• Biomedical Science Letters
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• v.27 no.4
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• pp.283-290
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• 2021

Severe acute respiratory syndrome coronavirus (SARS-CoV2) is a major coronavirus that infects humans with human Coronavirus (HuCoV)-229E, HCoV-OC43, HCoV-HKU1, HCoV-NL63, Severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle east respiratory syndrome coronavirus (MERS-CoV). SARS-CoV2 is currently a global pandemic pathogen. In this study, we developed conventional RT-PCR based diagnostic system for the detection of SARS-CoV2 which is relatively inexpensive but has high stability and a wide range of users. Three conventional RT-PCR primer sets capable of forming specific band sizes by targeting the ORF1ab [232 nucleotide (nt)], E (200 nt) and N (288 nt) genes of SARS-CoV2 were developed, respectively, and it were confirmed to be about 10~100 times higher detection sensitivity than the previously reported methods. In addition, a standard positive control that can generate specific amplicons by reacting with the developed RT-PCR primers and verify the false-positiv from contamination of the laboratory was produced. Therefore, the diagnostic system that uses the RT-PCR method is expected to be used to detect SARS-CoV2.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.53-53
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• 2003

The aim of this study was to determine the interactive effects of BSA and EGF on the viability and development of porcine diploid parthenotes developing in vitro. The addition of 0.1 and 0.4% BSA to the culture medium enhanced the development of 4-cell parthenotes to the blastocyst stage but EGF had no effect. However, while BSA also increased cell numbers, it did so only when EGF was also present. Either agent on its own had no effect. Similarly, apoptosis in the blastocysts was not influenced by either agent on its own but was reduced when both BSA and EGF were present. Furthermore, semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that EGF enhanced the mRNA expression of BclxL in the presence of 0.4% BSA but BSA and EGF alone had no effect. EGF and/or BSA did not influence Bak gene expression in the blastocyst stage parthenotes. These results suggest that BSA has both beneficial and detrimental effects on the viability of porcine diploid parthenotes developing in vitro and that exogenous EGF may block some of the detrimental effects of BSA, possibly by inhibiting the BSA-induced apoptosis by increasing Bcl-xL expression. This results in a net increase in cell numbers in porcine diploid parthenotes developing in vitro.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.47-47
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• 2003

The present study was conducted to determine the expression of the antioxidant enzyme(CuZn-SOD, Mn-SOD and GPX and apoptosis gene(caspase-3) for in vitro culture in in vitro maturation and in vitro fertilization(IVM/IVF) embryos in porcine. Porcine embryos derived from IVM/IVF were cultured in NCSU23 medium under 5% $CO_2$ in air at 38.5$^{\circ}C$. The patterns of gene expression for several antioxidant enzyme and apoptosis genes during preimplantion porcine embryo development were examined by the modified semi-quantitative single cell reverse transcriptase- polymerase chain reaction (RT-PCR). Preimplantation porcine embryos produced by IVM/IVF have expressed mRNAs for CuZn-SOD and GPX, whereas transcripts for Mn-SOD have not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell, 16 cell and morula stages. The fas ligand transcripts were detected in porcine blastocyst. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in in vitro culture of porcine IVM/IVF embryos.

• BMB Reports
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• v.57 no.1
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• pp.40-49
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• 2024

Prokaryotes encode clustered regularly interspaced short palindromic repeat (CRISPR) arrays and CRISPR-associated (Cas) genes as an adaptive immune machinery. CRISPR-Cas systems effectively protect hosts from the invasion of foreign enemies, such as bacteriophages and plasmids. During a process called 'adaptation', non-self-nucleic acid fragments are acquired as spacers between repeats in the host CRISPR array, to establish immunological memory. The highly conserved Cas1-Cas2 complexes function as molecular recorders to integrate spacers in a time course manner, which can subsequently be expressed as crRNAs complexed with Cas effector proteins for the RNA-guided interference pathways. In some of the RNA-targeting type III systems, Cas1 proteins are fused with reverse transcriptase (RT), indicating that RT-Cas1-Cas2 complexes can acquire RNA transcripts for spacer acquisition. In this review, we summarize current studies that focus on the molecular structure and function of the RT-fused Cas1-Cas2 integrase, and its potential applications as a directional RNA-recording tool in cells. Furthermore, we highlight outstanding questions for RT-Cas1-Cas2 studies and future directions for RNA-recording CRISPR technologies.

• Journal of Microbiology and Biotechnology
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• v.34 no.6
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• pp.1340-1347
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• 2024

Ehretia asperula is a medicinal plant of the Ehretiaceae family used to treat inflammatory disorders, but the underlying mechanisms are not fully elucidated. The anti-inflammatory potential was determined based on enzyme cyclooxygenase-2 (COX-2) inhibition, which showed that the 95% ethanol extract (95ECH) was most effective with a half-maximal inhibitory concentration (IC₅₀) value of 34.09 ㎍/mL. The effects of 95ECH on phagocytosis, NO production, gene, and protein expression of the cyclooxygenase 2/prostaglandin E2 (COX-2/PGE2) and inducible nitric oxide synthase/ nitric oxide (iNOS/NO) pathways in lipopolysaccharide (LPS)-induced RAW264.7 cells were examined using the neutral red uptake and Griess assays, reverse-transcriptase polymerase chain reactions (RT-PCR), and enzyme-linked immunosorbent assays (ELISA). The results showed that 95ECH suppressed phagocytosis and the NO production in activated macrophage cells (p < 0.01). Conversely, 95ECH regulated the expression levels of mRNAs for cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) as well as the corresponding proteins. In addition, PGE2 production was inhibited in a dose-dependent manner by 95ECH, and the expression of iNOS and COX-2 mRNAs was decreased in activated macrophage cells, as expected. Therefore, 95ECH from E. asperula leaves contains potentially valuable compounds for use in inflammation management.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.61-70
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• 1983

Synchronized cat kidney cells chronically infected with feline leukemia virus (FeLV) were used to study virus production, the synthesis of group specific antigen (gag) and envelope (env) proteins, the expression of env protein on the cell surface during the cell cycle, and the stability of viral RNA. As detecting method, we developed the radioimmunoassay (RIA) system using beta-emission of $^{131}I$ and demonstrated the validity of this system by comparison with routine RIA system using gamma-emission of $^{125}I$. The produced virus was analysed by developed RIA interval was determined by measuring reverse transcriptase activity. The results show that infected cells produce the complete virus particle containing products of gag, env and pol genes of FeLV, and maximum virus production occurs during mitosis of synchronized cells. Labeling of the cell surface of synchronized cells with $^{131}I$ shows that the amount of $gp70^{env}$ on the cell surface parallels cellular gorwth. Therefore, the cell cycle-dependent release of virus is not petition RIA of synchronized cells with $^{131}I$ labeled viral proteins synthesis during the cell cycle. The rate of synthesis of gag protein shows three peaks, corresponding to the $G_1,\;late\;S\;and\;late\;G_2$ phases of cell cycle. But the rate of synthesis of env protein dose not change, suggesting that in these cells the synthesis of these two gene products in controlled seperately. In Actionomycin D treated cells, the synthesis of viral proteins decreased sharply from 8 hours after treatment, and the late S and $G_2$ peaks of gag protein synthesis were disappeared. This shows the stability of viral RNA for about 6 hours in the absence of continuing viral RNA synthesis.

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.1
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• pp.91-100
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• 2003

In the present study, we investigated the protective effect of the Lycii Fructus water extracts (LFE) against CCl4-induced hepatotoxicity and the mechanism underlying these protective effects in the rats. The pretreatment of LFE has shown to possess a significant protective effect by lowering the serum alanine and aspartate aminoteansferase (AST and ALT) and alkaline phosphatase (ALP). This hepatoprotective action was confirmed by histological observation, In addition, the pretreatment of LFE prevented the elevation of hepatic malondialdehyde (MDA) formation and the depletion of reduced glutathione (GSH) content and catalase activity in the liver of CC1₄-injected rats. The LFE also displayed hydroxide radical scavenging activity in a dose-dependent manner (IC50 = 83.6 μg/ml), as assayed by electron spin resonance (ESR) spin-trapping technique. Moreover, the expression of cytochrome P450 2E1 (CYP2E1) mRNA, as measured by reverse transcriptase-polymerase chain reaction (RT-PCR), was significantly decreased in the liver of LFE-pretreated rats when compared with that in the liver of control group. Based on these results, it was suggested that the hepatoprotective effects of the LFE may be related to antioxidant effects and regulation of CYP2E1 gene expression.

• The Plant Pathology Journal
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• v.20 no.3
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• pp.212-219
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• 2004

Three isolates of Cucumber mosaic virus (CMV) from lily plants showing mosaic and distortion symptoms were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) using primers specific to Cucumovirus genus namely, LK-CMV, LK4-CMV, and LKS-CMV. Restriction enzymes patterns of the RT-PCR products revealed that the lily isolates belonged to subgroup IA of CMV. In terms of biological properties, the lily isolates have highly similar but distinct pathogenicity as reported in other lily strains and ordinary strains of CMV. To characterize the molecular properties, cDNAs containing coat protein (CP) gene and 3' non-coding region (NCR) of RNA3 for the isolates were cloned and their nucleotide sequences were determined. The CP similarity (218 amino acids) was highly homologous (>97％) with that of subgroup I CMV strains. However, an additional 20-nulcleotide long segment was only present in 3' NCR of lily isolates, which form an additional stem-loop RNA structure. By using chimeric construct exchange cDNA containing 3'NCR of LK-CMV into the full-length cDNA clone of RNA3 of Fny-CMV, this additional segment may prove to be significant in the identification and fitness of the virus in lily plants. The pathology of zucchini squash infected by F1F2L3-CMV, a pseudorecombinant virus was showed to change drastically the severe mosaic and stunting symptom into a mild chlorotic spot on systemic leave, compared with Fny-CMV. To delimit the sequence of RNA3 affected the pathology, various RNA3 chimeras were constructed between two strains of CMV. The symptom determinants of F1F2L3-CMV were mapped to the positions amino acid 234, 239, and 250 in 3a movement protein (MP). RNA3 chimeras changed the sequences encoding three amino acids were resulted in alteration of systemic symptom.

• Korean Journal of Agricultural Science
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• v.43 no.1
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• pp.21-27
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• 2016

Apple stem grooving virus (ASGV), Apple chlorotic leaf spot virus (ACLSV), and Apple stem pitting virus (ASPV) have been known to induce top working disease causing economical damage in apple. Occurrences of these three viruses in pome fruit trees, including apple, have been reported around the world. The transmission of the three viruses was reported by grafting, and there was no report of transmission through mechanical contact, insect vector, or seed except some herbaceous hosts of ASGV. As RNA extraction methods for fruit trees, Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and multiplex RT-PCR techniques have been improved for reliability and stability, and low titer viruses that could not be detected in the past have become detectable. We studied the seed transmission ability of three apple viruses through apple seedling diagnosis using RT-PCR. Nineteen seeds obtained from commercially grown apple were germinated and two of the resulting plants were ASGV positive. Seven clones of the amplified ASGV coat protein (CP) genes of these isolates were sequenced. Overall sequence identities were 99.84% (nucleotide) and 99.76% (amino acid). Presence of a previously unreported single nucleotide and amino acid variation conserved in all of these clones suggests a possible association with seed transmission of these 'S' isolates. A phylogenetic tree constructed using ASGV CP nucleotide sequences showed that isolate S sequences were grouped with Korean, Chinese, Indian isolates from apple and Indian isolates from kiwi.

• Korean Journal of Veterinary Research
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• v.37 no.2
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• pp.367-374
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• 1997

Bovine rotaviruses(A, 288, 55086 strains) isolated from fecal samples in Korea were propagated onto MA104 cells and were confirmed tentatively as G6, G8, and G10, respectively, by RFLP analysis. Full-length VP7 gene of these isolates was amplified by reverse transcriptase polymerase chain reaction(RT-PCR) using VP7 specific primers and cloned into TA vector. Nucleotide and deduced amino acid sequences of VP7 genes of the isolates were determined and compared with those of bovine rotavirus reference strains(NCDV; G6, UK; G6, Cody I-801; G8 and B223; G10). A, 288 and 55086 isolates showed high degree of nucleotide sequence homology with NCDV and UK(93% and 94%), Cody I-801(86%) and B223(97%), respectively, However, they showed 71~74% of nucleotide sequence homlogy with bovine rotavirus reference strains which belong to different serotypes. From the results of deduced amino acid sequence homology analysis, three isolates showed 94~96% of homology with the same serotype reference strains but 80~84% of homology with the different serotype reference strains. Three bovine rotavirus isolates, A, 288 and 55086 strains, were confirmed as G6, G8, and G10, respectively, by nucleotide and deduced amino acid sequence analysis.