• Journal of Microbiology and Biotechnology
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• v.33 no.11
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• pp.1506-1512
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• 2023

Quantitative analysis of adenosine triphosphate (ATP) has been widely used as a diagnostic tool in the food and medical industries. Particularly, the pathogenesis of a few diseases including inflammatory bowel disease (IBD) is closely related to high ATP concentrations. A bioluminescent D-luciferin/luciferase system, which includes a luciferase (FLuc) from the firefly Photinus pyralis as a key component, is the most commonly used method for the detection and quantification of ATP. Here, instead of isolating FLuc produced in recombinant Escherichia coli, we aimed to develop a whole-cell biocatalyst system that does not require extraction and purification of FLuc. To this end, the gene coding for FLuc was introduced into the genome of probiotic Saccharomyces boulardii using the CRISPR/Cas9-based genome editing system. The linear relationship (r² = 0.9561) between ATP levels and bioluminescence generated from the engineered S. boulardii expressing FLuc was observed in vitro. To explore the feasibility of using the engineered S. boulardii expressing FLuc as a whole-cell biosensor to detect inflammation biomarker (i.e., ATP) in the gut, a colitis mouse model was established using dextran sodium sulfate as a colitogenic compound. Our findings demonstrated that the whole-cell biosensor can detect elevated ATP levels during gut inflammation in mice. Therefore, the simple and powerful method developed herein could be applied for non-invasive IBD diagnosis.

• Journal of Animal Science and Technology
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• v.66 no.1
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• pp.103-114
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• 2024

This study was done to investigate the effects of the incorporation of Achyranthes japonica extracts (AJE) in diet on the production parameters of growing pigs. Exp 1: Total, 105 crossbred pigs (average body weight: 24.47 ± 2.46 kg) were used in a 6-week feeding trial. Pigs (seven replicates, five pigs per pen) were allotted randomly to three treatments. Dietary treatments: CON (basal diet); basal diet with 0.025% AJE, and basal diet + 0.050% AJE). Growth performance, nutrient digestibility, fecal microbial count, and fecal noxious gas were assessed in this study. Average daily gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) were not affected by the addition of up to 0.05% AJE. In the case of apparent total tract digestibility (ATTD), dry matter (DM), nitrogen (N), and digestible energy (DE) were not changed in 3rd and 6th weeks of the feeding trial through the addition of AJE up to 0.05% in the growing pig diet. In microbial count, Lactobacillus and Escherichia coli count at 3rd and 6th week was similar in all the treatment diets. The inclusion of AJE at levels up to 0.05% in growing pig diet had no effect on the production of NH3, H2S, acetic acid, and CO₂ in the feces. After ending the Exp 1, a total of nine pigs were divided into three treatment groups. Treatment diets were included, TRT1, basal diet + powder quercetin 30 g; TRT2, basal diet + powder quercetin 150 g; TRT3, basal diet + powder quercetin 300g. Rate of absorption in blood was increased with the higher dose of quercetin. The results suggested incorporation of AJE up to 0.05% has no significant effect on ADG, ADFI, and G:F, as well as DM, N, and DE digestibility, fecal microbial count, and fecal noxious gas emission in growing pigs, even though no negative effect was found.

• IMMUNE NETWORK
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• v.24 no.3
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• pp.27.1-27.14
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• 2024

The tumor microenvironment (TME) is formed by several immune cells. Notably, tumor-associated macrophages (TAMs) are existed in the TME that induce angiogenesis, metastasis, and proliferation of cancer cells. Recently, a point-mutated variant of IL-32θ was discovered in breast cancer tissues, which suppressed migration and proliferation through intracellular pathways. Although the relationship between cancer and IL-32 has been previously studied, the effects of IL-32θ on TAMs remain elusive. Recombinant human IL-32θ (rhIL-32θ) was generated using an Escherichia coli expression system. To induce M0 macrophage polarization, THP-1 cells were stimulated with PMA. After PMA treatment, the cells were cultured with IL-4 and IL-13, or rhIL-32θ. The mRNA level of M1 macrophage markers (IL-1β, TNFα, inducible nitric oxide synthase) were increased by rhIL-32θ in M0 macrophages. On the other hand, the M2 macrophage markers (CCL17, CCL22, TGFβ, CD206) were decreased by rhIL-32θ in M2 macrophages. rhIL-32θ induced nuclear translocation of the NF-κB via regulation of the MAPK (p38) pathway. In conclusion, point-mutated rhIL-32θ induced the polarization to M1-like macrophages through the MAPK (p38) and NF-κB (p65/p50) pathways.

• Journal of Life Science
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• v.29 no.6
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• pp.671-678
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• 2019

Antibacterial and antioxidant activities of plant sources have attracted a wide range of interest across the world over the last decade. This is due to the growing concern for safe and alternative sources of antibacterial and antioxidant agents. In this study, we focused on the antibacterial and antioxidant activities and the chemical composition of a methanol extract from Viburnum sargentii seeds. The chemical composition was determined by gas chromatography-mass spectroscopy (GC-MS), and the antibacterial activity was screened by a disc diffusion assay. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microbroth dilution and spread plate method, respectively. The V. sargentii extract showed growth inhibition activity on all tested Gram-positive (Listeria monocytogenes, Staphylococcus aureus, and Staphylococcus saprophyticus) and Gram-negative (Escherichia coli, Pseudomonas putida, and Proteus vulgaris) pathogenic bacteria. The MIC and MBC ranged from 0.156~1.25 mg/ml for Gram-positive and 0.625~5.0 mg/ml for Gram-negative tested bacteria. The GC-MS results revealed the presence of several phytochemicals such as ${\beta}-sitosterol$ and vitamin E, which are known for their pharmacological applications. The antioxidant activities of V. sargentii extract were investigated by three different methods: the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging assay, the reducing power assay, and the total antioxidant capacity assay. The results showed a concentration-dependent antioxidant potential for all three used methods. In sum, our findings suggest that the methanol extract of V. sargentii seeds has the potential to inhibit the growth of pathogenic bacteria and provide antioxidant compounds, making it therefore worthy of further investigation.

• Korean Journal of Food Science and Technology
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• v.38 no.5
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• pp.699-706
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• 2006

The antioxidative and antimicrobial activities of Euphorbia jolkini extracts were investigated. Total polyphenohc compounds extracted were approximately as follows: 162.08 mg/g from ethanol, 12.64 mg/g from n-hexane, 48.11 mg/g from dichloromethane, 544.08 mg/g from ethyl acetate, 176.42 mg/g from butanol, and 30.00 mg/g from water. The ethylacetate fraction of this extraction showed the highest antioxidative activity $(IC_{50})$: DPPH radical scavenging capacity was measured at $8.38\;{\mu}g/mL$, xanthine oxidase inhibitory activity was $466.01\;{\mu}g/mL$, superoxide radical scavenging capacity was $11.39\;{\mu}g/mL$, and nitric oxide scavenging capacity was $332.11\;{\mu}g/mL$. Antimicrobial activities were determined by paper disc method and minimum inhibitory concentration of E. jolkini extracts against food-borne pathogens and spoilage bacteria. The growth inhibition curves of E. jolkini extracts against Bacillus cereus, Listeria monocytogenes, and Escherichia coli were also determined. These results suggest that the ethylacetate fraction of E. jolkini has strong antimicrobial activity against the all species of microorganisms as well as strong antioxidant activity.

• Journal of the Korean Society of Food Science and Nutrition
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• v.36 no.9
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• pp.1106-1112
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• 2007

The solvent extracts of Euphorbia helioscopia, which were extracted by using several solvents with different polarities, were prepared for utility as natural preservatives. The E. helioscopia extract by 80% ethanol was sequentially fractionated with n-hexane, dichloromethane, ethylacetate, and butanol. In order to effectively screen for a natural preservatives agent, we first investigated the antioxidant activities such as DPPH radical scavenging capacity, superoxide radical scavenging capacity, and xanthine oxidase inhibitory activity of the E. helioscopia extracts. By the screening system, we found that ethylacetate fraction had the strongest antioxidant activity in a dose-dependent manner. The antimicrobial activities and cell growth inhibition were investigated for each strain with the different concentrations of E. helioscopia extracts. Antimicrobial activities were shown in ethylacetate fraction of E. helioscopia; however, ethanol, butanol and water fractions showed weak antimicrobial activity against the tested microorganisms. Among the five fractions, ethylacetate fraction showed the highest antimicrobial activities against microorganisms tested, such as Bacillus sublitis, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella Enteritidis and Salmonella Typhimurium. The polyphenol content from ethanol, n-hexane, dichloromethane, ethylacetate, butanol, and water fractions were 207.46 mg/g, 45.45 mg/g, 138.23 mg/g, 678.02 mg/g, 278.91 mg/g, and 63.76 mg/g, respectively. There seems to be a close relationship between antioxidant activities, and antimicrobial activities and polyphenol content in natural plant. From these results, it is suggested that E. helioscopia could be used for the ethylacetate fraction and could be suitable for the development of a food preservative.

• Journal of Life Science
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• v.16 no.3 s.76
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• pp.485-492
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• 2006

The PrrBA two-component system is one of the major regulatory systems that control expression of photosynthesis genes in response to changes in oxygen tension in the anoxygenic photosynthetic bacterium, Rhodobacter sphaeroides. The system consists of the PrrB histidine kinase and the PrrA response regulator. The N-terminal transmembrane domain of PrrB serves as a signal-sensing domain and comprises six transmembrane helices forming three periplasmic loops and two cytoplasmic loops. The $3^{rd}$ and $4^{th}$ transmembrane helices and the $2^{nd}$ periplasmic loop were suggested to play a crucial role in redox-sensory function. In this study we demonstrated that mutations of Asp-90, Gln-93, Leu-94, Leu-98, and Asn-106 in the $2^{nd}$ periplasmic loop and its neighboring region led to severe defects in PrrB sensory function, indicating that these amino acids might be related to the redox-sensing function of PrrB. The mutant forms (D90E, D90N, and D90A) of PrrB were heterologously overexpressed in Escherichia coli, purified by means of affinity chromatography and their autokinase activities were comparatively assessed. The D90N form of PrrB was shown to possess higher autokinase activity than the wild-type form of PrrB, whereas the D90E form of PrrB displayed lower autokinase activity than the wild-type form of PrrB. The D90A mutation led to the loss of PrrB autokinase activity.

• Korean Journal of Microbiology
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• v.47 no.4
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• pp.308-315
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• 2011

The purpose of this work was to examine the antimicrobial activity derived from the lactic acid bacterium, UK-3 isolated from the vaginas of women of childbearing age. Various physiological and biochemical properties of this strain were characterized. Both the BIOLOG system and phylogenetic analysis using 16S rRNA sequencing were utilized for identification, and the strain was designated as Lactobacillus plantarum UK-3, and registered in GenBank as [JK266589]. Growth rate, production of organic acids (e.g., lactic acid and acetic acid), and pH during growth were monitored. The maximum concentrations of lactic acid and acetic acid were approximately 684.11 mM and 174.26 mM, respectively, and pH changed from 7.0 to 3.7 after 72 h of incubation. High performance liquid chromatography was used to confirm lactic acid and acetic acid production. Significant antimicrobial activity of the concentrated supernatant was demonstrated against various Gram-positive (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Neisseria species., Listeria monocytogenes), Gram-negative bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis), and yeast (e.g., Candida albicans) by the plate diffusion method. As a result, the concentrated L. plantarum UK-3 cultures had lower acidity and inhibited the growth of all microorganisms tested, whereas the growth of L. acidophilus was not affected.

• Journal of Life Science
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• v.15 no.6 s.73
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• pp.916-922
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• 2005

Parkin, known as an E3 ubiquitin ligase, has essential role in protein quality control, and its severe dysfunction leads to neurodegenerative disorders. Human Parkin was excessively degraded when expressed in Escherichia coli under the conventional induction condition ($37^{\circ}C$ culture condition with 0.5 mM IPTG). To optimize the induction and culture conditions for recombinant human Parkin and develop a rapid method for the Parkin purification, we expressed Parkin by using PCEX system at the different culture temperatures and IPTC concentrations. The intact Parkin protein was purified to approximately $90\%$ purity with suitable amounts of protein under the optimal culture condition ($25^{\circ}C$E with 0.01 mM IPTG). Additionally, we constructed various parkin plasmids with different tagging systems and investigated their expression patterns in HEK293 cells. We found that the proteolytically sensitive site is localized within a ubiquitin-like domain of Parkin. This study developes a method for generating useful reagents to investigate biochemical properties of Parkin.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1189-1195
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• 2008

A study was conducted with 48 weaned barrows ($28{\pm}3d$, $8.45{\pm}0.14kg$) to determine the effect of Achyranthes bidentata polysaccharide (ABPS) supplementation on pig performance, immunological, adrenal and somatotropic responses following Escherichia coli lipopolysaccharide (LPS) challenge. The experiment was a $2{\times}2$ factorial design; the main factors included diet (supplementation with 0 or 500 mg/kg ABPS) and immunological challenge (LPS or saline). On d 14 and 21 of the trial, pigs were given an intraperitoneal injection with either $100{\mu}g/kg$ BW of LPS or an equivalent amount of sterile saline. Blood samples were obtained 3 h after injection for analysis of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), prostaglandin $E_2$ ($PGE_2$), cortisol, growth hormone (GH), insulin-like growth factor (IGF)-I and immunoglobulin G (IgG). On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was measured. LPS administration decreased average daily feed intake (ADFI) (p<0.05), had a tendency to decrease average daily gain (ADG) (p<0.10) during both the first and second challenge periods and increased (p<0.05) feed:gain ratio only during the first challenge period. ABPS tended to improve ADG (p<0.10) during the first challenge period, and improved ADG (p<0.05) and tended to improve ADFI (p<0.10) during the second challenge period. ABPS did not affect feed:gain ratio. An interaction (p<0.05) between LPS challenge and diet was observed for the plasma concentrations of TNF-${\alpha}$, $PGE_2$ and cortisol after both LPS challenges such that, among LPS-treated pigs, pigs fed the ABPS diet were lower for these indices than those receiving the control diet. In contrast, pigs fed the ABPS diet had higher IGF-I (p<0.05) compared with those fed the control diet. No effect of diet, LPS challenge or both on GH and IgG was observed after both LPS administrations. LPS challenge increased PBLP when these cells were incubated with $8{\mu}g/ml$ of LPS during both the challenge periods, and did likewise when incubated with $8{\mu}g/ml$ of concanavalin A only after the first challenge. ABPS had no effect on PBLP. These data demonstrate that ABPS alters the release of pro-inflammatory cytokines following an immunological challenge, which might enable pigs to achieve better performance.