• Microbiology and Biotechnology Letters
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• v.7 no.3
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• pp.165-173
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• 1979

Recovery component for cell division in UV-irradiated E. coli B was detected with use of the cell extract of E. coli B/r which is a resistant mutant of E. coli B against UV-irradiation. The active substance was non-dialyzable and increased the activity by adding B-NAD remarkably. One more factor for increasing or promoting the restoration recognized was magnesium. Magnesium was effective to stabilze the substance in procedure of isolation. Two active substances were obtained from sucrose gradient centrifugation. One of them was recovred from the botton area and the other from top area just below below surface. the former was not stabilized by magnesium, while the latter stabilized the activity by it remarkably. The former which did not require magnesium was insensitive to protease and the latter which required magnesium was sensitive to it. Both were insensitive to RNase and DNase. Recovery ratio was doubled by using nitrogen gas than aeration in purification process. DNA-ligase less mutant was revealed same activity on it's recovery ratio with the parent strain of E. coli K-12. The active substance stimulating the filament cell may exist as a complex which is inactivated easily in the dissociated state ana requrie B-NAD or magnesium.

• Biotechnology and Bioprocess Engineering:BBE
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• v.7 no.1
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• pp.1-5
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• 2002

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.386-392
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• 2015

A novel recombinant bacmid, bEasyBm, that enables the easy and fast generation of pure recombinant baculovirus without any purification step was constructed. In bEasyBm, attR recombination sites were introduced to facilitate the generation of a recombinant viral genome by in vitro transposition. Moreover, the extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of the Cotesia plutellae bracovirus early promoter to negatively select against the nonrecombinant background. The bEasyBm bacmid could only replicate in host insect cells when the barnase gene was replaced with the gene of interest by in vitro transposition. When bEasyBm was transposed with pDualBac-EGFP, the resulting recombinant virus, EasyBm-EGFP, showed high levels of EGFP expression efficiency compared with that of non-purified recombinant virus BmGOZA-EGFP, which was constructed using the bBmGOZA system. In addition, nonrecombinant backgrounds were not detected in unpurified EasyBm-EGFP stocks. Based on these results, a high-throughput system for the generation of multiple recombinant viruses at a time was established.

• BMB Reports
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• v.52 no.2
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• pp.133-138
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• 2019

Upon viral infection, the 2', 5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNaseL) system works to cleave viral RNA, thereby blocking viral replication. However, it is unclear whether OAS proteins have a role in regulating gene expression. Here, we show that OAS1 and OAS3 act as negative regulators of the expression of chemokines and interferon-responsive genes in human macrophages. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology was used to engineer human myeloid cell lines in which the OAS1 or OAS3 gene was deleted. Neither OAS1 nor OAS3 was exclusively responsible for the degradation of rRNA in macrophages stimulated with poly(I:C), a synthetic surrogate for viral double-stranded (ds)RNA. An mRNA sequencing analysis revealed that genes related to type I interferon signaling and chemokine activity were increased in $OAS1^{-/-}$ and $OAS3^{-/-}$ macrophages treated with intracellular poly(I:C). Indeed, retinoic-acid-inducible gene (RIG)-I- and interferon-induced helicase C domain-containing protein (IFIH1 or MDA5)-mediated induction of chemokines and interferon-stimulated genes was regulated by OAS3, but Toll-like receptor 3 (TLR3)- and TLR4-mediated induction of those genes was modulated by OAS1 in macrophages. However, stimulation of these cells with type I interferons had no effect on OAS1- or OAS3-mediated chemokine secretion. These data suggest that OAS1 and OAS3 negatively regulate the expression of chemokines and interferon-responsive genes in human macrophages.

• Tuberculosis and Respiratory Diseases
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• v.71 no.1
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• pp.8-14
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• 2011

Background: MicroRNAs (miRNAs) have demonstrated their potential as biomarkers for lung cancer diagnosis. In recent years, miRNAs have been found in body fluids such as serum, plasma, urine and saliva. Circulating miRNAs are highly stable and resistant to RNase activity along with, extreme pH and temperatures in serum and plasma. In this study, we investigated serum miRNA profiles that can be used as a diagnostic biomarker of non-small cell lung cancer (NSCLC). Methods: We compared the expression profile of miRNAs in the plasma of patients diagnosed with lung cancer using an miRNA microarray. The data from this assay were validated by quantitative real-time PCR (qRT-PCR). Results: Six miRNAs were overexpressed and three miRNAs were underexpressed in both tissue and serum from squamous cell carcinoma (SCC) patients. Sixteen miRNAs were overexpressed and twenty two miRNAs were underexpressed in both tissue and serum from adenocarcinoma (AC) patients. Of the four miRNAs chosen for qRT-PCR analysis, the expression of miR-23a was consistent with microarray results from AC patients. Receiver operating characteristic (ROC) curve analyses were done and revealed that the level of serum miR-23a was a potential marker for discriminating AC patients from chronic obstructive pulmonary disease (COPD) patients. Conclusion: Although a small number of patients were examined, the results from our study suggest that serum miR-23a can be used in the diagnosis of AC.

• The Journal of Korean Society of Virology
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• v.26 no.1
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• pp.77-90
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• 1996

Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the virus in the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western blotting. Therefore, the preparation of this protein in the level of higher quantity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA) virus and expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen. First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase $H^-$ AMV reverse transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplified specifically by Taq DNA polymrerase. The pT7blue (R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of BL21 (DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression. Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG was used and the expressed protein was characterized by Western blotting. The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from patients of HFRS as to be tested by ELISA and Western blotting.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.10
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• pp.1550-1557
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• 2018

Objective: Circular RNAs (circRNAs) are a newfound class of non-coding RNA in animals and plants. Recent studies have revealed that circRNAs play important roles in cell proliferation, differentiation, autophagy and apoptosis during development. However, there are few reports about muscle development-related circRNAs in livestock. Methods: RNA sequencing analysis was employed to identify and annotate circRNAs from longissimus dorsi of sheep. Reverse transcription followed by real-time quantitative (q) polymerase chain reaction (PCR) analysis verified the presence of these circRNAs. Targetscan7.0 and miRanda were used to analyse the interaction of circRNA-microRNA (miRNA). To investigate the function of circRNAs, an experiment was conducted to perform enrichment analysis hosting genes of circRNAs using gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. Results: About 75.5 million sequences were obtained from RNA libraries of sheep skeletal muscle. These sequences were mapped to 729 genes in the sheep reference genome. We identified 886 circRNAs, including numerous circular intronic RNAs and exonic circRNAs. Reverse transcription PCR (RT-PCR) and DNA sequencing analysis confirmed the presence of several circRNAs. Real-Time RT-PCR analysis exhibited resistance of sheep circRNAs to RNase R digestion. We found that many circRNAs interacted with muscle-specific miRNAs involved in growth and development of muscle, especially circ776. The GO and KEGG enrichment analysis showed that hosting genes of circRNAs was involved in muscle cell development and signaling pathway. Conclusion: The study provides comprehensive expression profiles of circRNAs in sheep skeletal muscle. Our study offers a large number of circRNAs to facilitate a better understanding of their roles in muscle growth. Meanwhile, we suggested that circ776 could be analyzed in future study.

• Horticultural Science & Technology
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• v.32 no.6
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• pp.912-916
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• 2014

Japanese apricot (Prunus mume Siebold and Zucc.) is a deciduous tree of the family Rosaceae, and it has long been used as a folk remedy for cough and dyspepsia. A new cultivar 'Okjoo' was developed from a cross between 'Gyokuei' and 'Rinsyu' carried out at the National Institute of Horticultural & Herbal Science in 1993. It w as s elected for good shape, large size and high yield capacity in 2006, and then it was granted official patent No. 4556 in 2013. It blooms 4 days and 2 days earlier than 'Gyokuei' and 'Rinsyu', respectively. Its flower petal color is pink, and the pollen amount is negligible. Its S-genotype, determined using Polymerase Chain Reaction with a S-RNase gene-specific primer pair, is $S_3S_6$. The average optimum harvest time of 'Okjoo' is late June. The fruit is round in shape and its suture is shallow. Average fruit weight is 18.5 g, and it contains total soluble solids $7.66^{\circ}Brix$ and titratable acidity at 4.81%. Fruit skin color is green. Sometimes only the light side of the fruits seems to develop blush. The incidence levels of scab (Cladosporium carpophilum Thumen) and bacterial shot hole (Xanthomonas arboricola pv. Pruni) are quite low. Consequently, 'Okjoo' seems to be a promising new cultivar for Japanese apricot growers.