• Bulletin of the Korean Chemical Society
• /
• 제34권3호
• /
• pp.735-742
• /
• 2013

Peptide nucleic acids (PNAs) that bind to complementary nucleic acid sequences with extraordinarily high affinity and sequence specificity can be used as antisense oligonucleotides against microRNAs, namely antagomir PNAs. However, methods for efficient cellular delivery must be developed for effective use of PNAs as therapeutic agents. Here, we demonstrate that antagomir PNAs can be delivered to hepatic cells by complementary DNA oligonucleotide and cationic liposomes containing galactosylated ceramide and a novel cationic lipid, DMKE (O,O'-dimyristyl-N-lysyl glutamate), through glycoprotein-mediated endocytosis. An antagomir PNA was designed to target miR-122, which is required for translation of the hepatitis C virus (HCV) genome in hepatocytes, and was hybridized to a DNA oligonucleotide for complexation with cationic liposome. The PNA-DNA hybrid molecules were efficiently internalized into hepatic cells by complexing with the galactosylated cationic liposome in vitro. Galactosylation of liposome significantly enhanced both lipoplex cell binding and PNA delivery to the hepatic cells. After 4-h incubation with galactosylated lipoplexes, PNAs were efficiently delivered into hepatic cells and HCV genome translation was suppressed more than 70% through sequestration of miR-122 in cytoplasm. PNAs were readily released from the PNA-DNA hybrid in the low pH environment of the endosome. The present study indicates that transfection of PNA-DNA hybrid molecules using galactosylated cationic liposomes can be used as an efficient non-viral carrier for antagomir PNAs targeted to hepatocytes.

• The Plant Pathology Journal
• /
• 제37권2호
• /
• pp.162-172
• /
• 2021

Soybean mosaic virus (SMV) is the predominant viral pathogen that affects the yield and quality of soybean. The natural host range for SMV is very narrow, and generally limited to Leguminosae. However, we found that SMV can naturally infect Pinellia ternata and Atractylodes macrocephala. In order to clarify the molecular mechanisms underlying the cross-family infection of SMV, we used double-stranded RNA extraction, rapid amplification of cDNA ends polymerase chain reaction and Gibson assembly techniques to carry out SMV full-length genome amplification from susceptible soybeans and constructed an infectious cDNA clone for SMV. The genome of the SMV Shanxi isolate (SMV-SX) consists of 9,587 nt and encodes a polyprotein consisting of 3,067 aa. SMV-SX and SMV-XFQ008 had the highest nucleotide and amino acid sequence identities of 97.03% and 98.50%, respectively. A phylogenetic tree indicated that SMV-SX and SMV-XFQ018 were clustered together, sharing the closest relationship. We then constructed a pSMV-SX infectious cDNA clone by Gibson assembly technology and used this clone to inoculate soybean and Ailanthus altissima; the symptoms of these hosts were similar to those caused by the virus isolated from natural infected plant tissue. This method of construction not only makes up for the time-consuming and laborious defect of traditional methods used to construct infectious cDNA clones, but also avoids the toxicity of the Potyvirus special sequence to Escherichia coli, thus providing a useful cloning strategy for the construction of infectious cDNA clones for other viruses and laying down a foundation for the further investigation of SMV cross-family infection mechanisms.

• Proceedings of the Korean Society of Plant Pathology Conference
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.123.1-123
• /
• 2003

The objective of this work was to analyze the population of sequence variants of citrus tristeza virus (CTV) isolates in Korea and to make the phylogeny trees of CTV in Korea. We also tried to analyze and find the mild strain of CTV to apply for the cross protection. The CTV isolates from yuzu (C. Junos) collected from different geographic areas of Southern provinces such as Namhae-Do, Kerche-Do, Bosung, Wan-Do and Koheung and Jeju-Bo, Korea were used for SSCP analysis. The SSCP profiles of the cDNAS obtained by RT-PCR with primers specifically designed for the p20 of the CTV population. The SSCP profiles obtained from 150 PCR products in yuzu contained two or three DNA bands, whereas, in some case, others contained four or more bands of similar intensity. The pathologically mild isolates of CTV usually yielded two DNA bands by SSCP profiles, whereas the SSCP profiles of the most virulent isolates contained more than two DNA bands. Plants shown severe stem pitting were corresponded to those plants with typical SSCP profiles of severe strains, and vice versa. This results indicate that the primers designed for SSCP analysis can be used for distinguishing the mild strains from severe strains of CTV.

• Journal of Microbiology and Biotechnology
• /
• 제18권6호
• /
• pp.1044-1049
• /
• 2008

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.

• The Korean Journal of Pain
• /
• 제18권2호
• /
• pp.210-213
• /
• 2005

Acute viral meningitis and myositis are rare complications of varicella-zoster virus (VZV) reactivation. A 71-years-old immunocompetent man, who presented with lower back pain radiating to the left lower extremities, developed vesicles on the L5 dermatomal area. The next day, he had complained of aberrant vesicles on the trunk, face and scalp, with generalized myalgia, headache and dizziness. He was confirmed with VZV meningitis and myositis, as demonstrated by the presence of VZV DNA in the blood and cerebral spinal fluid using a polymerase chain reaction (PCR) amplification. PCR has been used in patients with a VZV infection associated neurological symptoms, and provides a useful tool for the early diagnosis of VZV-associated neurological disease. The patient was treated with bed rest, with intravenous acyclovir for the VZV infection, and intravenous Patient-controlled Analgesia for pain management and the prevention of postherpetic neuralgia. When he visited the outpatient department 3 months later, the skin lesion, leg pain, headache and myalgia had all improved, without sequelae. Here, this case is reported, with a discussion of the relevant literature on its diagnosis and management.

• Korean Journal of Veterinary Research
• /
• 제31권3호
• /
• pp.311-318
• /
• 1991

The DNA fragment representing for Pseudorabies gp50 and gp60(Shope) was cloned by recombinant techniques. The viral DNA was extracted from the infected cells and digested with Bam HI. The 6.8 Kb of Bam HI fragment was isolated from agarose gel and further digested with Nde I followed by Klenow treatment. The blunt ended 4.9Kb fragment was cloned into pTZ18R plasmid vector. The upstream region of gp50 was further manipulated to remove its 5' promoter region and create EcoRl site for possible eukaryotic expression system. The result of partial sequencing of cloned DNA indicated that Shope strain showed 95% homology with gp50 of Rice strain.

• Molecules and Cells
• /
• 제27권2호
• /
• pp.243-250
• /
• 2009

Recent studies suggest a novel role of $HIF-1{\alpha}$ under nonhypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers $HIF-1{\alpha}$ activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of $HIF-1{\alpha}$ protein level as well as the increase in $HIF-1{\alpha}$ target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated $HIF-1{\alpha}$ knock-down. Interestingly, $HIF-1{\alpha}$ knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that $HIF-1{\alpha}$ activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response.

• Bulletin of the Korean Chemical Society
• /
• 제22권1호
• /
• pp.46-52
• /
• 2001

To evaluate the non-ionic polymer, poly(ethylene glycol) (PEG), as a component in cationic copolymers for non-viral gene delivery systems, PEG was coupled to polyethylenimine (PEI). We present the effects of different degrees and shapes of pegylation of PEI on cytotoxicity, water solubility and transfection efficiency. This work reports the synthesis and characterization of a series of cationic copolymers on the basis of the conjugates of PEI with PEG. The modified molecules were significantly less toxic than the original polymer. Moreover, the chemical modification led to enhancement of their solubility. The comparison of pegylated PEIs with different degrees of derivation showed that all the polymers tested reached comparable levels of transgene expression to that of native PEI. As assessed by agarose gel electrophoresis, even highly substituted PEI derivatives were still able to form polyionic complexes with DNA. However, aside from an increase in solubility and retention of the ability to condense DNA, methoxy-PEG-modified PEIs resulted in a significant decrease in the transfection activity of the DNA complexes. In fact, the efficiency of the copolymer was compromised even at a low degree of modification suggesting that the PEG action resulting from its shape is important for efficient gene transfer. The mode of PEG grafting and the degree of modification influenced the transfection efficiency of PEI.

• Microbiology and Biotechnology Letters
• /
• 제36권3호
• /
• pp.173-181
• /
• 2008

Bovine blood, cell, tissue, and organ are used as raw materials for manufacturing biologics such as biopharmaceuticals, tissue-engineered products, and cell therapy. Manufacturing processes for the biologics have the risk of viral contamination. Therefore viral validation is essential in ensuring the safety of the products. Bovine parvovirus (BPV) is one of the common bovine pathogens and has widely been known as a possible contaminant of biologics. In order to establish the validation system for the BPV safety of biologics, a real-time PCR method was developed for quantitative detection of BPV contamination in raw materials, manufacturing processes, and final products. Specific primers for amplification of BPV DNA were selected, and BPV DNA was quantified by use of SYBR Green 1. The sensitivity of the assay was calculated to be $1.3{\times}10^{-1}\;TCID_{50}/mL$. The real-time PCR method was validated to be reproducible and very specific to BPV. The established real-time PCR assay was successfully applied to the validation of Chinese hamster ovary (CHO) cell artificially infected with BPV. BPV DNA could be quantified in CHO cell as well as culture supernatant. Also the real-time PCR assay could detect $1.3{\times}10^0\;TCID_{50}/mL$ of BPV artificially contaminated in bovine collagen. The overall results indicated that this rapid, specific, sensitive, and robust assay can be reliably used for quantitative detection of BPV contamination during manufacture of biologics.

• Journal of fish pathology
• /
• 제27권1호
• /
• pp.1-9
• /
• 2014

The olive flounder, Paralichthys olivaceus is susceptible to viral hemorrhagic septicaemia virus (VHSV) at $15^{\circ}C$ but no mortality at $20^{\circ}C$ even though the virus can grow well in vitro at $20^{\circ}C$. Thus, we designed an experiment to know immune response of olive flounder against VHSV when the host reared at $15^{\circ}C$ or $20^{\circ}C$. cDNA microarray analysis was performed to compare the gene expression patterns of the kidney cells between the host reared at $15^{\circ}C$ or $20^{\circ}C$. The expression of MHC class I, IL-8, myeloperoxidae and endonuclease G-like having function for the antigen presentation and chemokine-factor were up-regulted both the $15^{\circ}C$ and $20^{\circ}C$ during VHSV infection. MHC class II gene existing on antigen-presenting cells and B cell lymphocytes, immunoglobulin (Ig) genes and phagocytosis related genes were down-regulated at $15^{\circ}C$ but highly expressed at $20^{\circ}C$. It can be thought that innate immune related antigen presentation by MHC class I and phagocytosis reaction against VHSV are efficiently occur both the temperature but macrophage or B cell related antigen presentation via MHC class II fails to induce downstream immune reactions (adaptive immunity) to make antibody, and it can be one of the reason that causes high mortality only at $15^{\circ}C$.