• Journal of Pharmaceutical Investigation
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• v.31 no.3
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• pp.185-190
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• 2001

Human papillomavirus (HPV) infection is known to cause cervical cancers. Human papillomavirus-like particles (VLP) have been studied as preventive vaccines of cervical cancers. To develop VLP as a therapeutic gene carrier, we studied the method to encapsulate cytokine genes in virus-like particles. HPV type 16 capsid L1 genes were amplified by polymerase chain reaction and cloned into T vector. L1 gene was then inserted into baculovirus transfer vector. The clone of baculovirus encoding L1 gene was isolated and used to express L1 protein in Sf 21 insect cells. VLP were purified by CsCl density gradient and ultracentrifugation. VLP were disassembled to capsomer units by treatment of a reducing agent. Given that interleukin-2 (IL-2) genes have been used in anticancer gene therapy and as a molecular adjuvant, IL-2 cytokine plasmids were chosen as a model gene. IL-2 plasmids were incubated with the disassembled capsomer suspension. To reassemble the particles, the mixture of capsomers and cytokine plasmids was dialyzed. The disassembly and reassembly of VLP were confirmed by transmission electron microscopy. The entrapment of cytokine plasmids in reassembled VLP was tested by the stability of plasmids against DNase I. After treatment of reassembled virus-like particles with DNase I, discrete IL-2 DNA band was observed. Our results indicate that IL-2 cytokine plasmid (3.5 kb size) can be encapsulated in the virus-like particles, suggesting the potential of VLP as a gene delivery system. Moreover, VLP containing the adjuvant cytokine plasmids might function as more effective subunit vaccines.

• Journal of Microbiology and Biotechnology
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• v.25 no.10
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• pp.1621-1628
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• 2015

Chlamydia possesses a conserved 7.5 kb plasmid that is known to play an important role in chlamydial pathogenesis, since some chlamydial organisms lacking the plasmid are attenuated. The chlamydial transformation system developed recently required the use of plasmid-free organisms. Thus, the generation and identification of plasmid-free organisms represent a key step in understanding chlamydial pathogenic mechanisms. A tricolor immunofluorescence assay for simultaneously detecting the plasmid-encoded Pgp3 and whole organisms plus DNA staining was used to screen C. muridarum organisms selected with novobiocin. PCR was used to detect the plasmid genes. Next-generation sequencing was then used to sequence the genomes of plasmid-free C. muridarum candidates and the parental C. muridarum Nigg strain. We generated five independent clones of plasmid-free C. muridarum organisms by using a combination of novobiocin treatment and screening plaque-purified clones with anti-Pgp3 antibody. The clones were confirmed to lack plasmid genes by PCR analysis. No GlgA protein or glycogen accumulation was detected in cells infected with the plasmid-free clones. More importantly, whole-genome sequencing characterization of the plasmid-free C. muridarum organism and the parental C. muridarum Nigg strain revealed no additional mutations other than loss of the plasmid in the plasmid-free C. muridarum organism. Thus, the Pgp3-based immunofluorescence assay has allowed us to identify authentic plasmid-free organisms that are useful for further investigating chlamydial pathogenic mechanisms.

• Korean Journal of Microbiology
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• v.25 no.2
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• pp.94-102
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• 1987

Rat liver LDH A-cDNA has been isolated from a .lambda.gt11-rat lover cDNA library and partially characterized. The size of the isolated rat liver LDH A-cDNA if shown to be 1.6Kb and restriction enzyme sites for the rat liver LDH A-cDNA are also mapped. 682-nucleotide sequence coding for 3'-end of rat liver LDH A-cDNA has been analyzed and compared to the nucleotide sequence of the same region of rat $C_6$-glioma cell LDH A-cDNA which has been cloned from the hormonally stimulated cell cultures. The result shows that 177 nucleotide sequences coding for the C-terminal 59-amino acids are identical but 505 nucleotide sequences of 3'-nontranslated region of the two LSH A-cDNA exhibit characteristic differences in thier nucleotide sequences. Computer analysis for the folding structures for 3'-end 400 nucleotide sequences of the two LDH A-cDNA shows a possibility implying that the two LDH A-mRNAs isolated from different tissues of rats may have different half life and therefore their translational efficiency may be different. It has been previously demonstrated that isoproterenol stimulated rat $C_6$ -glioma cell cultures produce LDH A-mRNA showing 2 to 3-fold longer half life in comparison to that of noninduced LHD A-mRNA. The result therefore support for the idea that hormonally stimulated rat $C_6$-glioma cells may produce LDH A-mRNA containing different nucleotide sequences at the 3'-end nontranslated region by which the hormonally induced LDH A-mRNA could have more stable secondary mRAN structure in comparison to that of noninduced LDH A-mRNA.

• BMB Reports
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• v.40 no.6
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• pp.921-927
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• 2007

UCP2 and UCP3 are members of the uncoupling protein family, which may play roles in energy homeostasis. In order to determine the regulation of the predominant expression of UCP3 in skeletal muscle, the effects of differentiation and myogenic regulatory factors on the promoter activities of the mouse UCP2 and UCP3 genes were studied. Reporter plasmids, containing approximately 3 kb of the 5'-upstream region of the mouse UCP2 and UCP3 genes, were transfected into C2C12 myoblasts, which were then induced to differentiate. Differentiation positively induced the reporter expression about 20-fold via the UCP3 promoter, but by only 2-fold via the UCP2 promoter. C2C12 myoblasts were cotransfected with expression vectors for myogenin and/or MyoD as well as reporter constructs. The simultaneous expression of myogenin and MyoD caused an additional 20-fold increase in the reporter expression via the UCP3 promoter, but only a weak effect via the UCP2 promoter. In L6 myoblasts, only MyoD activated the UCP3 promoter, but in 3T3-L1 cells neither factor activated the UCP3 promoter, indicating that additional cofactors are required, which are present only in C2C12 myoblasts. The expression of UCP2 and UCP3 is differentially regulated during muscle differentiation due to the different responsiveness of their promoter regions to myogenin and MyoD.

• Korean Journal of Plant Resources
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• v.6 no.1
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• pp.25-32
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• 1993

Many plants, which collected from Korea, were applied to antitumor and cytotoxic screeing tests against sarcom 180 a ascitec in mice, V-79 KB and P388 cultured cells. The results are summarixed as follows:1) The total packed cell volum method has been used for the antineoplastic screening for from natural higher plants in Korea. By this method, we have found out that the root, leaf and stem of Tripterygiu, regelii Spragne & Taketa having strong antineoplastic activity and also Rumex Japonicus Houtt. Eragrositis ferru-ginea Beauv. and Patrinia scabio-saefolia Fischer showed significant activity to anticancer tumor while cynanchum wilfordii Hemsley, and Rosa polyantha Sieb. et Zacc. showed slight activity to antitumor. 2) Among the 13 tested plants, the root and stem of Tripterygium regelii Spragne & Taketa and Amethystanthus excisus Nakai showed strong antitumor activity by the V79 cytotoxic cell screening test. 3) Twelve plants, which are glowing in mountainous area of Korea tested to anticancer activity. From the results, Eragrositis ferru-ginea Beauv., Angelica gigas Nakai, Geranium sibiricum L., Patrinia scabio-saefolia Fisher, Cynanchum wilfordii Hemsley, and Rubia akane Nakai have been proved to be anti-cancer plants by using P388 cell cultured method. 4) Tripterygiu, resgelii Spragne & Taketa, Eragrositis ferru-ginea Beauv., Patrinia scabio-saefoli Fisher, Cynanchum wilfordii Hemsley and Rasa polyantha Sieb. et Zacc., var. genuina Thunb. showed strong anti-tumor activity both total packed cell volume method and Cytotoxicity method.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.5
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• pp.1999-2002
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• 2012

Background: DNA polymerase is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. The encoded enzyme is a key to the base excision repair (BER) pathway. It is evident that pol beta has mutations in various cancer samples, but little is known about ovarian cancer. Aim: Identification of any variant form of $pol{\beta}$ cDNA in ovarian carcinoma and determination of association between the polymorphism and ovarian cancer risk in Indian patients. We used 152 samples to isolate and perform RT-PCR and sequencing. Results: A variant of polymerase beta (deletion of exon 4-6 and 11-13, comprising of amino acid 63-123, and 208-304) is detected in heterozygous condition. The product size of this variant is 532 bp while wild type pol beta is 1 kb. Our study of association between the variant and the endometrioid type shows that it is a statistically significant factor for ovarian cancer [OR=31.9 (4.12-246.25) with p<0.001]. The association between variant and stage IV patients further indicated risk (${\chi}^2$ value of 29.7, and OR value 6.77 with 95% CI values 3.3-13.86). The correlation study also confirms the association data (Pearson correlation values for variant/stage IV and variant/endometrioid of 0.44 and 0.39). Conclusion: Individuals from this part of India with this type of variant may be at risk of stage IV, endometrioid type ovarian carcinoma.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.277-286
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• 2001

Improvement and possible commercialization of a home-made electroporation apparatus(home-made) were further tried to establish a simple and effective introduction of foreign gene into sperm followed by in vitro fertilization. Expressions of introduced pJJ9 and pNT plasmids were shown in all fertilized eggs with electroporated spermatozoa. In particular, with this gene transfer system all the fry showed a consistently transient expression in the syncytium of the yolk sac. This fact is important since some required, minute quantity of human proteins can be produced from the established transient expression on the yolk sac of all fry derived from in vitro fertilization with electroporated spermatozoa. To explore tissue-specific expression in fish, which we will use a similar system later, we targeted the nerve tissue to see whether tissue-specific promoter is working in fish properly. pNT plasmid containing a nerve cell-specific tubulin promoter gene demonstrated consistently exact targeted expressions among the developing nerve cells in later stages of embryos and hatched fry. Finally, liver-specific genes are now being cloned by using already selected primers for useful human protein gene fusion.

• Microbiology and Biotechnology Letters
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• v.14 no.3
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• pp.245-250
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• 1986

Antibiotic resistant bacteria isolated from the river water in Cheongju City were studied on the transfer of their R-plasmids in water environment. The R-plasmids were transferred by conjugation between the isolates ai the frequencies of 1.1$\times$10$^{-6}$ to 1.2$\times$10$^{-7}$ under laboratory condition and 1.2$\times$10$^{-7}$ to 1.0$\times$10$^{-9}$ in natural environment. The R-plasmids isolated from those bacteria were also transferred into the recipient cells of E. coli HB101 at the frequencies of 1.7-6.7$\times$10$^{-6}$. The AP$^{r}$Cm$^{r}$Tc$^{r}$-plasmid of isolate T-44 which were transformation by conjugation and transformation was determined to be 9.01 kilobses in molecular size. When the AP$^{r}$Cm$^{r}$Tc$^{r}$-plasmid DNA was treated with restriction endonuclease, the plasmid appeared to have one restriction site for EroRI and BamHI, respectively, and three sites for Pst I endonuclease.

• The Journal of Korean Society of Virology
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• v.29 no.2
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• pp.107-118
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• 1999

To characterize the molecular nature of human immunodeficiency virus (HIV)-1, we determined the full-length HIV-1 sequences from cultured peripheral blood mononuclear cells (PBMC) of a Korean long-term nonprogressor (LTNP). Without antiretroviral therapy, the individual has maintained CD4+ T counts over $500/{\mu}l$ from 1989 to 1999. Plasma viral RNA copy was 992 U/ml in 1998. Culture supernatant showed positive from culture days 9. A series of 9 overlapping PCR products were amplified from cultured PBMC and cloned About 9.2 kb from R of 5' LTR to R of 3' LTR was determined by automated sequencing. The G-to-A hypermutations were shown throughout the entire region. As a result of G to A hypermutations, premature stop codon was found in integrase coding region. Though there was no recombination between subtypes over all genomes, TATA box in both LTRs was TAAAA which is detected in subtype E instead of TATAA in subtype B. And, there were nucleotide GC insertion between $NF-{\kappa}B$ I and Sp1 III, and duplication of $TCF-1{\alpha}$ in LTR. We could not find any deletion of amino acid in Nef, Gag, Pol and Env gene. This study is the first report on molecular nature of full genomes of HIV-1 isolated in Korea.

• Korean Journal of Veterinary Service
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• v.30 no.3
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• pp.393-406
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• 2007

Bovine tuberculosis is an important zoonosis worldwide. Mycobacterium bovis, the causative agent of this disease in cattle, is also a pathogen for humans and several economically important animals. The cases of tuberculosis are reported in two cow found at slaughter house located in Gwangju city. Histopathologically, in the lymph nodes, granulomas consisted of large areas of necrosis surrounded by variable thick bands of cellular infiltrate containing macrophages, Langhans-type multinucleated giant cells and lymphocytes. Lesions in the lung followed the same developmental pattern as did lesions in the lymph nodes with some exceptions. With the acid-fast staining, numerous mycobacteria were revealed in the lung and lymph nodes. M bovis was confirmed as a causative agent in these cattle using bacterial isolation and PCR and restriction fragment length polymorphism method based on a unique 12.7 kb fragment insertion sequence from the Mycobacterium tuberculosis genome and the pncA polymorphism, The insertion element IS6110 and IS1081 were present M bovis isolated from lungs and lymph nodes of cattle using PCR assay. These cases are interesting and important in public health aspect that M bovis-infected cattle were found during a routine post-mortem inspection at slaughter house.