• Korean Journal of Microbiology
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• v.44 no.2
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• pp.105-109
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• 2008

We studied on the function and localization of fission yeast Schizosaccharomyces pombe Nup97p, which is homologous to nucleoporin Nic96p in budding yeast Saccharomyces cerevisiae. There was no effect on growth and $poly(A)^{+}$ RNA distribution of cells when nup97 gene was overexpressed. However, the haploid ${\Delta}nup97::kan^{r}$ null mutants confirmed extensive $poly(A)^{+}$ RNA accumulation in the nucleus, abnormal DNA distribution, and cessation of growth when nup97 expression was repressed. We determined the subcellular localization of Nup97 tagged at the N terminus or the C terminus with GFP. Both fusions complemented growth defect of ${\Delta}nup97::kan^{r}$ null mutants. An integrated version of the nup97-GFP fusion was constructed at the nup97 locus. Nup97-GFP fusions expressed from its own promoter was localized at the nuclear periphery with a punctate appearance. These results suggest that Nup97p in fission yeast is also nucleoporin, which is involved in mRNA export.

• Korean Journal of Microbiology
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• v.44 no.1
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• pp.80-84
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• 2008

We constructed the null mutants of fission yeast Schizosaccharomyces pombe spDbp5 gene that is homologous to DEAD-box RNA helicase DBP5 in budding yeast Saccharomyces cerevisiae, which plays important roles in mRNA export out of nucleus. A null mutant in an $h^+/h^+$ diploid strain was constructed by replacing the spDbp5-coding region with an $ura4^+$ gene using one-step gene disruption method. Tetrad analysis showed that the spDbp5 is essential for vegetative growth. The haploid spDbp5 null mutants harboring pREP81X-spDbp5 plasmid showed extensive $poly(A)^+$ RNA accumulation in the nucleus and decrease in the cytoplasm after repression of spDbp5 expression. These results suggest that spDbp5 is also involved in mRNA export from the nucleus.

• Journal of Microbiology
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• v.42 no.1
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• pp.32-36
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• 2004

We have previously isolated three synthetic lethal mutants from Schizosaccharomyces pombe in order to identify mutations in the genes that are functionally linked to spmex67 with respect to mRNA export. A novel rsm1 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex1. The rsml gene contains no introns and encodes a 296 amino-add-long protein with the RING finger domain, a C3HC4 in the N-terminal half. The Δrsm1 null mutant is viable, but it showed a slight poly(A)$\^$＋/ RNA accumulation in the nucleus. Also, the combination of Δrsm1 and Δspmex67 mutations confers synthetic lethality that is accompanied by the severe poly(A)$\^$＋/ RNA export defect. These results suggest that rsm1 is involved in mRNA export from the nucleus.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.357-389
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• 1987

In an attempt to clarify the physiological functions of individual isoperoxidases, we have studied enzymatic and immunological properties as well as cellular distribution of isoperoxidases from tobacco callus and Korean radish. The gene expression patterns of isoperoxidases in shoot and non-shoot-forming tobbaco callus were also examined by rabbit reticulocyte lysatein vitro translation system. These results indicate that fraction of translatable poly(A)-isoperoxidase mRNA was increased considerably in shoots. At the present time, at least 6-7 isoperoxidases could be detected from the translation mixture of total cellular RNA, among which only one cell wall localized anodic isoperoxidase (named A3) mRNA was bimorphic mRNA. These data suggest the possible regulation of peroxidase activity during shoot formation by altering the polyadenylation state of mRNA. In case of Korean radish seedlings, poly(A)- peroxidase mRNA were also increased depending upon aging.

• The Korea Journal of Herbology
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• v.36 no.1
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• pp.97-102
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• 2021

Objectives : Virus infection through the respiratory tract causes various inflammatory diseases such as pneumonia, cystic fibrosis, and obstructive pulmonary disease, causing enormous social damage. Therefore, it is very important to develop a treatment and prevention of infectious diseases. In this study, we investigated the effect of water extracts of Liriope muscari (WELM), known to improve lung function, on the inflammatory response of lung carcinoma cell line A549 cells induced by the viral double stranded RNA mimetic Polyinosinic:polycytidylic acid (Poly I:C). Methods : The cell viability by WELM treatment was analyzed using MTS assay in A549 cells. After inducing an inflammatory response to WELM-treated A549 cells with Poly I:C, the degree of apoptosis was confirmed through bright field microscopy. Interferon beta (IFN-β) mRNA expression level in A549 cells was analyzed by quantitative reverse transcription PCR (qRT-PCR). Results : WELM treatment has no significant effect on cell viability of A549 cells. We confirmed that pre-treatment of WELM effectively reduces the Poly I:C-induced apoptotic cell death in A549 cells. In addition, it was confirmed that the mRNA expression level of IFN-β, a pro-inflammatory cytokine increased by Poly I:C treatment, was significantly suppressed by WELM treatment in A549 cells. Conclusions : These results provide the evidence that WELM is effective at inhibiting inflammation on respiratory viral infections and suggest that Liriope muscari might be a valuable natural substance in the prevention and treatment of infectious diseases.

• Korean Journal of Microbiology
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• v.42 no.2
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• pp.153-155
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• 2006

We constructed the null mutants of fission yeast Schizosaccharomyces pombe spSac3 gene that is homologous to budding yeast Saccharomyces cerevisiae SAC3 involved in mRNA export out of nucleus. Tetrad analysis showed that the spSac3 is essential for vegetative growth. The spSac3 mutants harboring pREP81X-spSac3 plasmid showed poly(A)+ RNA export defect in the presence of thiamine. These results suggest that spSac3 is also involved in mRNA export from the nucleus.

• Korean Journal of Microbiology
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• v.46 no.4
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• pp.401-404
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• 2010

Tho1 is a RNA-binding protein that assembles co-transcriptionally onto the nascent mRNA and is thought to be involved in mRNP biogenesis and mature mRNA export to cytoplasm in budding yeast. In fission yeast Schizosaccharomyces pombe, a homologue of THO1 (spTho1) was identified based on sequence alignment. A deletion mutant in a diploid strain was constructed by replacing one of spTho1-coding region with an ura4+ gene using one-step gene disruption method. Tetrad analysis showed that the spTho1 was not essential for growth. The spTho1 mutant did not show any defects of bulk mRNA export. However, over-expression of spTho1 from strong nmt1 promoter caused the growth defects and accumulation of poly(A)$^+$ RNA in the nucleus. These results suggest that spTho1 is involved in mRNA export from the nucleus to cytoplasm though it is not essential.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.43-48
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• 1993

These experiments were carried out to construct mouse testis cDNA library and to to seen H-Y Ag gene. Mouse testis was obtained from BALB/c inbreed mouse that was after-born 1 week. Isolation of mouse testis total RNA was carried out by guanidum/cesium choloride, poly(A+) mRNAs were purified by oligo d(T)-cellulose chromatography method. To investigate protein synthesis activity, in-vitro translation carried out by total RNA and poly(A+) mRNA. The products of in-vitro translation were identified in 12.5% PAGE. Single strand DNA and double strand DNA were synthesized from poly(A+) mRNA and purified using phenol/chloroform/isoamylalcohol. Synthesized cDNA was combined with cohesive Eco RI polylinker, its recombination efficiencies were identified by X-gal and IPTG. In the cDNA library, 1$\times$107 phagemids were screened with 32P labelled probe. Hybridization were carried on $65^{\circ}C$ for 16~20hours. And 1$\times$106 phagemids were screened with rabbit-anti-H-Y. In former, select 5 positive clones, and later, 1 positive clone. Its southern blot analysis showed various size of insert cDNA from 0.7kb to 3kb.

• Biomolecules & Therapeutics
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• v.28 no.3
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• pp.272-281
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• 2020

Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an "in vitro" model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G' and viscous G''), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D "in vitro" model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.

• Journal of Veterinary Science
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• v.24 no.3
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• pp.37.1-37.14
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• 2023

Background: Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. Objectives: The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). Methods: The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. Results: The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1β, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. Conclusions: The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.