• Journal of Fisheries and Marine Sciences Education
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• v.26 no.4
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• pp.808-822
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• 2014

넙치 (Paralichthys olivaceus)로부터 elastase-like serine protease (PoElSp)를 암호화하는 cDNA를 클로닝하여 그 서열을 분석한 결과, PoElSp 유전자는 269 아미노산을 암호화하는 978염기쌍으로 구성되었다. PoElSp 유전자의 조직 특이적 발현 양상을 RT-PCR법으로 조사한 결과, 간, 비장 및 소장에서 그 발현이 크게 나타났다. lipopolysaccharide (LPS)로 인위적 세균감염을 유도한 후, 1시간째에 콩팥에서, 3시간째에는 근육에서, PoElSp 유전자의 발현이 크게 증가하였다. 또한, 이 유전자의 발현은 비장에서 LPS 주입 후 1-24시간동안 점차로 증가하였다. pro-mature PoElSp (proPoElSp)에 해당하는 cDNA를 pET32a 벡터 시스템을 이용하여 대장균에서 발현시켰다. 이 재조합 proPoElSp 단백질의 활성은 gelatin zymography 방법과 합성형광 Z-Phe-Arg-AMC의 분해법을 이용하여 측정하였다. 단백질 분해효소 활성을 위한 최적 pH는 7.5였다. 실험결과들을 종합하면, PoElSp 단백질은 면역 반응에서 중추적 역할을 하리라 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.6
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• pp.758-763
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• 2012

As a member of a subclass of immunophilins, it is controversial that FKBP38 acts an upstream regulator of mTOR signaling pathway, which control the process of cell-growth, proliferation and differentiation. In order to explore the relationship between FKBP38 and mTOR in the Cashmere goat (Capra hircus) cells, a full-length cDNA was cloned (GenBank accession number JF714970) and expression pattern was analyzed. The cloned FKBP38 gene is 1,248 bp in length, containing an open reading frame (ORF) from nucleotide 13 to 1,248 which encodes 411 amino acids, and 12 nucleotides in front of the initiation codon. The full cDNA sequence shares 98% identity with cattle, 94% with horse and 90% with human. The putative amino acid sequence shows the higher homology which is 98%, 97% and 94%, correspondingly. The bioinformatics analysis showed that FKBP38 contained a FKBP_C domain, two TPR domains and a TM domain. Psite analysis suggested that the ORF encoding protein contained a leucine-zipper pattern and a Prenyl group binding site (CAAX box). Tissue-specific expression analysis was performed by semi-quantitative RT-PCR and showed that the FKBP38 expression was detected in all the tested tissues and the highest level of mRNA accumulation was detected in testis, suggesting that FKBP38 plays an important role in goat cells.

• Korean Journal of Veterinary Research
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• v.35 no.1
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• pp.95-105
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• 1995

The human homolog of position specific element of mouse Hoxa-7 was studied using transgene. It contains a 1.1 kb human DNA (HCR)- a homolog to the intergenic region between Hoxa-7 and -9, which directs the position specific expression of Hoxa-7-, tk promoter, LacZ (${\beta}$-galactosidase) gene as a reporter, and polyadenylation signal of SV40 large T antigen. It was injected into the mice embryos, and the resulting transgenic embryos were analysed through PCR as well as genomic Southern blotting with placenta DNA. Out of 20 embryos analysed, two were transgenic. Among them, one transgenic embryo expressed transgene when stained with X-gal. The expression pattern was in analogy to that of the mouse Hoxa-7, showing spatially restricted expression pattern, Since the expression of ${\beta}$-galactosidase is regulated by the upstream human HCR sequence, it implies that the HCR is the plausible position specific regulatory element of human.

• Animal cells and systems
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• v.7 no.2
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• pp.139-144
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• 2003

We analyzed seven Y chromosome binary markers (YAP, RPS4Y_711,\;M9,\;M175,\;LINE1,\;SRY_+465$ and 47z) in samples from a total of 254 males from Koreans and tow Mongolian ethnic groups (Buryat and Khalkh) to study the genetic relationship among these populations. We found eight distinct Y haplogroups constructed from the seven binary markers. Haplogroup DE-YAP was present at extremely low frequencies (∼2%) in the Korean and Mongolian populations. This result is consistent with earlier reports that showed the YAP+ chromosomes to be highly polymorphic only in populations from Japan and Tibet in east Asia. The observed high frequency of haplogroup $C-RPS4Y_711$ in the Mongolian populations (∼40%) is concordant with recent findings, showing that the $RPS4Y_711$-T chromosomes were distributed at high frequencies in Siberian and Mongolian populations compared with most other populations from east Asia. Thus, the relatively moderate frequency of haplogroup $C-RPS4Y_711$ in Korean (∼15%) can be seen as genetic evidence for probable interaction with Mongolian and/or Siberian populations. In contrast, the majority (∼75%) of modern Koreans studied here had high frequencies of Y chromosome lineages of haplogroup O-M175 and additional haplogroupts that define sublineage of O-M175, which are most likely related with modern populations in China. In conclusion, our data on the Y chromosome haplogroup distribution may provide evidence for interaction between Korean and Mongolian populations, but Korean tend to be much more related with those from southern-to-northern populations of China than to Mongolians in east Asia.

• Journal of Microbiology and Biotechnology
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• v.11 no.5
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• pp.825-830
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• 2001

Escherichia coli, which harbored the ars operon from a plasmid pMH12 of Klebsiella oxytoca D12, showed filamentation due to the expression of ars genes in the presence of arsenite. The continued DNA replication in the absence of cell division was revealed, since nucleoids abound with DAPI appeared to be arranged in chains. In contrast to overexpression of arsA, its frame-shift mutant and knock-out mutant lost filamentation in the presence of arsenite, which suggested that ars-induced division block was dependent on expression of arsA. ArsA-induced division inhibition was not a consequence of an inhibition of DNA replication, and the inability of arsenite to induce an SOS response indicated that arsA-mediated division inhibition was dependent on the expression of the gene product encoded by the minB operon. ArsA is a peripheral membrane protein with an ATP-binding domain, which is homologous to MinD that requires ATP-dependent efflux. These results suggested that ArsA could possibly recruit MinC to the membrane and modulate cytoplasmic FtsZ to block assembly at the middle of the cell.

• ALGAE
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• v.36 no.1
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• pp.1-12
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• 2021

As the global demand for the carrageenophyte Kappaphycus is steadily increasing, its overall productivity, carrageenan quality, and disease resistance are gradually declining. In the face of this dilemma, wild Kappaphycus populations are viewed as sources of new cultivars that could potentially enhance production; therefore, assessment of their diversity is crucial. This study highlights the morphological and genetic diversity of wild Kappaphycus species obtained from two sites in the Philippines. Nucleotide alignments of available 5' region of the mitochondrial cytochrome c oxidase subunit I (COI-5P) and cox2-3 spacer sequences of Kappaphycus confirmed the presence of K. alvarezii in Guiuan, Eastern Samar and K. striatus in Bolinao, Pangasinan. Based on the concatenated sequences of the COI-5P and the cox2-3 spacer, nine novel haplotypes were observed along with other published haplotypes. However, there was no relationship between haplotype and morphology. These newly recognized haplotypes indicate a reservoir of unutilized wild genotypes in the Philippines, which could be taken advantage of in developing new cultivars with superior traits. DNA barcodes generated from this study effectively expand the existing databank of Kappaphycus sequences and can provide insights in elucidating the genetic diversity of Kappaphycus species in the country.

• Journal of Life Science
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• v.19 no.11
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• pp.1529-1537
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• 2009

To elucidate further the antitumor effects of a natural L-arginine analogue, L-canavanine, the mechanism underlying apoptogenic activity of L-canavanine and its modulation by protein tyrosine kinase $p56^{lck}$ was investigated in human Jurkat T cells. When the cells were treated with 1.25 to 2.5 mM L-canavanine for 36 h, several apoptotic events including mitochondrial membrane potential (${\Delta\Psi}m$) loss, activation of caspase-9, -3, -8, and -7, poly (ADP-ribose) polymerase (PARP) degradation, and DNA fragmentation were induced without alteration in the levels of Fas or FasL. These apoptotic changes were more significant in $p56^{lck}$-deficient Jurkat clone JCaM1.6 than in $p56^{lck}$-positive Jurkat clone E6.1. The L-canavanine-induced apoptosis observed in $p56^{lck}$-deficient JCaM1.6 cells was significantly reduced by introducing $p56^{lck}$ gene into JCaM1.6 cells by stable transfection. Treatment of JCaM1.6/lck cells with L-canavanine caused a transient 1.6-fold increase in the kinase activity of $p56^{lck}$. Both FADD-positive wild-type Jurkat T cell clone A3 and FADD-deficient Jurkat T cell clone I2.1 exhibited a similar susceptibility to the cytotoxicity of L-canavanine, excluding involvement of Fas/FasL system in triggering L-canavanine-induced apoptosis. The L-canavanine-induced apoptotic sub-$G_1$ peak and activation of caspase-3, -8, and -7 were abrogated by pan-caspase inhibitor (z-VAD-fmk), whereas L-canavanine-induced activation of caspase-9 was not affected. These results demonstrated that L-canavanine caused apoptosis of Jurkat T cells via the loss of ${\Delta\Psi}m$, and the activation of caspase-9, -3, -8, and -7, leading to PARP degradation, and that the $p56^{lck}$ kinase attenuated the ${\Delta\Psi}m$ loss and activation of caspases, and thus contributed as a negative regulator to L-canavanine-induced apoptosis.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.31 no.3
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• pp.199-218
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• 2005

Purpose of Study: Peripheral nerve regeneration depends on neurotrophism of distal nerve stump, recovery potential of neuron, supporting cell like Schwann cell and neurotrophic factors such as BDNF. Peripheral nerve regeneration can be enhanced by the conduit which connects the both sides of transected nerve. The conduit maintains the effects of neurotrophism and BDNF produced by Schwann cells which can be made by gene therapy. In this study, we tried to enhance the peripheral nerve regeneration by using calcium phosphate coated porous conduit and BDNF-Adenovirus infected Schwann cells in sciatic nerve of rats. Materials and Methods: Microporous filter which permits the tissue fluid essential for nerve regeneration and does not permit infiltration of fibroblasts, was made into 2mm diameter and 17mm length conduit. Then it was coated with calcium phosphate to improve the Schwann cell adhesion and survival. The coated filter was evaluated by SEM examination and MTT assay. For effective allogenic Schwann cell culture, dorsal root ganglia of 1-day old rat were extracted and treated with enzyme and antimitotic Ara-C. Human BDNF cDNA was obtained from cDNA library and amplified using PCR. BDNF gene was inserted into adenovirus shuttle vector pAACCMVpARS in which E1 was deleted. We infected the BDNF-Ad into 293 human mammary kidney cell-line and obtained the virus plaque 2 days later. RT-PCR was performed to evaluate the secretion of BDNF in infected Schwann cells. To determine the most optimal m.o.i of BDNF-Ad, we infected the Schwann cells with LacZ adenovirus in 1, 20, 50, 75, 100, 250 m.o.i for 2 hours and stained with ${\beta}$-galactosidase. Rats(n=24) weighing around 300g were used. Total 14mm sciatic nerve defect was made and connected with calcium phosphate coated conduits. Schwann cells$(1{\times}10^6)$ or BDNF-Ad infected Schwann cells$(1{\times}10^6)$ were injected in conduit and only media(MEM) was injected in control group. Twelve weeks after surgery, degree of nerve regeneration was evaluated with gait analysis, electrophysiologic measurements and histomorphometric analysis. Results: 1. Microporous Millipore filter was effective conduit which permitted the adhesion of Schwann cells and inhibited the adhesion of fibroblast. We could enhance the Schwann cell adhesion and survival by coating Millipore filter with calcium phosphate. 2. Schwann cell culture technique using repeated treatment of Ara-C and GDNF was established. The mean number of Schwann cells obtained 1 and 2 weeks after the culture were $1.54{\pm}4.0{\times}10^6$ and $9.66{\pm}9.6{\times}10^6$. 3. The mRNA of BDNF in BDNF-Ad infected Schwann cells was detected using RT-PCR. In Schwann cell $0.69\;{\mu}g/{\mu}l$ of DNA was detected and in BDNF-Adenovirus transfected Schwann cell $0.795\;{\mu}g/{\mu}l$ of DNA was detected. The most effective infection concentration was determined by LacZ Adenovirus and 75 m.o.i was found the most optimal. Conclusion: BDNF-Ad transfected Schwann cells successfully regenerated the 14mm nerve gap which was connected with calcium phosphate coated Millipore filter. The BDNF-Ad group showed better results compared with Schwann cells only group and control group in aspect to sciatic function index, electrophysiologic measurements and histomorphometric analysis.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.157-161
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• 2002

A lactic acid bacteria with ${\beta}$-gal activity was isolated from Kimchi, a traditional fermented vegetable food in Korea. The isolate was identified as a Lactococcus lactis strain and named L. lactis A2. The gene encoding ${\beta}$-gal of L. lactis A2 was cloned as a 5.8 kb PstI fragment. DNA sequencing identified the complete lacA (galactoside acetyltransferase)-lacZ (${\beta}$-galactosidase) genes together with the 3' part of upstream galT (galactose-1-phosphate uridyltransferase), and the 5'region of downstream galE (UDP-galactose-4-epimerase) genes. L. lactis A2 had the same gal/lac operon structure as in L. lactis subsp. lactis 7962. Other genes of the Leloir pathway are most likely to be located in the 5'upstream of the 5.8 kb fragment on the A2 chromosome. Sequences downstream of galE were different from those of L. lactis subsp. lactis 7962.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.2
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• pp.103-109
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• 2005

In spite of the ongoing advances, standard therapies for oral cancer still has some limitations in efficacy and in ability to prolong survival rate of advanced disease and result in significant functional defect and severe cosmetic deformity. Currently gene therapy using tumor suppressor gene is considered as a potent candidate for new therapeutic approaches that can improve efficacy and reduce complications. The purpose of this research is to identify the role of adenoviral vector to transfer HCCS-1 tumor suppressor gene in oral cancer cells and to find out whether there is a possibility for it to serve in the field of gene therapy. The human SCC-25 cell line was used for transfection. To determine the efficiency of the adenovirus as a gene delivery vector cell line was transduced with LacZ gene and analysed with X-gal staining. Northern blot was performed to confirm the tranfection with HSCC-1 gene and cell viability was assessed by cell cytotoxicity assay. We had successfully construct the recombinant HSCC-1 adenovirus(Ad5CMV-HCCS-1). DNA extracted from Ad5CMV-HCCS-1 revealed HCCS-1 gene is incorporated. The transduction efficiencies were over than 50% of SCC-25 cells with a MOI of 2 and over 95% with a MOI of 50. Northern blot analysis showed that a single 0.6kb mRNA transcript was expressed in Ad5CMV-HCCS-1 transduced SCC-25 cells. There was no or very low transcription HCCS-1 mRNA in wild and Ad5CMV-LacZ transduced SCC-25 cells. Cells transduced with Ad5CMV-HCCS-1 showed significant growth inhibition. By day 6, Ad5CMV-HCCS-1 treated cell count was decreased to 30% of mock-infected cells, while that of Ad5CMV-LacZ treated cells was 90% of mock-infected cells (p<0.05). Finally, these result suggest that the Ad5CMV-HCCS-1 has potential as a gene therapy tool for oral cancer.