• 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.

• Journal of Microbiology and Biotechnology
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• v.4 no.3
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• pp.183-190
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• 1994

Cell-free extracts prepared from cultured insect cells, Spodoptera. frugiperda, were analyzed for activation of early gene transcription of an insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV). The template DNA used for in vitro transcription assays contained promoter sites for the baculovirus genes that have been classified as immediate early ($\alpha$) or early genes. These genes are located in the HindIII-K/Q region of the AcNPV genome. Nuclei isolated from the AcNPV-infected Spodoptera frugiperda cells were also used for in vitro transcription analysis by RNase-mapping the labeled RNA synthesized from in vitro run-on reaction in the isolated nuclei. The genes studied by this technique were p26 and pl0 genes which were classified as delayed early and late gene, respectively. We found that transcription of the genes from the HindIII-K region was accurately initiated and unique in the whole cell extract obtained from uninfected cells, although abundance of the in vitro transcripts was reverse to that of in vivo RNA. With isolated nuclei transcription of the p26 gene was inhibited by $\alpha$-amanitin suggesting that the p26 gene was transcribed by host RNA polymerase II. However, transcription of the pl0 gene in isolated nuclei was not inhibited by $\alpha$-amanitin, but rather stimulated by the inhibitor. We also found that the synthesis of $\alpha$-amanitin-resistant RNA polymerase was begun before 6 hr p.i., the time point at which the onset of viral DNA replication as well as the appearance of a-amanitin-resistant viral transcripts were detected. These studies give us strong evidence to support the previous data that early genes of AcNPV were transcribed by host RNA polymerease III, while transcription of late genes was mediated at least by a novel $\alpha$-amanitin-resistant RNA polymerase.

• Journal of Yeungnam Medical Science
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• v.20 no.2
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• pp.187-196
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• 2003

The cytokines are the hormone-like proteins, which are produced in the mononuclear cells. They have many roles, such as immune mediators, cell differentiations, angiogenesis. The chemokines have chemotactic effects which control the host immune response. There were few reports about the cytokines associated with musculoskeletal tumors. From late 1980s, the cytokine studies of bone tumors such as osteosarcoma were started, but most studies for benign and malignant musculoskeletal tumors were left to be explored. To evaluate the characteristics of the cytokines in variable musculoskeletal tumors, tissues were obtained from the seven patients who visited the Yeungnam University hospital from February to July 2000. They were lipoma (1 case), parosteal osteoma (1 case), enchondroma (2 cases), pigmented villonodular synovitis (1 case), ganglion (1 case), and metastaic squamous cell carcinoma (1 case). The gene experession of the cytokines were analyzed by RNase protection assay (RPA) and reverse transcription-polymerase chain reaction (RT-PCR). The lipoma and parosteal osteoma expressed MIP-$1{\beta}$, and IP-10 genes. The two enchondromas showed different results, one expressed all of MIP-$1{\alpha}$, MIP-$1{\beta}$ and IP-10 genes but the other expressed none of above. The pigmented villonodular synovitis strongly expressed MIP-$1{\alpha}$ and IP-10 when compared with the other cases. The ganglion did not express all of the chemokines mentioned above. And the metastatic squamous cell carcinoma expressed all of the chemokines and especially IP-10 was highly expressed. Even though this study has only a few cases, these results provide a basis for the cytokine mediating network study in musculoskeletal tumors.

• 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.

• The Korea Journal of Herbology
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• v.21 no.2
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• pp.9-13
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• 2006

Objectives : To determine the DNA sequence of the 18S rRNA gene of the Rehmannia glutinosa and analyze it phylogenetically Methods : Dried root of the Rehmannia glutinosa was ground with a mortar and pestle. Glass beads(0.5 mm in diameter), TE buffer and SDS solution were added to that. The mixture was vortexed vigorously and extracted with the mixture of phenol, chloroform and isoamyl alcohol and with the mixture of the chloroform and isoamyl alcohol. The nucleic acids were precipitated with ethanol and resuspended in TE buffer. Contaminating RNA was digested with RNAse A and the DNA was purified further with the Geneclean Turbo Kit. This DNA was used as a template for amplification of the 18S rRNA gene by PCR. The PCR product was cloned in the pBluescript SK II plasmid by blunt-end ligation and the DNA sequence of the insert was determined. This DNA sequence was analyzed phylogenetically by the BLAST program. Results and Conclusion : Vortexing the ground powder of the dried plant root with glass beads during cell lysis improved recovery of DNA. The DNA sequence of the Rehmannia glutinosa 18S rRNA gene was determined and deposited at the GenBank as the accession number DQ469606. Phylogenetic analysis of that sequence showed the relationship between the members of the family of Scrophulariaceae and also the close relationship of the Buddleja davidii to the members of the Scrophulariaceae family.

• Molecules and Cells
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• v.41 no.3
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• pp.214-223
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• 2018

Oligoadenylate synthetase (OAS) protein family is the major interferon (IFN)-stimulated genes responsible for the activation of RNase L pathway upon viral infection. OAS-like (OASL) is also required for inhibition of viral growth in human cells, but the loss of one of its mouse homolog, OASL1, causes a severe defect in termination of type I interferon production. To further investigate the antiviral activity of OASL1, we examined its subcellular localization and regulatory roles in IFN production in the early and late stages of viral infection. We found OASL1, but not OASL2, formed stress granules trapping viral RNAs and promoted efficient RLR signaling in early stages of infection. Stress granule formation was dependent on RNA binding activity of OASL1. But in the late stages of infection, OASL1 interacted with IRF7 transcripts to inhibit translation resulting in down regulation of IFN production. These results implicate that OASL1 plays context dependent functions in the antiviral response for the clearance and resolution of viral infections.

• Korean Journal of Veterinary Research
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• v.50 no.3
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• pp.221-229
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• 2010

Encephalomyocarditis virus (EMCV) belongs to the genus Cardiovirus within the family Picornaviridae. EMCV has been recognized either as a cause of mortality in young pigs, due to acute myocarditis, or of reproductive failure in sows. An EMCV K3 strain was isolated from the heart and brain in a mummified and aborted swine fetus in 1989. For the molecular characterization of the poly(C)-tract of EMCV Korean isolates, K3 strain, viral RNA was extracted and digested with RNase T1, and analyzed the length of the poly(C)-tract by polyacrylamide gel electrophoresis. The poly(C) regions also were amplified by RT-PCR and sequenced. The present study shows that K3 strain of EMCV had a short polymorphic poly(C) tracts (5 to 30 C's) with sequences consisting of $C_9$, $C_{10}$, $C_{13}$, $C_{14}$, $C_{16}$, $C_{20}$, $CUC_{11}$, $C_8UCUC_3UC_{10}$, $C_9UCUC_3UC_{10}$, $C_{10}UCUC_3UC_{10}$, etc. These polymorphism of poly(C)-tracts of EMCV K3 strain implies the historical information of in vivo and/or in vitro passage.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.1
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• pp.315-321
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• 2016

Background: Herbal medicine has becoming a potential source of treatment for different types of cancer including breast cancer. It has been shown that plants from the family Rhamnaceae possess anticancer activity. Objective: In this study, we determined the antiproliferative influence of Ziziphus spina christi- a species from this family- on the MCF-7 (human breast adenocarcinoma) cell line. Materials and Methods: The cytotoxicity of the total extract, ethanol, ethanol-aqueous (1:1) as well as aqueous fractions of Ziziphus spina christi leaves was evaluated through MTT assay against MCF-7 cell line. Cell cycle inhibition and apoptosis induction were assessed by flowcytometry cycle RNase/PI analysis and Annexin V-FLUOS, respectively. Apoptosis was also analyzed by immunoblotting assay. Results: Our results indicated that the ethanolic fraction had the lowest $IC_{50}$ value (0.02 mg/ml), induced cell cycle arrest at the G1/S phase as well as apoptosis after a 48h of treatment. Conclusions: This is the first report on anticancer effect of Ziziphus spina christi ethanolic fraction on breast cancer cells, providing a scientific basis for its utility in traditional medicine. However, further in-depth studies are needed to confirm the precise mechanisms.

• BMB Reports
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• v.29 no.4
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• pp.353-358
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• 1996

Bovine angiogenin (bAng) is a potent blood vessel inducing protein purified from cow In ilk. fluorescence spectroscopy has been used to study the interaction of bAng with actin in 50 mM Tris-HCl pH 7.5, and 1 mM $CaCl_2$ at $25^{\circ}C$. Actin contains four tryptophans but bAng contains no tryptophans. A 50% decrease in intrinsic fluorescence accompanied formation of the bAng/actin complex. By contrast, the interaction of RNase A, a homologous protein to bAng, with actin results in about 10% quenching of the fluorescence. Fluorescence titration experiments were performed by adding increasing concentrations of bAng (0~1.0 ${\mu}M$) to a constant concentration of actin (0.1 ${\mu}M$), and the dissociation constant $K_d$ for the bAng/actin complex and the stoichiometry n were measured as $20{\pm}1$ nM and $1.0{\pm}0.1$ respectively. These results suggest that the interaction between bAng with actin is specific and that quenching of actin fluorescence has occurred in the bAng/actin complex. The bAng binding sites of actin are discussed in the results of this study, and we propose that Trp-80 in the small domain of bovine actin is responsible for the bAng/actin binding.

• 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.