• Biomedical Science Letters
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• v.3 no.2
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• pp.107-114
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• 1997

Recent reports indicate that the clinical usefulness of prostate specific antigen (PSA), particulary in the differentiation of benign prostate hyperplasia from prostate cancer, can be improved by measuring the amount of free PSA in serum. Measuring free PSA is especially useful in attempts to improve diagnositc performance of PSA in the diagnostic gray zone of total PSA. The objective of this study was to develop free PSA assay kit using sandwich microplate enzyme immunoassay format. We chose a test format with polyclonal anti-PSA antibodies coated on the wells and monoclonal anti-free PSA antibodies for quantification to gain higher test sensitivity. We adpoted 10 uL of specimen and 2 hours of first incubation time with detecting antibody for free PSA EIA format using microplate. The within-day and between-day precision (%CV) in the high and low concentration ranges were below 4%. The correlation coefficient between in-house free PSA assay and commercial assay kit was r=0.9965 (slope=0.0984, y intercept=0.0173, N=27). No hook effect was found by 40 ng/mL and correlation coefficient (r) value of the fitted linear regression was over 0.995. The recovery tests were in the range of 98.9∼104.1％ for free PSA. In conclusion, in-house free PSA enzyme immune assay is cost effective, simple and rapid and could be useful for the prognosis after theraphy as well as for the differential diagnosis between prostate cancer and benign prostate hyperplasia.

• Journal of Veterinary Clinics
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• v.28 no.2
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• pp.183-189
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• 2011

The objective of this study was to examine the effect of fucoidan on the phagocytic capapcity and oxidative burst activity (OBA) of canine peripheral blood polymorphonuclear cells (PMNs). The phagocytic capacity and OBA of PMNs were evaluated simultaneously by using a flow cytometer. Fucoidan itself did not cause any direct effect on the phagocytic capacity and OBA of PMNs. However, the phagocytic capacity and OBA of PMNs were enhanced by the culture supernatant from PBMCs treated with fucoidan. The phagocytic capacity and OBA of PMNs were also increased by treatment with recombinant canine (rc) tumor necrosis factor (TNF)-${\alpha}$. The ability of the culture supernatant from fucoidan-treated PBMCs to stimulate the phagocytic capacity and OBA of PMNs was inhibited by addition of anti-rc TNF-${\alpha}$ polyclonal antibody (PAb) prior to the culture. The amount of TNF-${\alpha}$ in the culture supematant from PBMCs was shown to increase upon treatment of fucoidan as compared with that of vehicle-treated PBMCs culture supematant. The level of TNF-${\alpha}$ mRNA expression in PBMCs was also up-regulated by the fucoidan treatment. These results suggest that fucoidan has an immunoenhancing effect on the phagocytic capacity and OBA of canine PMNs, which is mainly mediated by TNF-${\alpha}$ released from fucoidan-stimulated PBMCs.

• Journal of Life Science
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• v.18 no.3
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• pp.409-413
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• 2008

The occurences of proteins relating to Antheraea pernyi arylphorin in haemolymph, fat body, integument, midgut and silkgland of the wild silkmoths, Antheraea yamamai, Antheraea pernyi, Samia cynthia pryeri and Actias gnoma in the 5th larval instar were investigated by immunoblot analysis using mouse polyclonal antibody against A. pernyi arylphorin as probe. In A. yamamai, A. pernyi, S. cynthia pryeri and A. gnoma, the major immunoreactive antigenic proteins with a molecular weight of 80 KDa against the antisera of the A. pernyi arylphorin were clearly observed in the haemolymph, but in the integument, fat body, midgut and silkgland of the corresponding wild silkmoths the presence of the immunoreactive proteins were very variable. These results suggest that the A. pernyi arylphorin has almost same immunological identity with those of the wild silkmoths, A. yamamai, S. cynthia pryeri and A. gnoma though the distribution of the corresponding antigenic arylphorins is different according to the tissues of the wild silkmoths.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1583-1590
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• 2006

T4 endonuclease V (T4 endo V) [EC 3. 1. 25. 1], found in bacteriophage T4, is responsible for excision repair of damaged DNA. The enzyme possesses two activities: a cyclobutane pyrimidine dimer DNA glycosylase (CPD glycosylase) and an apyrimidic/apurinic endonuclease (AP lyase). T4 denV (414 bp cDNA) encoding T4 en do V (138 amino acid) was synthesized and expressed using either an expression vector, pTriEx-4, in E. coli or a baculovirus AcNPV vector, pBacPAK8, in insect cells. The recombinant His-Tag/T4 endo V (rHis-Tag/T4 endo V) protein expressed from bacteria was purified using one-step affinity chromatography with a HiTrap Chelating HP column and used to make rabbit anti-His-Tag/T4 endo V polyclonal antibody for detection of recombinant T4 endo V (rT4 endo V) expressed in insect cells. In the meantime, the recombinant baculovirus was obtained by cotransfection of BacPAK6 viral DNA and pBP/T4 endo V in Spodoptera frugiperda (Sf21) insect cells, and used to infect Sf21 cells to overexpress T4 endo V protein. The level of rT4 endo V protein expressed in Sf21 cells was optimized by varying the virus titers and time course of infection. The optimal expression condition was set as follows; infection of the cells at a MOI of 10 and harvest at 96 h post-infection. Under these conditions, we estimated the amount of rT4 endo V produced in the baculovirus expression vector system to be 125 mg/l. The rT4 endo V was purified to homogeneity by a rapid procedure, consisting of ion-exchange, affinity, and reversed phase chromatographies, based on FPLC. The rT4 endo V positively reacted to an antiserum made against rHis-Tag/T4 endo V and showed a residual nicking activity against CPD-containing DNA caused by UV. This is the first report to have T4 endo V expressed in an insect system to exclude the toxic effect of a bacterial expression system, retaining enzymatic activity.

• Applied Biological Chemistry
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• v.38 no.1
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• pp.55-62
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• 1995

To understand the molecular structure of Korean garlic viruses, cDNA cloning of virus genomic RNA was attempted. Virus particles were isolated from virus-infected garlic leaves and a cDNA library was constructed from garlic virus RNA. One of these clones, S81, selected by random sequencing has been identified as a member of potexvirus group other than potyvirus and carlavirus. The clone is 873 bp long contains most of the coat protein (CP) coding region and 3'-noncoding region including poly(A) tail. A putative polyadenylation signal sequence (AAUAAA) and the hexanucleotide motif (ACUUAA), a replicational cis-acting element conserved in the 3'-noncoding region of potexvirus RNAs are noticed. The clone S81 shows about 30-40% identity in both nucleotide and amino acid sequences with CPs of potexviruses. The genome size of the virus was analysed to be 7.46 knt by Northern blot analysis, which was longer than those of other potexviruses. The open reading frame encoding CP was expressed as a fusion protein (S81CP) in Escherichia coli and the recombinant protein was purified by immobilized metal binding affinity chromatography. Polyclonal antibody was raised against S81CP in rabbit to examine the occurrence of garlic potexvirus in Korean garlic plants by immunoblot analysis. Two virus protein bands of Mr 27,000 and 29,000 from garlic leaf extract of various cultivars reacted with the antibody. It was shown that Mr 27,000 band might not be a degradation product of Mr 29,000 band, suggesting that two types of potexvirus different in size of coat protein could exist in Korean garlic plants.

• Tuberculosis and Respiratory Diseases
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• v.43 no.3
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• pp.420-433
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• 1996

Background : There have been many debates about the effects of nitric oxide on the neurogenic inflammation. The role of nitric oxide in the neurogenic inflammation of airways will be required a better understanding of the localization and types of nitirc oxide synthase(NOS) activity in the neurogenic inflammation of airways. Method : To investigate the role of nitric oxide in airway neurogenic inflammation, 1) the effects of neurokinin receptor antagonist (FK224) and nitric oxide synthase inhibitor, $N^{\omega}$-nitro-L-arginine (L-NNA) on plasma extravastion were evaluated in four groups of Sprague-Dawley rats ; sham operation group(sham NANC group), electrical vagal stimulation group(NANC2 group), intravenous pretreatment groups with FK224 (1mg/kg ; FK224 group), and L-NNA(1mg/kg ; L-NNA group) 15 minutes before vagal NANC stimulation. 2) NOS activity in trachea with neurogenic inflammation was localized by immunohistochemical stain. Immunohistochemical stain was performed by antibodies specific for inflammatory cells(iNOS), brain(bNOS), and endothelium (eNOS) on trachea obtained from sham NANC, NANC2, and FK224 groups. Results : The results are that plasma extravsation in neurogenic inflammation of rat airways was inhibited by FK224, but enhanced by L-NNA pretreatment(P<0.05). There was significantly increased infiltration of inflammatory cells in subepithelium of neurogenic inflammatory trachea, but the reduction of subepithelial infiltration of inflammatory cells was observed after pretreatment with FK224(P<0.05). Immunostaining with anti-iNOS antibody showed strong reactivity only in infiltrated inflammatory cells in neurogenic rat trachea, and these iNOS reactivity was reduced by pretreatment with FK224. bNOS immunoreactivity was significantly increased only in the nerves both of neurogenic inflammatory and FK224 pretreated trachea compared with sham NANC trachea(p<0.05). eNOS immunoreactivity was not significant change in endothelium in neurogenic inflammation of rat trachea. Conclusion : These results suggest that nitric oxide released from iNOS in infiltrated inflammatory cells has main role in neurogenic inflammation of rat trachea. The presence of bNOS immunoreactivity in the nerves indicates that nitric oxide may be released from the nerves in rat trachea with neurogenic inflammation.

• Parasites, Hosts and Diseases
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• v.25 no.2
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• pp.159-167
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• 1987

To analyse the antigen specificity of patients sera from 24 confirmed neurocysticercosis and a monoclonal antibody, SDS-PAGE using 10~15% linear gradient gel and EITB were done. Cystic fluid, saline extracts of scolex and of whole worm of C. cellulosae, saline extracts of sparganum, hydatid cyst fluid, saline extracts of Fasciola, Clonorchis and Paragonimus were used as antigen. Of protein bands in cystic fluid of C. cellulosae, patient sera reacted frequently to bands of 152, 94, 64, 48, 24, 15, 10 and 7kDa proteins. To saline extracts of scolex and whole worm of C. cellulosae, patients sera reacted frequently to 94, 64, 52, 39, 34, 15 and 10kDa bands. Two bands in sparganum extract (130 and 64kDa) and two bands in hydatid cyst fluid (52 and 27kDa) were cross-reacting bands with sera from cysticercosis patients. Saline extracts of Fasciola, ClonorchiJ and Paragonimus did 'not exhibit cross-reacting bands. Monoclonal antibody to cystic fluid of C. cellulosae was found to react with low molecular weight proteins of 15, 10 and 7kDa.

• Tuberculosis and Respiratory Diseases
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• v.59 no.2
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• pp.142-150
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• 2005

Background : Continuous growth stimulation by various factors, as well as chronic oxidative stress, may co-exist in many solid tumors, such as lung cancer. A new family of antioxidant proteins, the peroxiredoxins (Prxs), have been implicated in the regulation of many cellular processes, including cell proliferation, differentiation and apoptosis. However, a real pathophysiological significance of Prx proteins, especially in lung disease, has not been sufficiently defined. Therefore, this study was conducted to investigate the distribution and expression of various Prx isoforms in lung cancer and other pulmonary conditions. Method : Patients diagnosed with lung cancer, and who underwent surgery at the Ajou Medical Center, were enrolled. The expressions of Prxs, Thioredoxin (Trx) and Thioredoxin reductase (TR) were analyzed using proteomic techniques and the subcellular localization of Prx proteins was studied using immunohistochemistry on normal mouse lung tissue. Result : Immunohistochemical staining has shown the isoforms of Prx I, II, III and V are predominantly expressed in bronchial and alveolar lining epithelia, as well as in the alveolar macrophages of the normal mouse lung. The isoforms of Prx I and III, and thioredoxin were also found to be over-expressed in the lung cancer tissues compared to their paired normal lung controls. There was also an increased amount of the oxidized form of Prx I, as well as a putative truncated form of Prx III, in the lung cancer samples when analyzed using 2-dimensional electrophoresis. In addition, a 43 kDa intermediate molecular weight protein band, and other high molecular weight bands of over 20 kDa, recognized by the anti-Prx I antibody, were present in the tissue extracts of lung cancer patients on 1-Dimensional electrophoresis, which require further investigation. Conclusion : The over-expressions of Prx I and III, and Trx in human lung cancer tissue, as well as their possible chaperoning function, may represent an attempt by tumor cells to adjust to their microenvironment in a manner advantageous to their survival and proliferation, while maintaining their malignant potential.

• Tuberculosis and Respiratory Diseases
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• v.60 no.5
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• pp.523-531
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• 2006

Background : Cervical tuberculous lymphadenopathy is a very common disease with a similar incidence to pulmonary tuberculosis. Dendritic cells play a role of initial antigen presentation of this illness. Nevertheless, the precise role of these antigen-presenting cells according to the clinical features in unclear. The aim of this study was to determine the clinical implication of dendritic cell infiltration in the cervical lymph nodes. Methods : A review of the clinical characteristics was carried out retrospectively based on the clinical records and radiography. Immunohistochemical staining was performed on the available histology specimens of 72 cases using the S-100b polyclonal antibody for dendritic cells. The number of dendritic cells with tuberculous granuloma were determined. A $X^2$ test, unpaired T test and multiple logistic regression analysis were performed. Results : Thirty percent of subjects had previous or concurrent pulmonary TB. Twenty one percent of cases showed a positive reaction on the AFB stain. Within a granuloma, the number of infiltrated dendritic cells was $113.0{\pm}7.0$. The incidence of fever and cough decreased with increasing infiltration of dendritic cells Multivariate regression analysis showed that the infiltration of dendritic cells could significantly contribute to fever. Conclusion : Overall, dendritic cells can control a Mycobacterium tuberculosis infection and modulate the immune response, as well as resolve the clinical manifestations of TB lymphadenopathy.

• Clinical and Experimental Pediatrics
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• v.46 no.7
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• pp.695-701
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• 2003

Purpose : It had been suggested that pain arising from deep somatic body regions influences neural activity within periaqueductal gray(PAG) of midbrain via distinct spinal pathways. Aspirin is one of the popular non-steroidal anti-inflammatory drugs used in the management of pain. Fos expression was used as a marker for neuronal activity throughout central neurons following painful peripheral stimulation. This study was prepared to investigate changes of c-Fos immunoreactivity in midbrain by deep pain and effects of aspirin. Methods : Male Sprague-Dawley rats were injected with 0.1 mL of 5% formalin in the plantar muscle of the right hindpaw. For experimental group II, aspirin was injected intravenously before injection of formalin. An aspirin-untreated group was utilized as group I. Rats were sacrificed at 0.5, 1, 2, 6 and 24 hours after formalin injection. Rat's brains were removed and sliced in rat brain matrix. Brain slices were coronally sectioned at interaural 1.00-1.36 mm. Serial sections were immunohistochemically reacted with polyclonal c-Fos antibody. The numbers of c-Fos protein immunoreactive neurons in ventrolateral periaqueductal gray(VLPAG) and dorsomedial periaqueductal gray(DMPAG) were counted and analyzed statistically with Mann-Whitney U tests. Results : Higher numbers of c-Fos protein immunoreactive neurons were found in VLPAG. In both VLPAG and DMPAG of formalin-treated group, the numbers of c-Fos protein immunoreactive neurons were significantly higher at all time points than the formalin-untreated group, which peaked at two hours. The numbers of c-Fos immunoreactive neuron of the aspirin-treated group were less compared to the aspirin-untreated group at each time point. Conclusion : These results provide some basic knowledge in understanding the mechanism of formalin-induced deep somatic pain and the effects of aspirin.