• Research in Plant Disease
• /
• v.29 no.3
• /
• pp.286-294
• /
• 2023

Biological and molecular characterization of a Korean isolate of Orthotospovirus chrysanthinecrocaulis (formerly known as chrysanthemum stem necrosis virus, CSNV) isolated from Chrysanthemum morifolium was determined using host range and sequence analysis in this study. Twenty-three species of indicator plants inoculated mechanically CSNV-Kr was investigated for determination of host range. CSNV-Kr induced various local and systemic symptoms in the inoculated plant species. CSNV-Kr could not infect three plant species and induced symptomless in systemic leaves in Nicotiana tabacum cultivars, though the plant samples reacted positively with the antiserum to CSNV by double-antibody sandwich-enzyme-linked immunosorbent assay. The complete genome sequence of CSNV-Kr was determined. The L RNA of CSNV-Kr consists of 8,959 nucleotides (nt) and encodes a putative RNA-dependent RNA polymerase. The M RNA of CSNV-Kr consists of 4,835 nt and encodes the movement protein (NSm) and the glycoprotein precursor (Gn/Gc protein). The S RNA of CNSV-Kr consists of 2,836 nt and encodes NSs protein and N protein. The Gn/Gc and N sequence of CSNV-Kr were compared with those of previously published CSNV isolates originating from different countries at nucleotide and amino acid levels. The Gn/GC sequence of CSNV-Kr shared 98.8-99.5% identity with CSNV isolated from other countries and the N sequence of CSNV-Kr shared 98.8-99.6% identity. No particular region of variability could be found in either grouping of viruses. All of the CSNV isolates did not show any relationship according to geographical origins and isolation hosts, suggesting no distinct segregation of the CSNV isolates.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 2003.11a
• /
• pp.109-109
• /
• 2003

Taurine is present in a variety of tissue and exhibits many important physiological functions in many tissues. Although it is known that many tissues mediate taurine transport, its functions of taurine transport in bone have not been identified yet. In the present study, we investigated the expression of taurine transporter (TauT) and taurine uptake using mouse stromal ST2 cells and osteoblast-like MC3T3-El cells, which is bone related cells. Detection of TauT mRNA expression in these cells were performed by reverse transcription polymerase chain reaction (RT-PCR). The activity of TauT was assessed by measuring the uptake of ［$^3$H］taurine in the presence or absence of inhibitors. TauT mRNA was detected in these cells. ［$^3$H］Taurine uptake was dependent upon the presence of extracellular sodium, chloride and calcium ions, and inhibited by cold-taurine and ${\beta}$-alanine. These results suggest that taurine has biological functions in bone and some effect on the bone cells.

• Food Science and Biotechnology
• /
• v.17 no.4
• /
• pp.892-894
• /
• 2008

During dongchimi fermentation at 5 and $25^{\circ}C$, the pH lowered slowly and reached 4.03 at $5^{\circ}C$ after 30 days, whereas it lowered dramatically and reached 3.59 at $25^{\circ}C$ after 2 days. The predominant bacteria were Leuconostoc (Leu.) mesenteroides at $25^{\circ}C$ until day 2 which changed into Lactobacillus (Lb.) plantarum at day 3, analyzed by a culture dependent method with partial 16S rRNA gene sequencing, whereas Leu. mesenteroides occupied predominantly at $5^{\circ}C$ until day 7. In a culture-independent method using a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) with partial 16S rRNA gene sequencing, Lb. algidus was predominant at $5^{\circ}C$ until day 7 and Lb. plantarum occupied predominantly at $25^{\circ}C$ until day 3, which is different from the results of the culture based method, indicating the both methods need to be combined for accuracy. Based on the culture-dependent method, Leu. mesenteroides might be responsible for the early and mid-phase of dongchimi fermentation.

• The Plant Pathology Journal
• /
• v.29 no.1
• /
• pp.31-41
• /
• 2013

The presence of Citrus tristeza virus (CTV) has previously been reported in citrus growing regions of Turkey. All serologically and biologically characterized isolates including I$\breve{g}$d${\i}$r, which was the first identified CTV isolates from Turkey, were considered mild isolates. In this study, molecular characteristics of the I d r isolate were determined by different methods. Analysis of the I$\breve{g}$d${\i}$r isolate by western blot and BD-RT-PCR assays showed the presence of MCA13 epitope, predominantly found in severe isolates, in the I$\breve{g}$d${\i}$r isolate revealing that it contains a severe component. For further characterization, the coat protein (CP) and the RNA-depen-dent RNA polymerase (RdRp) genes representing the 3' and 5' half of CTV genome, respectively, were amplified from dsRNA by RT-PCR. Both genes were cloned separately and two clones for each gene were sequenced. Comparisons of nucleotide and deduced amino acid sequences showed that while two CP gene sequences were identical, two RdRp clones showed only 90% and 91% sequence identity in their nucleotide and amino acid sequences, respectively, suggesting a mixed infection with different strains. Phylogenetic analyses of the CP and RdRp genes of I$\breve{g}$d${\i}$r isolate with previously characterized CTV isolates from different citrus growing regions showed that the CP gene was clustered with NZRB-TH30, a resistance breaking isolate from New Zealand, clearly showing the presence of severe component. Furthermore, two different clones of the RdRp gene were clustered separately with different CTV isolates with a diverse biological activity. While the RdRp-1 was clustered with T30 and T385, two well-characterized mild isolates from Florida and Spain, respectively, the RdRp-2 was most closely related to NZRB-G90 and NZRB-TH30, two well-characterized resistance breaking and stem pitting (SP) isolates from New Zealand confirming the mixed infection. These results clearly demonstrated that the I$\breve{g}$d${\i}$r isolate, which was previously described as biologically a mild isolate, actually contains a mixture of mild and severe strains.

• Animal cells and systems
• /
• v.3 no.3
• /
• pp.311-317
• /
• 1999

Prolactin (PRL) was reported to be locally synthesized in many brain areas including the hypothalamus, thalamus (TH) and hippocampus (HIP). In the pituitary lactotrophs, PRL synthesis is dependent upon a pituitary-specific transcription factor, Pit-1. In the present study, we attempted to identify Pit-1 or Pit-1-like protein in brain areas known as the synthetic sites of PRL. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis showed the same Pit-1 transcripts in brain areas such as the medial basal hypothalamus (MBH), preoptic area (POA), TH, and HIP with the Pit-1 transcripts in the anterior pituitary (AP). Electrophoretic mobility shift assay (EMSA) was run with nuclear protein extracts from brain tissues using a double strand oligomer probe containing a putative Pit-1 binding domain. Shifted bands were found in EMSA results with nuclear proteins from MBH, POA, TH and HIP. Specific binding of the Pit-1-like protein was further confirmed by competition with an unlabeled cold probe. Antisense Pit-1 oligodeoxynucleotide (Pit-1 ODN), which was designed to bind to the Pit-1 translation initiation site and block Pit-1 biosynthesis, was used to test Pit-1 dependent brain PRL transcription. Two nmol of Pit-1 ODN was introduced into the lateral ventricle of a 60-day old male rat brain. RNA blot hybridization and in situ hybridization indicated a decrease of PRL mRNA signals by the treatment of Pit-1 ODN. Taken together, the present study suggests that Pit-1 may play an important role in the transcriptional regulation of local PRL synthesis in the brain.

• Microbiology and Biotechnology Letters
• /
• v.49 no.3
• /
• pp.283-288
• /
• 2021

This study investigated the anticancer activity of methanol extract from Platycarya strobilacea leaf in AGS human gastric cancer cells. We determined the cell viability effect of P. strobilacea using MTS assay. Apoptosis induction and cell cycle arrest were confirmed by fluorescein isothiocyanate and propidium iodide staining using cellometer K2. The mRNA expression levels of the Bcl-2 family were confirmed by reverse transcription-polymerase chain reaction. The cell viability was decreased in a dose-dependent manner treated with different concentrations of P. strobilacea. Total, early, and late apoptotic cells were dramatically increased, and the cell cycle was arrested at the sub-G1 phase. The mRNA expressions of Bcl-2 and Bcl-xL were reduced, whereas pro-apoptotic factors, Bax and Bak, were increased in a dose-dependent manner. These results suggested that P. strobilacea leaf extract induced significant apoptotic activity through an intrinsic mitochondria pathway.

• The Journal of Korean Medicine
• /
• v.20 no.2
• /
• pp.108-120
• /
• 1999

In this study, body weight levels of glucose, insulin and triglyceride in blood and glucosidase activity of the small intestine were investigated to determine the effect of Sangbaegpitang and Supungsungiwhan on the glucose metabolism of db/db mice. The GLUT4 mRNA of muscle tissue and the Acetyl CoA Carboxylase and the activation rate of GLUT2 mRNA of liver tissue were measured by the reverse transcription-polymerase chain reaction method and by the vitro transcription. The results were obtained as follows: 1. In the Sangbaegpitang administration group, (1) The level of triglyceride was decreased significantly and the glucosidase activity of the small intestine was inhibited remarkably, (2) The amounts of the GLUT4 mRNA in muscle tissue and Acetyl CoA Carboxylase mRNA in liver tissue were increased significantly. (3) Though glucose level in both fasting and non-fasting, were decreased and the insulin level in blood was increased, the results showed no statistical significance. 2. In the Supungsungiwhan administration group, (1) The levels of glucose and triglyceride were decreased significantly in the blood of non-fasting animals. (2) The glucosidase activity of small intestine was inhibited markedly and the amounts of GLUT4 mRNA of muscle tissue and GLUT2 mRNA of liver tissue were increased significantly. (3) The glucose levels in the fasting group were reduced, while insulin level was increased but showed no statistical significance, Based on the above results, our conclusions are as follows: Sangbaegpitang & Supungsungiwhan are thought to be capable of inhibiting the activity glucosidase, the enzyme which influences carbohydrate metabolism in the small intestine of db/db mice(the experimental diabetic model) and delaying the absorption of carbohydrate, thus proving effective on inhibiting the increase of non-fasting glucose level effectively. Futhermore Sangbaegpitang and Supungsungiwhan are though: to be capable of preventing the composition of free fatty acids by restoring the production of GLUT4 mRNA of muscle tissues and GLUT2 mRNA of liver tissues. Those results suggests that above prescriptions can be applied to non-insulin dependent diabetes mellitus in order to improve insulin resistance.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2017.06a
• /
• pp.20-20
• /
• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.

• Proceedings of the Korean Society of Crop Science Conference
• /
• 2017.06a
• /
• pp.97-97
• /
• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.

• Biomedical Science Letters
• /
• v.18 no.3
• /
• pp.201-209
• /
• 2012

Apoptosis is a physiological programmed cell death process. Tubercle bacilli inhibit apoptosis of alveolar macrophages and phagolysosome fusion. We investigated whether the Bcl-2 family anti-apoptotic member, Bfl-1/A1, plays an important role in the anti-apoptotic process during mycobacterial infection. PMA-treated human monocytoid THP-1 cells were infected with mycobacteria (H37Rv, BCG, and K-strain) at a multiplicity of infection (MOI) of 10 for 0, 1.5, 3, 6, 9, 12, 18, 24, 48, or 72 h. In addition, PMA-treated THP-1 cells were pretreated with specific inhibitors for 45 min before stimulation with mycobacteria at an MOI of 10 for 4 h. After the indicated time, the cells were subject to reverse transcription-polymerase chain reaction (RT-PCR) analysis, and a Bfl-1/A1-specific Western blot was performed. In PMA-differentiated THP-1 cells, the expression level of Bfl-1/A1 mRNA was increased by Mycobacterium tuberculosis (MTB) H37Rv infection. The mRNA level of Bfl-1/A1 peaked 3 h after MTB infection, then declined gradually until 9 h. However, Bfl-1/A1 mRNA induction gradually re-increased from 24 h to 72 h after MTB infection. No difference in Bfl-1/A1 expression was detected following infection with MTB H37Rv, K-strain, or M. bovis BCG. These results were not dependent on mycobacterial virulence. Moreover, mRNA levels of other anti-apoptotic molecules (Mcl-1, Bcl-2, and Bcl-xL) were not increased after MTB H37Rv or K-strain infection. These results suggest that mycobacteria induce the innate immune host defense mechanisms that utilize Bfl-1/A1 molecules at early time points, regardless of virulence.