• IMMUNE NETWORK
• /
• v.3 no.4
• /
• pp.302-309
• /
• 2003

Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-$dhfr^-$ cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.3
• /
• pp.941-945
• /
• 2015

Semaphoring is a transmembrane receptor which participates in many cytokine-mediated signal pathways that are closely related to the angiogenesis, occurrence and development of carcinoma. The present study was designed to access the effect of mono-antibody (mAb) guided radioimmunotherapy (RIT) on skin carcinoma and investigate the potential mechanisms. Semaphoring mAb was acquired from mice (Balb/c), purified with rProtein A column; purity, concentration and activity were tested with SDS-PAGE and indirect ELISA; specificity and expression on the cutanuem carcinoma line and tissue were tested by Western blotting; morphology change was assessed by microscopy. MTT assay and colony inhibition tests were carried out to test the influence on the proliferation of tumor cells; Western blotting was also carried out for expression of apoptosis-associated (caspase-3, Bax, Bcl-2) and proliferation-related (PI3K, p-Akt, Akt, p-ERK1/2, ERK1/2) proteins and analyse the change in signal pathways (PI3K/Akt and MEK/ERK). The purity of purified semaphorin mAb was 96.5% and the titer is about $1{\times}10^6$. Western blotting showed semaphoring mAb to have specifically binding stripes with semaphoring b1b2 protein, B16F10, and A431 cells at 39KDa, 100KDa and 130KDa, respectively. Positive expression was detected both in cutanuem carcinoma line and tissue and it mostly located in cell membranes. MMT assay revealed dose-relate and time-relate inhibitory effect of semaphorin mAb on A431 and B16F10. Colony inhibition tests also showed dose-relate inhibitory effects. Western blotting demonstrated the expression of apoptosis and proliferation-related protein and changes in signal pathway. In conclusion, we demonstrated that semaphorin is highly expressed on the tumor cell-surfaces and RIT with semaphorin mAb has effect in i nhibiting proliferation and accelerating apoptosis of tumor cells.

• Restorative Dentistry and Endodontics
• /
• v.25 no.4
• /
• pp.587-598
• /
• 2000

Urea in the oral cavity is hydrolyzed mainly by bacterial ureases to ammonia, which in turn, raises pH of the oral environment, maintaining oral pH homeostasis, thereby inhibiting dental caries. Streptococcus salivarius has been shown to be a major contribution to oral ureolysis. Synthesis of urease by S. salivarius appears to be constitutive, but can be greatly enhanced in the acidic environment. It has been presumed that ureolytic activity of S. salivarius strains isolated from caries-active site is greater than that of strains from caries-free site. However, no in vivo study has supported the presumption. The present study was performed to observe the ureolytic activity of S. salivarius strains isolated from different environments in the same individual, finding out whether the ureolytic activity is related to dental caries. For the purpose, S. salivarius strains were isolated from caries-active site (>C2), a caries-free site of the tooth, and the dorsum of the tongue of each of 50 patients having decayed teeth. The strains isolated from the patients who harbored S. salivarius in more than two sites were selected and then their ureolytic activities were measured. In order to examine clonal diversity of the strains, their ureC genes were amplified by polymerase chain reaction (PCR) and then restricted with EcoRV, and the protein profiles of the strains were compared by SDS-PAGE. The results were as follows: 1. Of 50 patients, 13 patients harbored S. salivarius in more than two sites; a total of 61 S. salivarius strain were isolated from the patients and selected for the study. 2. Of 17 isolates from the caries-active site of 9 patients harboring S. salivarius in more than two sites including carious lesion, 10 (58.8%) showed a high ureolytic activity (> 200 ${\mu}mol/min/mg$). While, 19 out of 44 isolates (43.2%) from the caries-free site of the teeth and the dorsum of the tongues of 13 patients were the strains with a high ureolytic activity. 3. Of 9 patients harboring S. salivarius in more than two sites including caries-active site. 6 patients were found to have the strains in the caries-active site showing a lower ureolytic activity than the strains in the other sites. 4. Of 34 isolates with ureolytic activity higher than 40 ${\mu}mol/min/mg$, 32 isolates produced 0.54-Kbp PCR products regardless of the sites of bacterial collection. In contrast, of 27 isolates with ureolytic activity lower than 40${\mu}mol/min/mg$, 26 isolates yielded 1.3-Kbp PCR products or none regardless of the sites. 5. Different clonal types of S. salivarius with relatively higher and lower ureolytic activities were found in the same individuals and even in the same sites. 6. None of strains showing different ureolytic activity appeared to be the same clonal type. The overall results suggest that ureolytic activity of the isolates does not appear to be related to differences of the environments but related to their own genetic traits.

• Microbiology and Biotechnology Letters
• /
• v.31 no.2
• /
• pp.103-110
• /
• 2003

The regulation of pyrimidine nucleotide synthesis has been proved to be controlled by a regulatory protein PyrR-mediated attenuation in the Gram-positive bacteria. After several bacterial genome sequencing projects, we have discovered the PyrR orthologues in the databases for Haemophilus influenzae and Synechocystis and sp. PCC6803 genome sequences. To investigate whether these PyrR orthologue proteins regulate pyrimidine nucleotide synthesis as well as the cases of Bacillus, the PyrR regions of each strains were amplified by PCR and cloned with pUC19 or T-vector in Escherichia coli and with a shuttle vector pHPS9 for E. coli and B. subtilis. For the regulation test of the PyrR orthologues, the aspartate-transcarbamylase (ATCase) assay was carried out. From the results of the ATCase assay, it was confirmed that Synechocystis sp. PCC6803 could not restore by pyrimidines to a B. subtilis, PyrR but H. influenzae PyrR could. For Purification of PyrR orthologue proteins, PyrR orthologue genes were cloned into the expression vector (pET14b). Over-expressed product of PyrR orthologue genes was purified and analyzed by the SDS-PACE. The purified PyrR orthologue proteins from H. influenzae and Synechocystis sp. PCC6803 turned out to be molecular mass of 18 kDa and 21 kDa, respectively. The result of uracil phosphoribosyl transferase (UPRTase) assay with purified PyrR orthologue proteins showed that H. influenzae PyrR protein only has UPRTase activity. In addition, we could predict several regulatory mechanisms that PyrR orthologue proteins regulate pyrimidine de novo synthesis in bacteria, through phylogenetic analysis for PyrR orthologue protein sequences.

• Journal of the Daesoon Academy of Sciences
• /
• v.25_1
• /
• pp.25-85
• /
• 2015

In this study, source criticism (an establishment of authentic text) of the Hyun-Mu Sutra(玄武經) among different editions is studied and an attempt of a new interpretation appropriate to that is attempted. The Hyun-Mu Sutra, a scripture written in 1909, began to communicate with the world through the religions of Jeungsanism. In particular, it was remarkable that The Hyun-Mu Sutra was absorbed as canon textbooks Jeonkyung(典經), the Scriptures of Daesoonjinrihoe, The Fellowship of Daesoon Truth(大巡眞理) from a loner and secret pull-out of heritage traditions. However, this scripture though written in 1909 and more than 100 years has passed, remained in a state unestablished authentic text. The Hyun-Mu Sutra is the scripture consisted of 25 pages by the religions of Jeungsanism[Gang Il-sun 姜一淳(1871~1909)]. 33 page type of Hyun-Mu Sutra has been distributed in the world until now the authentic text of The Hyun-Mu Sutra. However, as a result of the examination, diagnostic scripture(病勢文) was found to have been added by descendants. After a review of authentic text of The Hyun-Mu Sutra, it concluded that there is no diagnostic scripture in primary The Hyun-Mu Sutra. Though The Hyun-Mu Sutra is a booklet of a small amount, the notation and expression is so unique, it has been in secrecy to read its contents. Interpretation way of The Hyun-Mu Sutra up to now can be summarized in two as follows. 1) approaches by I-ching 2) approaches by ten celestrial stemps and twelve earthly branches(10干12支). Approaches by I-ching among this sometimes was supplemented with Buddhist classification methods. Nevertheless, these studies can be evaluated limited because it fails to secure authentic text of The Hyun-Mu Sutra. In this study, the contents of The Hyun-Mu Sutra was examined itemized by focusing on the following four points. 1) The icon of The Hyun-Mu Sutra(玄武經符) is similar as normal talisman(符籍) but it has other features. 2) 'Reverse Fonts'(反書體)[the opposite view of the standard fonts(正書體), reflected in the mirror fonts] and size or location used in text is not in uniform. 3) letters in scripture were pointed and points were stamped in the left and upper and lower characters. 4) "Spiritual poem" (詠歌, the Korean traditional music with a view of elegance as an origin of eco), and the music with the Five-Sounds[宮Gung, 商Sang, 角Gak, 徵Chi, 羽Wu) were related. As a result, content analysis of The Hyun-Mu Sutra is carried out in the next four points. 1) The icon of The Hyun-Mu Sutra (玄武經符) has been primarily developed by Jeungsan. 2) 'Reverse Fonts'(反書體)[the opposite view of the standard fonts(正書體), reflected in the mirror fonts] and reverse location such as '宙宇' [the reverse of '宇宙'] represents based on a new world based on a forward and reverse I-ching(正易). 3) Dot and neighbor points is a symbolic map that guides the position of lateral new world(後天) and era(人尊) 4) Spiritual poem is the entrance to achieve the Realization of Do(道通). The above can be considered as the results of this study.

• 한국노년학
• /
• v.32 no.2
• /
• pp.559-576
• /
• 2012

This research aims to address the effect of Guided Autobiography(GAB) on ego-integrity, depression and life-satisfaction in the elderly and to investigate psychological changes and experiences on the senior subject. 20 subjects participated weekly autobiography writing sessions in a senior academy in the S city for 13 weeks. In this research, we carried out examination on ego-integrity, depression test, and life-satisfaction survey were performed before and after the writing sessions for a quantitative analysis, which was later investigated through a 'corresponding sample T-test'. Based on the results of the above mentioned tests, the qualitative analysis was conducted through an individual sessions with 5 selected participants. The results of this research are summarized as following. First, ego-integrity showed satisfactorily meaningful difference between pre- and post-GAB writing. The participants recollected the past repent and revisited unresolved issues in their lives. The subjects were able to accept these past misdemeanors and appreciate their lives. GAB indeed helped improving ego-integrity. Second, the hypothesis that GAB writing will help decrease depression was accepted. 2-page weekly writing assignments enabled the participants think of joyful moments in their past, and showed decrease the symptoms of depression. Third, this study revealed that GAB writing improved life- satisfaction. The participants learned to express gratitude and peace in their mind. The happy feeling and optimistic thoughts enhanced their satisfaction of life in turn. In addition, it turned out that the effects of GAB were more drastic in group sessions than in individual writings. Interpersonal interactions in group sessions encouraged the exchange of positive feedback, thereby helping them reflect themselves positively.

• Journal of Korean Society of Forest Science
• /
• v.81 no.3
• /
• pp.273-279
• /
• 1992

Protoplasts isolated from leaf mesophyll tissues of Populus koreana ${\times}$ P. nigra var. italica were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana ${\times}$ P. nigra var. italica and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v), respectively, The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of $Ca^{2+}$ in eluting solution at pH 10.5. Dividing cells and/or mint-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, $0.45{\mu}M$ 2, 4-D, and $0.5{\mu}M$ BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing $5.0{\mu}M$ zeatin. To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein band patterns compared with those of the original source plants.

• Horticultural Science & Technology
• /
• v.29 no.4
• /
• pp.366-373
• /
• 2011

To analyze the genetic relationships among Korean potato cultivars and to develop cultivar identification method using DNA markers, we carried out genotyping using simple sequence repeats (SSR) analysis and developed multiplex-SSR set. Initially, we designed 92 SSR primer combinations reported previously and applied them to twenty four Korean potato cultivars. Among the 92 SSR markers, we selected 14 SSR markers based on polymorphism information contents (PIC) values. PIC values of the selected 14 markers ranged from 0.48 to 0.89 with an average of 0.76. PIC value of PSSR-29 was the lowest with 0.48 and PSSR-191 was the highest with 0.89. UPGMA clustering analysis based on genetic distances using 14 SSR markers classified 21 potato cultivars into 2 clusters. Cluster I and II included 16 and 5 cultivars, respectively. And 3 cultivars were not classified into major cluster group I and II. These 14 SSR markers generated a total of 121 alleles and the average number of alleles per SSR marker was 10.8 with a range from 3 to 34. Among the selected markers, we combined three SSR markers, PSSR-17, PSSR-24 and PSSR-24, as a multiplex-SSR set. This multiplex-SSR set used in the study can distinguish all the cultivars with one time PCR and PAGE (Polyacrylamide gel electrophoresis) analysis and PIC value of multiplex-SSR set was 0.95.

• Journal of Life Science
• /
• v.25 no.11
• /
• pp.1280-1289
• /
• 2015

Fish-meat gel is being produced mostly relying on surimi and raw materials imported from Southeast Asia and North America and present in small amount in local markets. In this study, common farmed local fishes were examined as stable and reliable sources of surimi for fish-meat gel production. For testing, five main farmed-fish of Korea, namely; Bastard halibut (Paralichthys olivaceus), Red sea bream (Pagrus major), Korean rockfish (Sebastes schlegeli), Common mulle (Mugil cephalus), and Finespotted flounder (Pleuronichthys cornutus) were used following a traditional washing process. The quality of the surimi was determined by the values of water content, whiteness index, gel strength and impurity. Accordingly, fish-meat gel and surimi quality experiments were carried out by measuring compressive and texture properties, expressible moisture content, Hunter color scale values and SDS-page protein patterns. Also gel characteristics were compared with that of FA and RA grade surimi (Alaska Pollock). Fish-meat gels were prepared by salt mincing the farmed-fish surimi with NaCl (2% w/w) and moisture adjustment to 84% by ice water adding. Prepared fish-meat paste was filled into 20-25 cm long polyvinylidene chloride casings and heated at 90℃ for 20 min. The whiteness values of fish-meat gels produced from surimi were increased by using farmed-fish and became comparable to that of FA Alaska Pollock gel. Among all tested farmed-fish, P. olivaceus and P. major exhibited better properties than RA Alaska Pollock and similar properties to FA Alaska Pollock. Therefore, current data suggests that fish farming can be an efficient and sustainable fish-meat source for fish-meat gel production in Korea.

• The Journal of Korean Society of Virology
• /
• v.27 no.2
• /
• pp.239-255
• /
• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.