• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.369-377
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• 1998

Rotavirus is a major cause of severe gastroenteritis in young children and animals throughout the world. The VP7 of rotavirus is thought to induce the synthesis of neutralizing antibodies and to be responsible for determining viral serotypes. The cDNA coding for the VP7 capsid protein of human rotavirus, obtained from Korean patients (HRV-Y14), was cloned and its nucleotide sequence was determined. Comparative analysis of the nucleotide sequences between VP7 of Y14 and that of other foreign isolates showed $92.7~95.2\%$ homology to G1 serotypes (RV-4, KU, K8, WA), $74.2\%$ homolgy to G2 serotype HU-5, $76.4\%$ homology to G3 serotype SA-11, and $77.6\%$ homology to G4 serotype A01321. These data suggest that HRV-Y14 can be classified as a G1 serotype. cDNA coding for VP7 of HRV-YI4 was subcloned into the baculovirus vector and the VP7 glycoprotein was expressed in insect cells. The expressed proteins in Sf9 cell extract and tissue culture fluid were separated on SDS-PAGE, and Western blot analysis with monoclonal antibody raised against the synthetic peptide containing 21 amino acids within the VP7 conserved region was performed. The molecular weight of recombinant VP7 was estimated to be 36 kDa which is about the same size as the native VP7. Addition of tunicamycin in the culture media caused a reduction of the molecular weight of the recombinant VP7 indicating that the expressed protein was glycosylated.

• Proceedings of the Korean Institute of Navigation and Port Research Conference
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• v.29 no.1
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• pp.399-404
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• 2005

In this paper, An immunized PID(I-PID) controller based on cell mediated immune response is proposed to improve the control performance of the controller with PID scheme. And it is applied to the vibration of the building structure in the port with active damper systems. The immune system of organism in the real body regulates the antibody and T-cells to protect the attack from the foreign materials which are virus, germ cell, and other antigens. It has similar characteristics that are the adaptation and robustness to overcome disturbances and to control the plant of engineering application. At firstly, we build a model of the T-cell regulated immune response mechanism. We have also designed an I-PID controller focusing on the T-cell regulated immune response of biological immune system. Finally, we show that some computer simulations of the vibraton control for the building structure system with wind force excitation. These results for the proposed method also show that is has performance than other conventional controller design method.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.116-120
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• 2004

The medicinal properties of Ganoderma lucidum have been recognized in China for many centuries. Active pharmaceutical components include triterpenes. To elucidate the molecular regulation of triterpene biosynthesis in this mushroom, a 57-base pair DNA fragment encoding the fourth conserved domain SQ-4 (SMGLFLQKTNIIRDYNEDL) of squalene synthase was synthesized and cloned into the expression vector pET-32a(＋). The recombinant fusion protein induced by IPTG (isopropyl-$\beta$-D-thiogalactopyranoside) was overexpressed in the Escherichia coli. Using the purified recombinant fusion protein of 20.9 kDa, a specific polyclonal antibody was obtained from immunized rabbit. Expression of squalene synthase at different development stages of Ganoderma lucidum was analyzed.

• Biomolecules & Therapeutics
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• v.26 no.5
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• pp.464-473
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• 2018

Cripto is a small glycosylphosphatidylinositol-anchored signaling protein that can detach from the anchored membrane and stimulate proliferation, migration, differentiation, vascularization, and angiogenesis. In the present study, we demonstrated that Cripto positively affected proliferation and survival of mesenchymal stem cells (MSCs) without affecting multipotency. Cripto also increased expression of phosphorylated janus kinase 2 (p-JAK2), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), 78 kDa glucose-regulated protein (GRP78), c-Myc, and cyclin D1. Notably, treatment with an anti-GRP78 antibody blocked these effects. In addition, pretreatment with STAT3 short interfering RNA (siRNA) inhibited the increase in p-JAK2, c-Myc, cyclin D1, and BCL3 levels caused by Cripto and attenuated the pro-survival action of Cripto on MSCs. We also found that incubation with Cripto protected MSCs from apoptosis caused by hypoxia or $H_2O_2$ exposure, and the level of caspase-3 decreased by the Cripto-induced expression of B-cell lymphoma 3-encoded protein (BCL3). These effects were sensitive to down-regulation of BCL3 expression by BCL3 siRNA. Finally, we showed that Cripto enhanced expression levels of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). In summary, our results demonstrated that Cripto activated a novel biochemical cascade that potentiated MSC proliferation and survival. This cascade relied on phosphorylation of JAK2 and STAT3 and was regulated by GRP78. Our findings may facilitate clinical applications of MSCs, as these cells may benefit from positive effects of Cripto on their survival and biological properties.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.273-277
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• 1997

The sesquiterpene cyclase (SC) was induced and its products were accumulated in the culture media of Hyoscyamus muticus hairy roots by addition of Rhizoctonia solani extracts. The cumulative production of solavetivone was nearly doubled by integrated extraction of the products from the media during the 24 h accumulation period. Western blots with monoclonal antibodies against SC show that the enzyme levels are the same for both extracted and non-extracted cultures. SC activities measured in vitro with radioactive substrate are not significantly different. These results suggest that productivity is controlled by substrate availability within the terpenoid pathway, and feedback regulation precedes the branch-point enzyme sesquiterpene cyclase.

• Reproductive and Developmental Biology
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• v.42 no.2
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• pp.7-12
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• 2018

In this review, we have tried to summarize the evidence and molecular characterization indicating that $20{\alpha}$-hydroxysteroid dehydrogenase ($20{\alpha}$-HSD) is a group of the aldo-keto reductase (AKR) family, and it plays roles in the modulation and regulation of steroid hormones. This enzyme plays a critical role in the regulation of luteal function in female mammals. We have studied the molecular expression and regulation of $20{\alpha}$-HSD in cows, pigs, deer, and monkeys. The specific antibody against bovine $20{\alpha}$-HSD was generated in a rabbit immunized with the purified recombinant protein. The mRNA expression levels increased gradually throughout the estrous cycle, the highest being in the corpus luteum (CL) 1 stage. The mRNA was also specifically detected in the placental and ovarian tissues during pregnancy. The $20{\alpha}$-HSD protein was intensively localized in the large luteal cells and placental cytotrophoblast villus, glandular epithelial cells of the endometrium, syncytiotrophoblast of the placenta, the isthmus cells of the oviduct, and the basal part of the primary chorionic villi and chorionic stem villus of the placenta and large luteal cells of the CL in many mammalian species. Further studies are needed to determine the functional significance of the $20{\alpha}$-HSD molecule during ovulation, pregnancy, and parturition. This article will review how fundamental information of these enzymes can be exploited for a better understanding of the reproductive organs during ovulation and pregnancy.

• Molecules and Cells
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• v.38 no.9
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• pp.773-780
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• 2015

3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and $10{\mu}g$ 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 $LD_{50}$ PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% ($5{\mu}g$) and 47% ($10{\mu}g$). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.

• Korean Chemical Engineering Research
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• v.60 no.2
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• pp.300-307
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• 2022

Time-resolved serial femtosecond crystallography (TR-SFX) is a powerful technique for determining temporal variations in the structural properties of biomacromolecules on ultra-short time scales without causing structure damage by employing femtosecond X-ray laser pulses generated by an X-ray free electron laser (XFEL). The mixing rate of reactants and biomolecule samples, as well as the hit rate between crystal samples and x-ray pulses, are critical factors determining TR-SFX performance, such as accurate image acquisition and efficient sample consumption. We here develop two distinct sample delivery systems that enable ultra-fast mixing and on-demand droplet injecting via pneumatic application with a square pulse signal. The first strategy relies on inertial mixing, which is caused by the high-speed collision and subsequent coalescence of droplets ejected through a double nozzle, while the second relies on on-demand pneumatic jetting embedded with a 3D-printed micromixer. First, the colliding behaviors of the droplets ejected through the double nozzle, as well as the inertial mixing within the coalesced droplets, are investigated experimentally and numerically. The mixing performance of the pneumatic jetting system with an integrated micromixer is then evaluated by using similar approaches. The sample delivery system devised in this work is very valuable for three-dimensional biomolecular structure analysis, which is critical for elucidating the mechanisms by which certain proteins cause disease, as well as searching for antibody drugs and new drug candidates.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.1971-1982
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• 2017

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.