• The Korean Journal of Zoology
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• v.39 no.4
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• pp.378-386
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• 1996

We have developed a couple of new luciferase reporter plasmida with very low background reporter actlvltlea. One can be used to measure the promoter strength, after insertion of some promoter fragment into the reporter plasmid, and the other, with very low basal promoter actlvltlea, allis In studying eukaryotic transcriptional regulators. The latter reporter plasmid contains such cli elements as a 17 nucleotide long inftlator, Spl.blndIng sftes, GAL4 binding sltea, and bInding sitea for a certain Drosophila homeodomain proteins. In an attempt to construct an improved reporter plaimid by fadlltating transcriptional termination and minimizing any interference by cryptic promoters which may be preaent in the reporter pleamld DNA, we have inserted transsrlptional termination-related signals, a three tandem repeat of SV4O polyadenylatlon signal (AAA) and the putative transcrtptional termination signal (UMS) of the mouse c-mos gene, Into just upstream of the initIator, and the promoter actlvitiea were measured by a transIent expression assay employing the Drosophila Schneider line 2 cells. As expected, the basal promoter activitIes decreased maximally when both transcription termination related elements were inserted. Moreover, the reporter plasmld with the two elements allowed more sensitive measurement of transcriptional activation than the reporter piasmid without them. Theae reporter plasmids can be used for studying transcriptional regulators of higher organisms Including mammals as well as Droiophlla melanogaiter.

• Korean Journal of Microbiology
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• v.54 no.2
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• pp.126-135
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• 2018

The characteristics of enzyme and gene for mannanase B had been reported from Cellulosimicrobium sp. YB-43 producing some kind of mannanase. A gene coding for the enzyme, named mannanase C (ManC), was expected to be located downstream of the manB gene. The manC gene was cloned by polymerase chain reaction and sequenced completely. From this nucleotide sequence, ManC was identified to consist of 448 amino residues and contain a carbohydrate binding domain CBM2 besides a catalytic domain, which was homologous to mannanase belonging to the glycosyl hydrolase family 5. The catalytic domain of ManC showed the highest amino acid sequence similarity of 55% with the mannanases from Streptomyces sp. SirexAA-E (55.8%; 4FK9_A) and S. thermoluteus (57.6%; BAM62868). The His-tagged ManC (HtManC) lacking N-terminal signal peptide with hexahistidine at C-terminus was produced and purified from cell extract of recombinant Escherichia coli. The purified HtManC showed maximal activity at $65^{\circ}C$ and pH 7.5, with no significant change in its activity at pH range from 7.5 to 10. HtManC showed more active on konjac and locust bean gum (LBG) than guar gum and ivory nut mannan (ivory nut). Vmax and Km values of the HtManC for LBG were 68 U/mg and 0.45 mg/ml on the optimal condition, respectively. Mannobiose and mannotriose were observed on TLC as major products resulting from the HtManC hydrolysis of mannooligosacharides. In addition, mannobiose and mannose were commonly detected as the hydrolyzed products of LBG, konjac and ivory nut.

• Microbiology and Biotechnology Letters
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• v.48 no.2
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• pp.158-166
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• 2020

A gene coding for the xylanase was cloned from Paenibacillus woosongensis, followed by determination of its complete nucleotide sequence. This xylanase gene, designated as xyn10A, consists of 1,446 nucleotides encoding a polypeptide of 481 amino acid residues. Based on the deduced amino acid sequence, Xyn10A was identified to be a modular enzyme composed of a catalytic domain highly homologous to the glycosyl hydrolase family 10 xylanase and a putative carbohydrate-binding module (CBM) in the C-terminus. By using DEAE-sepharose and phenyl-sepharose column chromatography, Xyn10A was purified from the cellfree extract of recombinant Escherichia coli carrying a P. woosongensis xyn10A gene. The N-terminal amino acid sequence of the purified Xyn10A was identified to exactly match the sequence immediately following the signal peptide predicted by the Signal5.0 server. The purified Xyn10A was a truncated protein of 33 kDa, suggesting the deletion of CBM in the C-terminus by intracellular hydrolysis. The purified enzyme had an optimum pH and temperature of 6.0 and 55-60℃, respectively, with the kinetic parameters V_max and K_m of 298.8 U/mg and 2.47 mg/ml, respectively, for oat spelt xylan. The enzyme was more active on arabinoxylan than on oat spelt xylan and birchood xylan with low activity for p-nitrophenyl-β-xylopyranoside. Xylanase activity was significantly inhibited by 5 mM Cu²⁺, Mn²⁺, and SDS, and was noticeably enhanced by K⁺, Ni²⁺, and Ca²⁺. The enzyme could hydrolyze xylooligosaccharides larger than xylobiose. The predominant products resulting from xylooligosaccharide hydrolysis were xylobiose and xylose.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.1
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• pp.34-41
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• 2019

Platelet aggregation is essential for the formation of a hemostatic plug in the case of blood vessel damage. On the other hand, excessive platelet aggregation may cause cardiovascular disorders, such as thrombosis, atherosclerosis, and myocardial infarction. Scopoletin, which found in the root of plants in the genus Scopolia or Artemisia, has anti-coagulation and anti-malaria effects. This study examined the effects of scopoletin on human platelet aggregation induced by collagen. Scopoletin had anti-platelet effects via the down-regulation of thromboxane $A_2$ ($TXA_2$) production and intracellular $Ca^{2+}$ mobilization ($[Ca^{2+}]_i$), which are aggregation-inducing molecules produced in activated platelets. On the other hand, scopoletin increased both the cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels, which are known as intracellular $Ca^{2+}$-antagonists and aggregation-inhibiting molecules. In particular, scopoletin increased the potently cAMP level more than cGMP, which led to suppressed fibrinogen binding to ${\alpha}IIb/{\beta}_3$ in collagen-induced human platelet aggregation. In addition, scopoletin inhibited collagen-elevated adenosine triphosphate (ATP) release in a dose-dependent manner. The results suggest that aggregation amplification through granule secretion is inhibited by scopoletin. Therefore, scopoletin has potent anti-platelet effects and may have potential for the prevention of platelet-derived vascular diseases.

• Animal Bioscience
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• v.36 no.1
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• pp.29-42
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• 2023

Objective: Pigs, an ideal biomedical model for human diseases, suffer from about 50% early embryonic and fetal death, a major cause of fertility loss worldwide. However, identifying the causal variant remains a huge challenge. This study aimed to detect single nucleotide polymorphisms (SNPs) and candidate genes for the number of mummified (NM) piglets using the imputed whole-genome sequence (WGS) and validate the potential candidate genes. Methods: The imputed WGS was introduced from genotyping-by-sequencing (GBS) using a multi-breed reference population. We performed genome-wide association studies (GWAS) for NM piglets at birth from a Landrace pig populatiGWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase on. A total of 300 Landrace pigs were genotyped by GBS. The whole-genome variants were imputed, and 4,252,858 SNPs were obtained. Various molecular experiments were conducted to determine how the genes affected NM in pigs. Results: A strong GWAS peak located on SSC11: 0.10 to 7.11 Mbp (Top SNP, SSC11:1,889,658 bp; p = 9.98E-13) was identified in cyclin dependent kinase 8 (CDK8) gene, which plays a crucial role in embryonic retardation and lethality. Based on the molecular experiments, we found that Y-box binding protein 1 (YBX1) was a crucial transcription factor for CDK8, which mediated the effect of CDK8 in the proliferation of porcine ovarian granulosa cells via transforming growth factor beta/small mother against decapentaplegic signaling pathway, and, as a consequence, affected embryo quality, indicating that this pathway may be contributing to mummified fetal in pigs. Conclusion: A powerful imputation-based association study was performed to identify genes associated with NM in pigs. CDK8 was suggested as a functional gene for the proliferation of porcine ovarian granulosa cells, but further studies are required to determine causative mutations and the effect of loci on NM in pigs.

• Proceedings of the Plant Resources Society of Korea Conference
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• 2023.04a
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• pp.17-17
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• 2023

Chrysanthemum zawadskii (C. zawadskii) is used in traditional East Asian medicine for the treatment of various diseases, including inflammatory disease. However, it has remained unclear whether extracts of C. zawadskii inhibit inflammasome activation in macrophages. The present study assessed the inhibitory effect of an ethanol extract of C. zawadskii (CZE) on the activation of the inflammasome in macrophages and the underlying mechanism. Bone marrow[-derived macrophages (BMDMs) were obtained from wild-type C57BL/6 mice. The release of IL-1β and lactate dehydrogenase in response to nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome activators, such as ATP, nigericin and monosodium urate (MSU) crystals, was significantly decreased by CZE in lipopolysaccharide(LPS)-primed BMDMs. Western blotting revealed that CZE inhibited ATP-induced caspase-1 cleavage and IL-1β maturation. To investigate whether CZE inhibits the priming step of the NLRP3 inflammasome, we confirmed the role of CZE at the gene level using RT-qPCR. CZE also downregulated the gene expression of NLRP3 and pro-IL-1β as well as NF-κB activation in BMDMs in response to LPS. Apoptosis associated speck-like protein containing a caspase-recruitment domain (CARD) oligomerization and speck formation by NLRP3 inflammasome activators were suppressed by CZE. By contrast, CZE did not affect NLR family CARD domain containing protein 4 (NLRC4) or absent in melanoma 2 (AIM2) inflammasome activation in response to Salmonella typhimurium and poly(dA:dT) in LPS-primed BMDMs, respectively. The results revealed that three key components of CZE, namely linarin, 3,5-dicaffeoylquinic acid and chlorogenic acid, decreased IL-1β secretion in response to ATP, nigericin and MSU. These findings suggest that CZE effectively inhibited activation of the NLRP3 inflammasome.

• Journal of Microbiology and Biotechnology
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• v.34 no.3
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• pp.547-561
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• 2024

In this study, we aim to investigate the precise alterations in the gut microbiota during the onset and advancement of diabetic nephropathy (DN) and examine the impact of Ruminococcus gnavus (R. gnavus) on DN. Eight-week-old male KK-Ay mice were administered antibiotic cocktails for a duration of two weeks, followed by oral administration of R. gnavus for an additional eight weeks. Our study revealed significant changes in the gut microbiota during both the initiation and progression of DN. Specifically, we observed a notable increase in the abundance of Clostridia at the class level, higher levels of Lachnospirales and Oscillospirales at the order level, and a marked decrease in Clostridia_UCG-014 in DN group. Additionally, there was a significant increase in the abundance of Lachnospiraceae, Oscillospiraceae, and Ruminococcaceae at the family level. Moreover, oral administration of R. gnavus effectively aggravated kidney pathology in DN mice, accompanied by elevated levels of urea nitrogen (UN), creatinine (Cr), and urine protein. Furthermore, R. gnavus administration resulted in down-regulation of tight junction proteins such as Claudin-1, Occludin, and ZO-1, as well as increased levels of uremic toxins in urine and serum samples. Additionally, our study demonstrated that orally administered R. gnavus up-regulated the expression of inflammatory factors, including nucleotide-binding oligomerization domain-like receptor pyrin domain-containing protein 3 (NLRP3) and Interleukin (IL)-6. These changes indicated the involvement of the gut-kidney axis in DN, and R. gnavus may worsen diabetic nephropathy by affecting uremic toxin levels and promoting inflammation in DN.

• Journal of Life Science
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• v.30 no.11
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• pp.947-955
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• 2020

The foot-and-mouth disease virus (FMDV), a member of the Aphthovirus genus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. During replication of the FMDV RNA (ribonucleic acid) genome, FMDV-encoding RNA polymerase 3D acts in a highly location-specific manner. This suggests that specific RNA structures recognized by 3D polymerase within non-coding regions of the FMDV genome assist with binding during replication. One such region is the cis-acting replication element (CRE), which functions as a template for RNA replication. The FMDV CRE adopts a stem-loop conformation with an extended duplex stem, supporting a novel 15-17 nucleotide loop that derives stability from base-stacking interactions, with the exact RNA nucleotide sequence of the CRE producing different RNA secondary structures. Here, we show that CRE sequences of FMDVs isolated in Korea from 2010 to 2017 exhibit A and O genotypes. Interestingly, variations in the RNA secondary structure of the Korean FMDVs are consistent with the phylogenetic relationships between these viruses and reveal the specificity of FMDV infections for particular host species. Therefore, we conclude that each genetic clade of Korean FMDV is characterized by a unique functional CRE and that the evolutionary success of new genetic lineages may be associated with the invention of a novel CRE motif. Therefore, we propose that the specific RNA structure of a CRE is an additional criterion for FMDV classification dependent on the host species. These findings will help correctly analyze CRE sequences and indicate the specificity of host species for future FMDV epidemics.

• Restorative Dentistry and Endodontics
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• v.25 no.4
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• pp.561-574
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• 2000

Poly-P has been used to prevent decomposition of foods and has been shown to have inhibitory effect on the growth of gram positive bacteria. The purpose of this study was to evaluate the effect of poly-P on the growth of Porphyromonas endodontalis, a gram negative obligate anaerobic rod, endodontopathic bacterium. P. endodontalis ATCC 35406 was in BHI broth containing hemin and vitamin K with or without poly-P. Inhibitory effect of each poly-P which was added at the beginning(lag phase) or during(exponential phase) the culture, MIC(minimum inhibitory concentration) was determined by measuring the optical density of the bacterial cell at 540nm. Viable cell counts were measured to determined whether poly-P has a bactericidal effect. Leakage of intracellular nucleotides from P. endodontalis was determined at 260nm and morphological change of P. endodontalis was observed under the TEM(transmission electron microscope). Binding of 32P-labeled poly-P to P. endodontalis was examined. SDS-polyacrylamide gel electrophoresis and zymography were performed to observe the changes in protein and enzyme profiles of P. endodontalis, respectively. The results from this study were as follows : 1. The minimal inhibitory concentration(MIC) of poly-P to P. endodontalis appeared to be 0.04~0.05%. 2. Poly-P added to the P. endodontalis culture during the exponential phase of P. endodontalis was as much effective as poly-P added at the begining of the culture, suggesting that the antibacterial effect of poly-P is not much dependent on the initial inoculum size of P. endodontalis. 3. Poly-P are bactericidal to P. endodontalis, demonstrating the decrease of the viable cell counts. 4. Intracellular nucleotide release from the P. endodontalis, was not increased in the presence of poly-P and was not reversed by the addition of divalent cations like $Ca^{2+}$ and $Mg^{2-}$. 5. Under the TEM, it was observed that fine electro-dense materials were prominent in the poly-P grown P. endodontalis, appearing locally in the cell, and the materials were more abundant and more dispersed in the cell as the incubation time with poly-P increased. In addition, highly electron dense granules accumulated in many poly-P grown cells, most of which were atypical in their shape. 6. Binding of 32P-labeled poly-P to P. endodontalis appeared to be 32.8 and 45.5 and 53.4% at 30 minutes, 1 hours and 2 hours, respectively. 7. In the presence of poly-P. the synthesis of proteins with apparent molecular masses of 25, 27, 35, 45 was lost or drastically decreased whereas expression of a protein with an apparent molecular mass of 75 was elevated. 8. Proteolytic activity of P. endodontalis was decreased by poly-P. The overall results suggest that use of poly-P may affect the growth of P. endodontalis, and the anti-bacterial activity of poly-P seems largely bactericidal. Changes in shape, protein expression, and proteolytic activity of P. endodontalis by poly-P may be directly and indirectly attributed to the antibacterial effect of poly-P. Further studies will be needed to confirm the effect of poly-P.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.96-105
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• 2005

For screening of autoregulator receptor gene from Streptomyces longwoodensis, PCR was performed with primers of receptor gene designed on the basis of amino acid sequences of autoregulator receptor proteins with known function. PCR products were subcloned into the BamHIsite of pUC19 and transformed into the E. coli $DH5{\alpha}$. The isolated plasmid from transformant contained the fragment of 100 bp, which was detected on $2\%$ gel after BamHI treatment. The insert, 100 bp PCR product, was confirmed as the expected internal segment of gene encoding autoregulator receptor protein by sequencing. Southern and colony hybridizations with the 100 bp fragment as a probe allowed to select a genomic clone of S. longwoodensis, pSLT harboring a 4.4 kb SphI fragment. Nucleotide sequencing analyses revealed a 651 bp open reading frame(ORF) were isolated protein showing moderate homology ($35{\sim}46\%$) with the ${\Gamma}$-butyrolactone autoregulator receptors from Streptomyces sp., and this ORF was named sltR The sltR/pET-17b plasmid was constructed to overexpress the recombinant SltR protein (rSltR) in E. coli BL21 (DE3)/pLysS, and the rSltR protein was purified to homogeneity by DEAE-Sephacel column chromatography, and DEAE-5PW chromatography (HPLC). The molecular mass of the purified rSltR protein was 55 kDa by HPLC gel-filtration chromatography and 28 kDa by SDS-PAGE, indicating that the rSltR protein is present as a dimer. A binding assay with tritium-labeled autoregulators revealed that the rSltR has clear binding activity with a A-factor type autoregulator as the most effective ligand.