• pp.19-26
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• 2006

Objective: In order to elucidate the retrogressive degeneration of orofacial cleft, the fissured tissues of prenatal and postnatal cleft lip and palate were examined by histological and immunohistochemical methods. Design: Totally 42 cases of prenatal (n=17) and postnatal (n=25) cleft lip and/or palate were examined in comparison with 10 cases of normal lip and oral mucosa using immunohistochemical stainings of MMP-3, MMP-9, MMP-10, cathepsin G, PCNA, E-cadherin, TGase 2, HSP-70, vWF, and VEGF. Main Outcome Measures: In the fissured tissue the sebaceous glands were strongly positive for PCNA and grew into the underlying fibromuscular tissue (24/42). Some hyperplastic sebaceous glands of prenatal cleft lip produced infundibular follicular cyst (9/17). The skin and mucosal epithelia from the postnatal cleft lip and palate (10/25) showed severe basal hyperplasia (11/25) and melanocyte infiltration (7/25). Results: The immunostaining of MMP-3 and HSP-70 were strongly positive in the hyperplastic sebaceous glands and nearby atrophying muscle bundles of the fissured tissue, while MMP-9, MMP-10, and cathepsin G were almost negative. The immunoreactions of the other antibodies used in this study were similar between in the fissured tissues and in the normal controls. Conclusions: These data suggest that the over-expression of MMP-3 is closely related to the sebaceous gland hyperplasia, epithelial dysplasia, and the muscle degeneration, and that the over-expression of MMP-3 in the fissured tissue may continuously aggravate the cleft condition in the later life.

• Journal of Periodontal and Implant Science
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• v.31 no.4
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• pp.661-668
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• 2001

Anti-P. gingivalis immune sera were obtained from mice immunized with either P. gingivalis alone, or F. nucleaturm followed by P. gingivalis. Two groups of immune sera were examined for binding capacity to P. gingivalis biofilm by confocal laser scanning microscope, Antibody avidity index was also determined for each immune sera. The results indicated that prior immunization of mice with F. nucleaturm impaired P. gingivalis-specific immune sera in binding capacity to biofilm and antibody avidity to P. gingivalis. Elevated antibody responses in patients with destructive periodontal disease has often been related to suboptimal level of protective antibody $(opsonophagocytosis)^{1-3)}$ while post-immune sera obtained with experimental animals using a single periodontal pathogen demonstrated satisfactory levels of protective function against the homologous bacterial $challenge^{4,5)}$.The reason is unclear why elevated IgG responses in periodontal patients to periodontal pathogens do not necessarily reflect their protective function. Such an immune deviation might be derived from the fact that destructive periodontal disease is cumulative result of immunopathologic processes responding to an array of different colonizing microorganisms sequentially infecting in the subgingival environmental niche. Fusobacterium nucleaturm is one of the key pathogens in gingivitis, in the transitional phase of conversion of gingivitis into destructive periodontitk, and in adult $periodontitis^{6-8)}$. It also plays a central role in coaggregation with other important microbial species in subgingival $area^{6,9,10)}$ as well as in $biofilm^{11)}$, especially with Porphyromonas gingjvalis in synergism of virulence in human periodontal disease or in animal $models^{12-14)}$. This organism has also been reported to have immune modulating activity for secondary immune response to Actinobacillus $actinomycetemcomitans^{15)}$. It is presumed that sequential colonization and intermicrobial coaggregation between intermediate and late colonizers could potentially modulate the immune responses and development of specific T cell phenotypes in periodontal lesions. We have recently demonstrated the skewed polarization of P. gingivalis-specific helper T cell clones in mice immunized with F. nucleaturm followed by P. $gingivalis.^{16)}$. Consequently F. nucleaturm may initially prime the immune cells and modify their responses to the successive organism, P. gingivalis. This could explain why one frequently observes non-protective serum antibodies to P. gingivalis in periodontal patients in contrast with those obtained from animals that were immunized with $P.gingivalis\;alone^{17)}$. The present study was performed to investigate the immune modulating effect of F. nucleatum on serum binding to experimental biofilms and the avidity of anti-P. gingivalis antibody.

• Animal cells and systems
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• v.1 no.1
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• pp.157-164
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• 1997

Ly-6E.1 antigen was proposed as a regulatory molecule of T lymphocyte activation, a hematopoietic stem cell marker, a memory cell marker, and an adhesion molecule. Though there were several reports suggesting the presence of Ly-6 ligand, the characterization of the ligand was not yet performed, As an attempt to screen the expression of Ly-6E.1 ligand, we prepared a probe for detecting Ly-6E.1 ligand by producing a fusion protein between Ly-6E.1 and $hlgC_{r1}$, A mammalian cell expression vector with Ly-6E.$1/hlgC_{r1}$ chimeric cDNA was transfected in SP2/0-Ag14 myeloma cells, and stable transfectants were selected. The fusion protein was produced as a dimer and maintained the epitopes for monoclonal antibodies specific for Ly-6E.1 and for anti-human lgG antibody. The purified fusion protein through Gammabind G column was used for FACS analyses for the expression of Ly-6E.1 ligand. The fusion protein interacted with several cell lines originating from B cells, T cells, or monocytes. The fusion Protein also strongly stained bone marrow, lymph node, and spleen cells, but thymic cells weakly, if any. The staining was more obvious in C57BL/6 $(Ly-6^b)$ than Balb/c $(Ly-6^a)$ mice. These results suggest that the interaction of Ly-6E.1 with Ly-6E.1 ligand may function both in the stem cell environment and in the activation of mature lymphocytes. The fusion protein may be a valuable tool in characterization of biochemical properties of the Ly-6E.1 ligand and, further, in isolating its cDNA.