• Biomolecules & Therapeutics
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• v.23 no.4
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• pp.320-326
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• 2015

The clinical benefits of oncogenic BRAF inhibitor therapies are limited by the emergence of drug resistance. In this study, we investigated the role of a negative regulator of the MAPK pathway, Spry2, in acquired resistance using BRAF inhibitor-resistant derivatives of the BRAF-V600E melanoma (A375P/Mdr). Real-time RT-PCR analysis indicated that the expression of Spry2 was higher in A375P cells harboring the BRAF V600E mutation compared with wild-type BRAF-bearing cells (SK-MEL-2) that are resistant to BRAF inhibitors. This result suggests the ability of BRAF V600E to evade feedback suppression in cell lines with BRAF V600E mutations despite high Spry2 expression. Most interestingly, Spry2 exhibited strongly reduced expression in A375P/Mdr cells with acquired resistance to BRAF inhibitors. Furthermore, the overexpression of Spry2 partially restored sensitivity to the BRAF inhibitor PLX4720 in two BRAF inhibitor-resistant cells, indicating a positive role for Spry2 in the growth inhibition induced by BRAF inhibitors. On the other hand, long-term treatment with PLX4720 induced pERK reactivation following BRAF inhibition in A375P cells, indicating that negative feedback including Spry2 may be bypassed in BRAF mutant melanoma cells. In addition, the siRNA-mediated knockdown of Raf-1 attenuated the rebound activation of ERK stimulated by PLX4720 in A375P cells, strongly suggesting the positive role of Raf-1 kinase in ERK activation in response to BRAF inhibition. Taken together, these data suggest that RAF signaling may be released from negative feedback inhibition through interacting with Spry2, leading to ERK rebound and, consequently, the induction of acquired resistance to BRAF inhibitors.

• Korean Journal of Soil Science and Fertilizer
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• v.44 no.6
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• pp.1169-1175
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• 2011

The soil chemical properties, microbial community structures and biochemical properties of lettuce or cucumber-cultivated greenhouse soil samples were analyzed to assess soil health and characterize microbial distribution in 8 locations in Korea. Although most of chemical properties were within the soil management guidelines, the available phosphate, and the contents of exchangeable potassium and calcium were higher than those of recommended levels. In the culture-dependent analysis, 841 bacterial strains were isolated from the greenhouse soils and were identified at the genus level by 16S rRNA gene sequences analysis. The dominant bacterial genera were Bacillus (35.7%), Microbacterium (9.3%), Arthrobacter (5.7%) and Lysobacter (5.1%). The abundance of pseudomonads was highly variable depending on the soil samples. In the culture-independent analysis, soil microbial community was investigated by using phospholipid fatty acid (PLFA) method. Principal component analysis (PCA) showed that a specific grouping for microbial community structure in the greenhouse soils was not observed based on cultivated crops and investigated sites. The results revealed that the greenhouses soils examined are relatively sound managed in terms of soil chemical contents and microbial properties.

• Development and Reproduction
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• v.11 no.3
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• pp.187-193
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• 2007

Ethane 1,2-dimethane sulfonate(EDS), a Leydig cell specific toxicant, has been widely used to create the reversible testosterone withdrawal rat model. Though the maintenance of epididymal structure and function is highly dependent on the testosterone secreted from testis, its derivatives, dihydroxytestosterone(DHT) and estrogen, might have crucial roles. The aim of present study was to monitor the expression patterns of sex steroid receptors, cytochrome P450 aromatase(P450arom) and $5{\alpha}$-reductase in the rat epididymis up to 7 weeks after EDS injection. Adult male rats($350{\sim}400g$) were injected with a single does of EDS(75 mg/kg i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. The transcriptional activities of the target genes were evaluated by semi-quantitative RT-PCRs. The transcript level of estrogen receptor alpha($ER{\alpha}$) in EDS group was significantly higher than control level on week 1(P<0.01). After week 2, there was no significant difference in $ER{\alpha}$ levels between EDS group and control. The transcript level of estrogen receptor beta($ER{\beta}$) in EDS group was significantly higher than control level on week 1(P<0.05), lowered on weeks 2 and 3(P<0.05 and P<0.01, respectively), fluctuated during weeks 4 and 6, and elevated on week 7(P<0.05). The androgen receptor (AR) message levels increased significantly week 2(P<0.01), then returned to control level on week 3. In contrast, expression of cytochrome P450 aromatase(P450arom) decreased sharply during weeks $1{\sim}3$(P<0.01 on weeks 1 and 2; P<0.05 on week 3), then went back to control level on week 4. The mRNA level of $5{\alpha}$-reductase type 2($5{\alpha}$-RT2) increased significantly on week 4(P<0.01), then returned to control level. The present study indicated that EDS administration could induce reversible alterations in the transcriptional activities of sex steroid hormone receptors and androgenconverting enzymes in rat epididymis. EDS injection model will be useful to clarify the regulation mechanism of mammalian epididymal physiology.

• Molecules and Cells
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• v.27 no.6
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• pp.635-640
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• 2009

Testis-derived germline stem (GS) cells can undergo reprogramming to acquire multipotency when cultured under appropriate culture conditions. These multipotent GS (mGS) cells have been known to differ from GS cells in their DNA methylation pattern. In this study, we examined the DNA methylation status of the H19 imprinting control region (ICR) in multipotent adult germline stem (maGS) cells to elucidate how epigenetic imprints are altered by culture conditions. DNA methylation was analyzed by bisulfite sequencing PCR of established maGS cells cultured in the presence of glial cell line-derived neurotrophic factor (GDNF) alone or both GDNF and leukemia inhibitory factor (LIF). The results showed that the H19 ICR in maGS cells of both groups was hypermethylated and had an androgenetic pattern similar to that of GS cells. In line with these data, the relative abundance of the Igf2 mRNA transcript was two-fold higher and that of H19 was three fold lower than in control embryonic stem cells. The androgenetic DNA methylation pattern of the H19 ICR was maintained even after 54 passages. Furthermore, differentiating maGS cells from retinoic acid-treated embryoid bodies maintained the androgenetic imprinting pattern of the H19 ICR. Taken together these data suggest that our maGS cells are epigenetically stable for the H19 gene during in vitro modifications. Further studies on the epigenetic regulation and chromatin structure of maGS cells are therefore necessary before their full potential can be utilized in regenerative medicine.

• International Journal of Industrial Entomology and Biomaterials
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• v.13 no.1
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• pp.31-35
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• 2006

The ubiquitin conjugating enzyme 2 (E2) is core component of ubiquitin proteasome pathway (UPP) which represents a selective mechanism for intracellular proteolysis in eukaryotic cells. The E2 has been implicated in the intracellular transfer of ubiquitin to target protein. We show here the involvement of E2 in antiviral immune of Bombyx mori to Bombyx mori nuclear polyhedrosis virus (BmNPV). In this study, mRNA fluorescent differential display PCR (FDD-PCR) was performed with BmNPV highly resistant silkworm strain NB and susceptible silkworm strain 306. At 24 h post BmNPV infection, FDD-PCR with the arbitrary primer AP34 showed that one cDNA band was down-regulated in the midgut of resistant strain, but highly expressed in susceptible strain. The deduced amino acid sequence of this cDNA clone share 99% identity with the recently published B. mori ubiquitin conjugating enzyme E2 (Genbank NO: DQ311351). Fluorescent quantitative PCR corroborated down regulation of E2 in resistant strain. We there conclude that BmNPV infection evokes strong response of susceptible strain including activation of UPP. BmNPV may evolve escape mechanisms that manipulate the UPP in order to persist in the infected host. In addition, the identification of down-regulation of E2 in resistant strain, as well as structure data, are essential to understanding how UPP operates in silkworm antiviral immune to BmNPV disease.

• Journal of Society of Preventive Korean Medicine
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• v.14 no.2
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• pp.1-12
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• 2010

The individual differences in disease development and susceptibility have been researched primarily on the subject of genes, environment or the interaction between genes and the environment respectively. However, there have been limitations in explaining complex diseases, and the differences in health and diseases in monozygotic and dizygotic twins. Fortunately, thanks to active research on the relationship between genes and the environment, and epigenetics, there has been much progress in the understanding of body's reactions and changes. Epigenetics is referred to as a study of gene expression through the interactions of DNA methylation, chromatin's histone and the change of structure in tail, RNA editing without any change in DNA sequence. In this paper, we introduce the basic concepts and mechanisms of epigenetics. The result of the epigenetics is heritable ; can regulate gene expressions ; is reversible ; and has many variable forms depending on cell types. The influences of epigenetics occur throughout life, but it is mainly determined in utero during early pregnancies. Diseases occur or the risk rises if these influences continue after birth until adult life when problems occur in excess/lack of nutrition, environmental plasticity, or already inputted data. Therefore, there is a need for change and innovation, especially in interest and investment in health education for young women near pregnancies and correct treatment of epigenetic-related diseases.

• Journal of Microbiology and Biotechnology
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• v.30 no.2
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• pp.196-205
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• 2020

In this study, the acidic lipase from Aspergillus niger (ANL) was homologously expressed in A. niger. The expression of ANL was significantly improved by the expression of the native ANL with the introns, the addition of the Kozak sequence and the optimization of the signal sequences. When the cDNA sequence of ANL fused with the glaA signal was expressed under the gpdA promoter in A. niger, no lipase activity could be detected. We then tried to improve the expression by using the full-length ANL gene containing three introns, and the lipase activity in the supernatant reached 75.80 U/ml, probably as a result of a more stable mRNA structure. The expression was further improved to 100.60 U/ml by introducing a Kozak sequence around the start codon due to a higher translation efficiency. Finally, the effects of three signal sequences including the cbhI signal, the ANL signal and the glaA signal on the lipase expression were evaluated. The transformant with the cbhI signal showed the highest lipase activity (314.67 U/ml), which was 1.90-fold and 3.13-fold higher than those with the ANL signal and the glaA signal, respectively. The acidic lipase was characterized and its highest activity was detected at pH 3.0 and a temperature of 45℃. These results provided promising strategies for the production of the acidic lipase from A. niger.

• BMB Reports
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• v.40 no.3
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• pp.426-431
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• 2007

To investigate the possible mechanisms of high-altitude native animals in adapting to high altitude, we cloned hemoglobin alpha-chain (alpha-chain Hb) gene from Pantholops hodgsonii, an animal species that indigenously lives at elevations of 3700-5500 m on the Qinghai-Tibetan plateau. Using reverse transcription polymerase chain reaction (RT-PCR) technique, the alpha-chain Hb gene was amplified from total RNA in the liver of the Pantholops hodgsonii. TA cloning technique was used and the PCR product was cloned into pGEM-T vector. The DNA sequence of the gene was highly homologous with sheep (99.1%), goat (98.6%), cattle (95.6%) and human (86.5%). The alpha-chain Hb gene encoded a 142-amino acid protein that could be identified with the homology of alpha-chain Hb protein in sheep (98%), goat (96%), cattle (91%) and human (87%). However, 18 alternations were detected when compared with the alpha-chain Hb gene in human, and 2 in sheep. Moreover, the alterations of a117 GluAsp and $\alpha$132 AsnSer in important regions were noted in human and sheep, respectively. Phylogenetic analysis suggested that the structure of alpha-chain Hb was highly similar to that in sheep. This study provided essential information for elucidating the possible roles of hemoglobin in adapting to extremely high altitude in Pantholops hodgsonii.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.11
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• pp.1591-1598
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• 2004

To isolate the $\beta$-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of $\beta$-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72$^{\circ}C$, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding $\beta$-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of $\beta$-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase(KNOUC112$\beta$-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112$\beta$-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.12
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• pp.1726-1735
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• 2014

This study investigated the effects of acarbose addition on changes in ruminal fermentation characteristics and the composition of the ruminal bacterial community in vitro using batch cultures. Rumen fluid was collected from the rumens of three cannulated Holstein cattle fed forage ad libitum that was supplemented with 6 kg of concentrate. The batch cultures consisted of 8 mL of strained rumen fluid in 40 mL of an anaerobic buffer containing 0.49 g of corn grain, 0.21 g of soybean meal, 0.15 g of alfalfa and 0.15g of Leymus chinensis. Acarbose was added to incubation bottles to achieve final concentrations of 0.1, 0.2, and 0.4 mg/mL. After incubation for 24 h, the addition of acarbose linearly decreased (p<0.05) the total gas production and the concentrations of acetate, propionate, butyrate, total volatile fatty acids, lactate and lipopolysaccharide (LPS). It also linearly increased (p<0.05) the ratio of acetate to propionate, the concentrations of isovalerate, valerate and ammonia-nitrogen and the pH value compared with the control. Pyrosequencing of the 16S rRNA gene showed that the addition of acarbose decreased (p<0.05) the proportion of Firmicutes and Proteobacteria and increased (p<0.05) the percentage of Bacteroidetes, Fibrobacteres, and Synergistetes compared with the control. A principal coordinates analysis plot based on unweighted UniFrac values and molecular variance analysis revealed that the structure of the ruminal bacterial communities in the control was different to that of the ruminal microbiota in the acarbose group. In conclusion, acarbose addition can affect the composition of the ruminal microbial community and may be potentially useful for preventing the occurrence of ruminal acidosis and the accumulation of LPS in the rumen.