• Journal of Microbiology and Biotechnology
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• v.31 no.9
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• pp.1323-1329
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• 2021

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

• BMB Reports
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• v.57 no.2
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• pp.86-91
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• 2024

The fatty acids content of castor (Ricinus communis L.) seed oil is 80-90% ricinoleic acid, which is a hydroxy fatty acid (HFA). The structures and functional groups of HFAs are different from those of common fatty acids and are useful for various industrial applications. However, castor seeds contain the toxin ricin and an allergenic protein, which limit their cultivation. Accordingly, many researchers are conducting studies to enhance the production of HFAs in Arabidopsis thaliana, a model plant for oil crops. Oleate 12-hydroxylase from castor (RcFAH12), which synthesizes HFA (18:1-OH), was transformed into an Arabidopsis fae1 mutant, resulting in the CL37 line producing a maximum of 17% HFA content. In addition, castor phospholipid:diacylglycerol acyltransferase 1-2 (RcPDAT1-2), which catalyzes the production of triacylglycerol by transferring HFA from phosphatidylcholine to diacylglycerol, was transformed into the CL37 line to develop a P327 line that produces 25% HFA. In this study, we investigated changes in HFA content when endogenous Arabidopsis PDAT1 (AtPDAT1) of the P327 line was edited using the CRISPR/Cas9 technique. The successful mutation resulted in three independent lines with different mutation patterns, which were transmitted until the T4 generation. Fatty acid analysis of the seeds showed that HFA content decreased in all three mutant lines. These findings indicate that AtPDAT1 as well as RcPDAT1-2 in the P327 line are involved in transferring and increasing HFAs to triacylglycerol.

• Pediatric Infection and Vaccine
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• v.14 no.1
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• pp.83-90
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• 2007

Purpose : Staphylococcal scalded skin syndrome (4S) is a well known disease defined by clinical, microbiological and histological criteria caused by Staphylococcus aureus. This disease is uncommon but has been increasingly recognized. We investigated the clinical features of staphylococcal scalded skin syndrome. Methods : We reviewed retrospectively medical records of 53 patients diagnosis of staphylococcal scalded skin syndrome who were admitted to Changwon Fatima hospital from February 2002 to December 2005. These patients were divided into 3 clinical types; generalized type, intermediate type, abortive type. Age, sex ratio, clinical manifestations, laboratory findings, response to therapy and prognosis were investigated. Result : 1)The mean age of patients was 2.8 years, ranging from 20 days to 7 years. Male-to-female ratio was 1.9:1. 2) By clinical types, 6 patients were in the generalized type (11%), 29 patients in the intermediate type (55%), 18 patients in the abortive type (34%). The coexisting diseases were variable, including conjunctivitis (25 cases), atopic dermatitis (11 cases), otitis media (1 case). On laboratory findings, most of patients didn't have leukocytosis or increased C-reactive protein. 4) A total of fifteen Methicillin Resistant Staphylococcal Aureus (MRSA) strains were isolated from September 2003 through December 2005. Fourteen strains were positive for exfoliative toxin B gene by PCR and negative for enterotoxin, toxic shock syndrome toxin and Panton-Valentine leukocidin genes. 5) The mean duration of admission was 7.3 days. Patients were treated with vancomycin or amoxacillin/clavulanate or ampicillin/sulbactam or cefuroxime without significant sequelaes. Conclusion : Recently, Staphylococcal scalded skin syndrome caused by exfoliative toxin B produced by MRSA in the Changwon area has been increasing.

• The Korean Journal of Physiology and Pharmacology
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• v.5 no.4
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• pp.287-297
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• 2001

Contraction of smooth muscle is initiated by an increase in cytosolic $Ca^{2+}$ leading to activation of $Ca^{2+}$/ calmodulin-dependnet myosin light chain (MLC) kinase and phosphorylation of MLC. The types of contraction and signaling mechanisms mediating contraction differ depending on the region. The involvement of these different mechanisms varies depending on the source of $Ca^{2+}$ and the kinetic of $Ca^{2+}$ mobilization. $Ca^{2+}$ mobilizing agonists stimulate different phospholipases $(PLC-{\beta},\;PLD\;and\;PLA_2)$ to generate one or more $Ca^{2+}$ mobilizing messengers $(IP_3\;and\;AA),$ and diacylglycerol (DAG), an activator of protein kinase C (PKC). The relative contributions of $PLC-{\beta},\;PLA_2$ and PLD to generate second messengers vary greatly between cells and types of contraction. In smooth muscle cell derived form the circular muscle layer of the intestine, preferential hydrolysis of $PIP_2$ and generation of $IP_3$ and $IP_3-dependent\;Ca^{2+}$ release initiate the contraction. In smooth muscle cells derived from longitudinal muscle layer of the intestine, preferential hydrolysis of PC by PLA2, generation of AA and AA-mediated $Ca^{2+}$ influx, cADP ribose formation and $Ca^{2+}-induced\;Ca^{2+}$ release initiate the contraction. Sustained contraction, however, in both cell types is mediated by $Ca^{2+}-independent$ mechanism involving activation of $PKC-{\varepsilon}$ by DAG derived form PLD. A functional linkage between $G_{13},$ RhoA, ROCK, $PKC-{\varepsilon},$ CPI-17 and MLC phosphorylation in sustained contraction has been implicated. Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to $M_2$ muscarinic receptors activating at least three intracellular phospholipases, i.e. phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD) and the high molecular weight (85 kDa) cytosolic phospholipase $A_2\;(cPLA_2)$ to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic $M_3$ receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the $G_{q/11}$ type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate $(PIP_2),$ producing inositol 1, 4, 5-trisphosphate $(IP_3)$ and DAG. $IP_3$ causes release of intracellular $Ca^{2+}$ and formation of a $Ca^{2+}$-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.1
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• pp.201-208
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• 2013

Objective: To investigate changes in the invasive capacity of gastric cancer cells in vitro after expression inhibition of T lymphoma invasion and metastasis inducing factor 1 (Tiam 1) and underlying mechanisms. Methods: Using adhesion selection, two subpopulations with high ($M_H$) or low ($M_L$) invasive capacity were separated from the human gastric cancer cell line MKN-45 ($M_0$). Tiam 1 antisense oligodeoxynucleotide (ASODN) was transfected into $M_H$ cells with liposomes, and expression of Tiam 1 mRNA and protein was determined by RT-PCR and quantitative cellular-ELISA. Changes in the cytoskeleton, invasive capacity in vitro and expression of ras-related $C_3$ botulinum toxin substrate 1 (Rac 1), integrin ${\beta}1$ and matrix metalloproteinase 2 (MMP 2) between Tiam 1 ASODN transfected $M_H$ cells and non-transfected cells were observed by HE staining, cytoskeletal protein staining, scanning electron microscopy, Boyden chamber tests and cyto-immunohistochemistry. Results: A positive correlation existed between the expression level of Tiam l mRNA or protein and the invasion capacity of gastric cancer cells. After ASODN treatment ($0.43{\mu}M$ for 48 h), Tiam 1 mRNA transcription and protein expression in $M_H$ cells were decreased by 80% and 24% respectively (P < 0.05), compared with untreated controls, while invasive capacity in vitro was suppressed by 60% (P < 0.05). Morphologic and ultrastructural observation also showed that ASODN-treated $M_H$ cells exhibited smooth surfaces with obviously reduced filopodia and microspikes, which resembled $M_0$ and $M_L$ cells. Additionally, cytoskeletal distribution dramatically altered from disorder to regularity with reduced long filament-like structure, projections, pseudopodia on cell surface, and with decreased acitn-bodies in cytoplasm. After Tiam 1 ASODN treatment, the expression of Rac 1 and Integrin ${\beta}1$ in $M_H$ cells was not affected (P > 0.05), but that of MMP 2 in $M_H$ cells was significantly inhibited compared with untreated cells (P < 0.05). Conclusion: Over-expression of Tiam-1 contributes to the invasive phenotype of gastric cancer cells. Inhibition of Tiam 1 expression could impair the invasive capacity of gastric cancer cells through modulating reconstruction of the cytoskeleton and regulating expression of MMP 2.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• BMB Reports
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• v.52 no.8
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• pp.496-501
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• 2019

Conventionally, immunotoxins have been produced as a single polypeptide from fused genes of an antibody fragment and a toxin. In this study, we adopted a unique approach of chemical conjugation of a toxin protein and an antibody fragment. The two genes were separately expressed in Escherichia coli and purified to high levels of purity. The two purified proteins were conjugated using a chemical linker. The advantage of this approach is its ability to overcome the problem of low recombinant immunotoxin production observed in some immunotoxins. Another advantage is that various combinations of immunotoxins can be prepared with fewer efforts, because the chemical conjugation of components is relatively simpler than the processes involved in cloning, expression, and purification of multiple immunotoxins. As a proof of concept, the scFv of trastuzumab and the PE24 fragment of Pseudomonas exotoxin A were separately produced using E. coli and then chemically crosslinked. The new immunotoxin was tested on four breast cancer cell lines variably expressing HER2. The chemically crosslinked immunotoxin exhibited cytotoxicity in proportion to the expression level of HER2. In conclusion, the present study revealed an alternative method of generating an immunotoxin that could effectively reduce the viability of HER2-expressing breast cancer cells. These results suggest the effectiveness of this method of immunotoxin crosslinking as a suitable alternative for producing immunotoxins.

• The Korean Journal of Physiology
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• v.30 no.2
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• pp.209-218
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• 1996

Acetylcholine (ACh) activates the inwardly rectifying muscarinic $K^{+}$ channel in rat atrial cells via pertussis toxin (PTX)-sensitive G-protein ($G_k$) coupled with the muscarinic receptor (mAChR). Although this $K^{+}\;(K_{ACh})$ channel function has reported to be modulated by the phosphorylation process, a kinase and phosphatase involved in these processes are still unclear. Since either PKA or PKC was not effective on this ATP-modulation, the present study examined the possible involvement of the protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) in the function of the $K_{ACh}$ Channel. In the inside-out (I/O) patch preparation excised from the adult rat atrial cell, when activated by 10 ${\mu}M$ ACh in the pipette and 100 ${\mu}M$ GTP in the bath, the mean open time (${\tau}_{o}$) and the channel activity ($K_{ACh}$) was 1.13 ms (n=5) and 0.19 (n=6), respectively. Following the application of 1 mM ATP into the bath, ${\tau}_{o}$ increased by 34% (1.54 ms, n=5) and $K_{ACh}$ by 66% (0.28, n=6). Channel function elevated by ATP was lasted after washout of ATP. However, this ATP-induced increase in the $K_{ACh}$ channel function did not occur in pretreated cells with genistein ($50{\sim}100 {\mu}M$), a selective PTK inhibitor, but occurred in pretreated cells with equimolar daidzein, a negative control of the genistein. On the contrary, PTP which acts on tyrosine residue conversely reversed both ATP-induced increased ${\tau}_{o}$ by 32% (1.20 ms, n=3) and $K_{ACh}$ by 41% (0.15, n=3), respectively. Taken together, these results suggest that $K_{ACh}$ channel may, at least partly, be regulated by the tyrosyl phosphorylation, although it is unclear where this process exerts on the muscarinic signal transduction pathway comprising the mAChR-$G_{k}$-the $K_{ACh}$ channel.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.11
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• pp.1644-1651
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• 2014

Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine ${\beta}$-casein gene locus using a knock-in vector for the ${\beta}$-casein gene locus. We developed the knock-in vector on the porcine ${\beta}$-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5' arm and 3' arm from the porcine ${\beta}$-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using ${\beta}$-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous ${\beta}$-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.

• Korean Journal of Microbiology
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• v.50 no.1
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• pp.1-7
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• 2014

Site-specific incorporation of unnatural amino acids (SSIUA) into protein can be achieved in vivo by coexpression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to develop the SSIUA technique in Escherichia coli, here we established a new 2-step screening system that can be used for selecting an ARS variant(s) that ligates an unnatural amino acid to a suppressor tRNA. A positive selection system consists of chloramphenicol acetyl transferase gene containing an amber mutation at the $27^{th}$ residue, and efficiently concentrated amber suppressible ARS with a maximum enrichment factor of $9.0{\times}10^5$. On the other hand, a negative selection system was constructed by adding multiple amber codons in front of a lethal gene encoding the control of cell death B toxin (ccdB) which acts as an inhibitory protein of bacterial topoisomerase II. Amber suppression of ccdB by an orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (TyrRS) and an amber suppressor tRNA significantly inhibits bacterial growth. This selection system was also able to efficiently remove amber suppressible ARS which could ligate natural amino acids to the suppressor tRNA. Thus, sequential combination of these two selection systems might be able to function as a powerful tool for selecting an ARS variant that specifically ligates an unnatural amino acid to the suppressor tRNA from an ARS mutant pool.