• IMMUNE NETWORK
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• v.4 no.1
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• pp.16-22
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• 2004

Background: To develop a novel treatment strategy for hepatitis B virus infection, a major cause of liver chirosis and cancer, we aimed to make human monoclonal antibodies inhibiting RNase H activity of P protein playing in important role in HBV replication. In this regard, phage display technology was employed and demonstrated as an efficient cloning method for human monoclonal antibody. So this study analysed the usability of human monoclonal antibody as protein based gene therapy. Methods: RNase H of HBV was expressed as fusion protein with maltose binding protein and purified with amylose resin column. Single chain Fv (scFv) phage antibody library was constructed by PCR cloning using total RNAs of PBMC from 50 healthy volunteers. Binders to RNase H were selected with BIAcore 2000 from the constructed library, and purified as soluble antibody fragment. The affinity and sequences of selected antibody fragments were analyzed with BIAcore and ABI automatic sequencer, respectively. And finally RNase H activity inhibiting assay was carried out. Results: Recombinant RNase H expressed in E. coli exhibited an proper enzyme activity. Naive library of $4.46{\times}10^9cfu$ was screened by BIAcore 2000. Two clones, RN41 and RN56, showed affinity of $4.5{\times}10^{-7}M$ and $1.9{\times}10^{-7}M$, respectively. But RNase H inhibiting activity of RN41 was higher than that of RN56. Conclusion: We cloned human monoclonal antibodies inhibiting RNase H activity of P protein of HBV. These antibodies can be expected to be a good candidate for protein-based antiviral therapy by preventing a replication of HBV if they can be expressed intracellularly in HBV-infected hepatocytes.

• Biomedical Science Letters
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• v.6 no.1
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• pp.11-17
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• 2000

SNAP-25 was first investigated as a neuron-specific protein preferentially expressed in CA3 pyramidal neurons of mouse hippocampus. It is a presynaptic plasma membrane protein in the nerve cell and plays an important role in the synaptic vesicle membrane docking and fusion pathway. We have recently isolated SNAP-25 cDNA from a rat brain cDNA library using a probe of Z2 cDNA. It consisted of 2,101 bp and an open reading frame (ORF) was identified between nucleotides (nt) 209 and 827. The AUG codon (nt 209∼211) was surrounded by CTACCATGG, which corresponded to the consensus sequence of ribosomal binding site. The ORF was terminated by TAA (nt 827∼829) to encode a polypeptide of 206 amino acid residues. The 3'-untranslated region contained two extensive stretches of repeated (CA)28 and (CA)19 at positions 925∼980 and 1645∼1682. It is noteworthy that cysteine residues were clustered in the span of amino acid residues 84∼991 : Cys-Gly-Leu-Cys-Val-Cys-Pro-Cys. Rat SNAP-25 showed 88% and 97% identity in nucleotide sequences to that of human and mouse, respectively. Amino acid sequence of rat SNAP-25 showed 100% identity to that of mouse and human SNAP-21.

• Journal of Life Science
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• v.27 no.12
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• pp.1415-1420
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• 2017

The metallothionein (MT) gene (IbMT3) was selected from an EST library of suspension-cultured sweet potato cells. The MT gene, which is one of abundant ESTs in the library, is involved in stress regulation of cells and tissues. A full-length IbMT3 cDNA was obtained and analysis of its nucleotide sequence revealed that IbMT3 encoded a type 3 MT protein, based on its structural characteristics. The function of type 3 MT in plants is not yet known. Northern blot analysis showed stronger expression of IbMT3 in suspension-cultured cells than in sweet potato plant leaves. Since cell culture is known to impose a state of oxidative stress on cells, sweet potato plants were subjected to oxidative stress to investigate the transcriptional regulation of IbMT3. When the herbicide methyl viologen (MV) was administered for 6, 12, and 24 hr, IbMT3 transcription rapidly increased at 6 hr and then decreased. A cold treatment at $15^{\circ}C$ for 24 and 48 hr resulted in a gradual increase in IbMT3 expression. These findings indicate that IbMT3 expression is regulated in response to environmental and oxidative stress. IbMT3 isoform is expected to have antioxidant effects in sweet potato plants and may play an important role in cellular adaptation to oxidative stress.

• 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.

• Proceedings of the Korean Aquaculture Society Conference
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• 2003.10a
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• pp.44-44
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• 2003

Gene expression in five tissues of the abalone (Haliotis discus hannai) was investigated using an expressed sequence tag (EST) analysis. Randomly selected clones were obtained from cDNA libraries constructed with gill (GI), digestive diverticula(DD), hepatopancreas (HP), foot/mucus (FM) and rectangular muscle (RM). Of 1,235 clonesanalyzed (288 clones for GI, DD, HP each,166 for FM, and 205 for RM), 741 (60.0%) clones in total turned out to share significant similarity with the sequences from NCBI GenBank (less than 10/sup -3/ of e-values), 423 sequences showed poor similarity (> 10/sup -3/), and 71 sequences didn't match with any sequences in GenBank. The percent unique sequence (singleton) was ranged from 56.1% (RM) to 74.7% (FM) among libraries. On the other hand, overall percent singleton was 55.3% when all the ESTs from five libraries were assembled into contigs. Analysis of the organisms represented by the best hit for each EST (e-values < 10/sup -3/) showed that 23.8% matched with mammalian entries, 24.0% with mollusks, 14.4% with insects, 11.6% with fish and 26.2% with others. The expressed patterns differed among the tissues when judged by the categorization of the sequences from each library into 10 broad functional classes. In all the libraries, the class I (no hit o. poor similarity) was the largest category with an average of 40.1%. This largest class was followed by class V (general metabolisms) in DD (21.9%), GI (14.6%) and HP (16.7%), while the 'cell structure and motility'(class VI) was the second largest class in remaining two libraries (31.2% for RM and 9.6% for FM). The class IX (cell division and proliferation) was the smallest class in all the libraries (less than 3%). This report provides the first tissue-specific lists of expressed abalone genes, which could be a fundamental basis for genomics program of abalone species.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3719-3724
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• 2013

Background: RASSF1A has been reported to be a candidate tumor suppressor in non-small cell lung cancer (NSCLC). However, the association between RASSF1A promoter methylation and NSCLC remains unclear, particularly in regarding links to clinicopathologic features. Methods: Eligible studies were identified through searching PubMed, EMBASE, Cochrane Library and China National Knowledge Infrastructure (CNKI) databases. Studies were pooled and odds ratios (ORs) with corresponding confidence intervals (CIs) were calculated. Funnel plots were also performed to evaluate publication bias. Results: Nineteen studies involving 2,063 cases of NSCLC and 1,184 controls were included in this meta-analysis. A significant association was observed between RASSF1A methylation and NSCLC in the complete data set (OR = 19.42, 95% CI: 14.04-26.85, P < 0.001). Pooling the control tissue subgroups (heterogeneous/autologous) gave pooled ORs of 32.4 (95% CI, 12.4-84.5) and 17.7 (95% CI, 12.5-25.0) respectively. Racial subgroup (Caucasian/Asian) analysis gave pooled ORs of 26.6 (95% CI, 10.9-64.9) and 20.9 (95% CI, 14.4-30.4) respectively. The OR for RASSF1A methylation in poorly-differentiated vs. moderately/well-differentiated NSCLC tissues was 1.88 (95% CI, 1.32-2.68, P<0.001), whereas there were no significant differences in RASSF1A methylation in relation to gender, pathology, TNM stage and smoking behavior among NSCLC cases. Conclusion: This meta-analysis suggests a significant association between RASSF1A methylation and NSCLC, confirming the role of RASSF1A as a tumor suppressor gene. Large-scale and well-designed case-control studies are needed to validate the associations identified in the present meta-analysis.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.5
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• pp.2499-2506
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• 2016

Background: Gastric cancer (GC) is the second leading cause of cancer-related death worldwide. Several environmental, genetic and epigenetic factors have been suggested to have a role in GC development. Epigenetic mechanisms like histone changes and promoter hyper-methylation are now being increasingly studied. Associations between methylation of many gene promoters with the risk of gastric cancer have been investigated worldwide. Such aberrant methylation may result in silencing of specific genes related to cell cycling, cell adhesion, apoptosis and DNA repair. Thus this molecular mechanism might have a key role in proliferation and migration of cancerous cells. Materials and Methods: In this review article we included studies conducted on DNA methylation and gastric cancer in Iranian populations. Using Science direct, Pubmed/PMC, Springer, Wiley online library and SciELO databases, all published data until 31 January 2016 were gathered. We also searched Science direct data base for similar investigations around the world to make a comparison between Iran and other countries. Results: By searching these databases, we found that the association between methylation of seven gene promoters and gastric cancer had been studied in Iran until 31 January 2016. These genes were p16, hLMH1, E-cadherin, CTLA4, $THR{\beta}$, mir9 and APC. Searching in science direct database also showed that 92 articles had been published around the world till January 2016. Our investigation revealed that despite the importance of GC and its high prevalence in Iran, the methylation status of only a few gene promoters has been studied so far. More studies with higher sample numbers are needed to reveal the relation of methylation status of gene promoters to gastric cancer in Iran. Conclusions: Further studies will be helpful in identifying associations of DNA methylation in candidate genes with gastric cancer risk in Iranian populations.

• Korean Journal of Environmental Biology
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• v.29 no.4
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• pp.312-320
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• 2011

In order to estimate a better methodological factor to understand phytoplankton ecology between abundance and bio-volume of phytoplankton, each 1,160 phytoplankton data, including abundance, classification and chlorophyll $a$ concentration were collected in Korean coastal waters of Incheon (Yellow sea), Tongyeong (South sea), and Ulsan (East sea). Based on these data, phytoplankton bio-volume can be calculated through a geometric model. The correlation coefficient between abundance and chlorophyll $a$ concentration was higher than the coefficient between biovolume and chlorophyll $a$ concentration, because a small size phytoplankton has relatively dense chlorophyll contents compared with the proportion of chlorophyll in a large size phytoplankton. Thus, the interpretation using abundance to understand phytoplankton ecology in Korean coastal waters may be more effective than that using bio-volume.

• BMB Reports
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• v.32 no.5
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• Microbiology and Biotechnology Letters
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• v.33 no.4
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• pp.255-259
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• 2005

IgE-dependent histamine-releasing factor (HRF) is found extracellularly to regulate the degranulation process of histamine in mast cells and basophils and known to play a predominant role in the pathogenesis of chronic allergic disease. HRF has been also identified in the intracellular region of the cell. Previously, we reported that HRF interacts with the 3rd cytoplasmic domain of the alpha subunit of Na,K-ATPase. To understand the molecular mechanism of the regulation of Na, K-ATPase activity by HRF, we investigated the interaction between HRF and TPI since TPI was obtained as HRF-interacting protein in HeLa cDNA library, using yeast two hybrid screening. Domain mapping study of the interaction between HRF and TPI revealed that the C-terminal region of the residue 156-249 of TPI is involved in the interaction with HRF. The interaction between HRF and TPI was confirmed by immunoprecipitation from HeLa cell extracts. Our results suggest that TPI is a HRF-binding protein and the interaction between HRF and TPI nay thus affect Na, K-ATPase activity.