• Proceedings of the Microbiological Society of Korea Conference
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• 2008.05a
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• pp.62-64
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• 2008

A major hurdle to the development of RNA interference as therapy for HIV infection is the delivery of siRNA to T lymphocytes which are difficult cells to transfect even in vitro. We have employed a single chain antibody to the pan T cell surface antigen CD7 was conjugated to an oligo-9-arginine peptide (scFvCD7-9R) for T cell-specific siRNA delivery in NOD/SCIDIL2${\gamma}$-/- mice reconstituted with human peripheral blood lymphocytes (Hu-PBL). Using a novel delivery, we first show that scFvCD7-9R efficiently delivered CD4 siRNA into human T cells in vitro. In vivo administration to Hu-PBL mice resulted in reduced levels of surface CD4 expression on T cells. Mice infected with HIV-1 and treated on a weekly basis with scFvCD7-9R-siRNA complexes targeting a combination of viral genes and the host coreceptor molecule CCR5 successfully maintained CD4/CD3 T cell ratios up to 4 weeks after infection in contrast to control mice that displayed a marked reduction in CD4 T cell numbers. p24 antigen levels were undetectable in 3 of the 4 protected mice. scFvCD7-9R/antiviral siRNA treatment also helped maintain CD4 T cell numbers with reduced plasma viral loads in Hu-PBL mice reconstituted with PBMC from donors seropositive for HIV, indicating that this method can contain viral replication even in established HIV infections. Our results show that scFvCD7-9R could be further developed as a potential therapeutic for HIV-1 infection.

• BMB Reports
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• v.36 no.4
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• pp.379-386
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• 2003

A mutant herpes simplex virus 1, mtHSV, was constructed by inserting the E. coli beta-galactosidase gene into the loci of icp34.5, the apoptosis-inhibiting gene of HSV. The mtHSV replicated in and lysed U251 (human glioma cells), EJ (human bladder cells), and S-180 (mice sarcoma cells), but not Wish (human amnion cells) cells. With its intact tk (thymidine kinase) gene, mtHSV exhibited susceptibility to acyclovir (ACV), which provided an approach to control viral replication. An in vivo test with mtHSV was conducted in immune-competent mice bearing sarcoma S-180 tumors, which were treated with a single intratumoral injection of mtHSV or PBS. Tumor dimensions then were measured at serial time points, and the tumor volumes were calculated. Sarcoma growth was significantly inhibited with prolonged time and reduced tumor volume. There was microscopic evidence of necrosis of tumors in treated mice, whereas no damage was found in other organs. Immunohistochemical staining revealed that virus replication was exclusively confined to the treated tumor cells. HSV-1 DNA was detected in tumors, but not in the other organs by a polymerase chain reaction analysis. From these experiments, we concluded that mtHSV should be a safe and promising oncolytic agent for cancer treatment.

• Journal of Veterinary Science
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• v.21 no.3
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• pp.49.1-49.14
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• 2020

Background: Coinfection with avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) is common in chickens, and the molecular mechanism of the synergistic pathogenic effects of the coinfection is not clear. Exosomes have been identified as new players in the pathogenesis of retroviruses. The different functions of exosomes depend on their cargo components. Objectives: The aim of this study was to investigate the function of co-regulation differentially expressed proteins in exosomes on coinfection of ALV-J and REV. Methods: Here, viral replication in CEF cells infected with ALV-J, REV or both was detected by immunofluorescence microscopy. Then, we analyzed the exosomes isolated from supernatants of chicken embryo fibroblast (CEF) cells single infected and coinfected with ALV-J and REV by mass spectrometry. KEGG pathway enrichment analyzed the co-regulation differentially expressed proteins in exosomes. Next, we silenced and overexpressed tripartite motif containing 62 (TRIM62) to evaluate the effects of TRIM62 on viral replication and the expression levels of NCK-association proteins 1 (NCKAP1) and actin-related 2/3 complex subunit 5 (ARPC5) determined by quantitative reverse transcription polymerase chain reaction. Results: The results showed that coinfection of ALV-J and REV promoted the replication of each other. Thirty proteins, including TRIM62, NCK-association proteins 1 (NCKAP1, also known as Nap125), and Arp2/3-5, ARPC5, were identified. NCKAP1 and ARPC5 were involved in the actin cytoskeleton pathway. TRIM62 negatively regulated viral replication and that the inhibition of REV was more significant than that on ALV-J in CEF cells coinfected with TRIM62. In addition, TRIM62 decreased the expression of NCKAP1 and increased the expression of ARPC5 in coinfected CEF cells. Conclusions: Collectively, our results indicated that coinfection with ALV-J and REV competitively promoted each other's replication, the actin cytoskeleton played an important role in the coinfection mechanism, and TRIM62 regulated the actin cytoskeleton.

• IMMUNE NETWORK
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• v.3 no.2
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• pp.126-135
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• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• Journal of Aquaculture
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• v.8 no.3
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• pp.209-220
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• 1995

The nuclear matrix was isolated from Misgumus mizolepis liver nuclei by low salt extraction and restriction enzyme treatment. The structure was digested with proteinase K. After centrifugation, matrix attachment regions (MARs) were obtained by RNase treatment and phenol-chloroform extraction. The result leads to the appearance of smeared bands in the range of about 0.3-15 kb. pURY19 vector was constructed by inserting 2.13 kb Eco47 III fragment of the yeast uracil 3 gene into the unique Ssp I site of pUC19 plasmid vector as a selection marker. This vector is unable to be maintained in Sacrharomyces cerevisiae by itself since it cannot replicate as an extrachromosomal element. Using this system, we attempted cloning the ARS (autonomously replicating sequence) from M. mizelepis to develop an efficient expression vector for the transgenic fish. pURY19N_{l-62}$ were constructed by inserting MARs in pURY19 plasmid vector and transformation of E. coli $DH5\alpha$. Replication origins (ARS) of M. mizolepis were isolated, which enabled the vector to replicate autonomously in S. cerevisiae. The cloned DNA fragments were sequenced by Sanger's dideoxy-chain termination method. All clones were AT-rich. $pURY19N_6$, one of the clones, expecially contained ARS consensus sequence, Topoisomerase II consensus, near A-box and T-box.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.75.2-75
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• 2003

To develop an effective protection strategy against tomato bushy stunt virus (TBSV), tobacco plants expressing single-chain Fv antibodies (scFv), were established. A previous had shown that the replication activity of viral replicase was inhibited by the selected scFvs. Moreover, no systemic symptom was found after virus inoculation on leaves of wt N. benthamiana infiltrated with an Agrobacterium suspension resulting i3l expression of the scFvs. However, control plants showed systemic symptoms. In this study the localization of the scFvs within two transgenic plant lines, (CP28H3, CP-P55) was demonstrated using immunogold labelling. The gold particles, indicating the presence of scFv, were mostly found In the cytoplasm of the plant cells including chloroplasts and in the cell walls. However, they were hardly found in the vacuole, nucleoplasm and intercellular spaces. Gold particles often accumulated in either the cytosol or chloroplasts showing a specific labeling, There was no difference in type of gold labeling between both transgenic lines. The localization of the scFv in the cytoplasm further conforms the inhibition of the RNA-dependent RNA polymerase (RdRp) by the selected scFv because it is known that the RdRp is localized to membraneous cytosolic structures.

• Tribology and Lubricants
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• v.29 no.5
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• pp.304-309
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• 2013

Shark skin has functionalities such as self-cleaning and antifouling; it also exhibits excellent drag reduction owing to a hierarchical structure of microgrooves and nanometer-long chain mucus drag reduction interfaces around the shark body. In this study, the wettability of a shark skin surface and its replicas are evaluated. First, a shark skin template is taken from a real shark. Then, shark skin replicas are produced directly from a shark skin template, using a micromolding technique. The quantitative replication precision of the shark skin replicas is evaluated by comparing the geometry of the shark skin template to the replica using 2D surface profiles. Contact angles at the solid-air-water interfaces are evaluated for the shark skin template and its replicas under two conditions: with and without hydrophobic coating. The results show that the microriblets on shark skin improve the hydrophobic feature and play a critical role in self-cleaning.

• Journal of Microbiology and Biotechnology
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• v.15 no.5
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• pp.1146-1151
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• 2005

The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Previously, we reported that the yeast-to-hypha transition in this organism is suppressed by farnesoic acid, a morphogenic autoregulatory substance that accumulates in the medium as the cells proliferate. In this study, using a differential display reverse transcription polymerase chain reaction (DDRT-PCR) technique, we have identified several genes induced in C. albicans by farnesoic acid treatment. These observations indicate that farnesoic acid can alter the expressivity of multiple genes, including the DNA replication machinery and cell-cycle-control proteins.

• Bulletin of the Korean Chemical Society
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• v.32 no.5
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• pp.1569-1574
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• 2011

The presence of replication-competent adenovirus (RCA) subpopulations in adenoviral vector products raises a variety of safety issues for development of therapies based on gene therapy. To analyze the differing effects of adenoviral vector and RCA in vivo, we examined alterations in amino acids (AAs) using rat plasma following injection of ${\beta}$-galactosidase expressing recombinant adenovirus (designated rAdLacZ) or RCA. Plasma AAs were examined by gas chromatography-mass spectrometry. A total of 16 AAs were positively measured. In the rAdLacZ group compared to the control group, the level of aspartic acid was significantly increased (Student's t-test), while the level of glutamic acid was significantly reduced. Additionally, in the RCA group compared to the control group, the level of four AAs, valine, leucine, and isoleucine as branched-chain amino acids, and proline were significantly increased, whereas the levels of three AAs, glycine, threonine, and glutamic acid were significantly reduced. Altered plasma free AA metabolic patterns in rAdLacZ and RCA groups, compared with the control group, may explain the disturbance of AA metabolism related to viral infection.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.74.2-74
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• 2003

Arabidopsis infected with beet curly top virus (BCTV) has the systemic symptoms like stunting of Plant growth, curling of leaves and shoot tips, and callus induction. The regulation of sucrose metabolism by BCTV infection is essential for obtaining the energy source in the process of virus replication and symptom development. Sucrose metabolism-associated gene expression and biochemical enzyme activity were analyzed with the rossette leaves and inflorescencestems of BCTV infected Arabidopsis by the time course of 1, 7, 14, 21 day postinoculation. The expression of invertase and sucrose synthase genes ( encoding sucrose-cleaving enzymes )was increased and reversely the level of Atkin10a ( sucrose non-fermenting gene ) was decreased, resulting by semi-quantitative reverse transcription polymerase chain reaction. The biochemical analysis of invertase and sucrose synthase activity was performed. The activity of neutral invertase in the inflorescence stems was elevated remarkably. The photosynthetic response in the source of sucrose metabolism was consistent with the down-regulation of ribulose 1,5 bisphosphate carboxylase gene, and lower activity than mock-inoculated plants. The levels of genes pertaining to the cell cycle, hormone, and biotic stress-related pathway showed an increase or a decrease dependent on viral symptoms. Therefore, sucrose sensing by BCTV infection can regulate the expression of sucrose metabolism-related key enzymes such as invertase and Atkin10a, and these gene products might influence to symptom development.