• Title/Summary/Keyword: Structural activity

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Directed Evolution of Soluble α-1,2-Fucosyltransferase Using Kanamycin Resistance Protein as a Phenotypic Reporter for Efficient Production of 2'-Fucosyllactose

  • Jonghyeok Shin;Seungjoo Kim;Wonbeom Park;Kyoung Chan Jin;Sun-Ki Kim;Dae-Hyuk Kweon
    • Journal of Microbiology and Biotechnology
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    • v.32 no.11
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    • pp.1471-1478
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    • 2022
  • 2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered Escherichia coli is often hampered owing to the insolubility and instability of α-1,2-fucosyltransferase (the rate-limiting enzyme). In this study, we aimed to enhance 2'-FL production by increasing the expression of soluble α-1,2-fucosyltransferase from Helicobacter pylori (FucT2). Because structural information regarding FucT2 has not been unveiled, we decided to improve the expression of soluble FucT2 in E. coli via directed evolution using a protein solubility biosensor that links protein solubility to antimicrobial resistance. For such a system to be viable, the activity of kanamycin resistance protein (KanR) should be dependent on FucT2 solubility. KanR was fused to the C-terminus of mutant libraries of FucT2, which were generated using a combination of error-prone PCR and DNA shuffling. Notably, one round of the directed evolution process, which consisted of mutant library generation and selection based on kanamycin resistance, resulted in a significant increase in the expression level of soluble FucT2. As a result, a batch fermentation with the ΔL M15 pBCGW strain, expressing the FucT2 mutant (F#1-5) isolated from the first round of the directed evolution process, resulted in the production of 0.31 g/l 2'-FL with a yield of 0.22 g 2'-FL/g lactose, showing 1.72- and 1.51-fold increase in the titer and yield, respectively, compared to those of the control strain. The simple and powerful method developed in this study could be applied to enhance the solubility of other unstable enzymes.

SKF96365 impedes spinal glutamatergic transmission-mediated neuropathic allodynia

  • Qiru Wang;Yang Zhang;Qiong Du;Xinjie Zhao;Wei Wang;Qing Zhai;Ming Xiang
    • The Korean Journal of Physiology and Pharmacology
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    • v.27 no.1
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    • pp.39-48
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    • 2023
  • Spinal nerve injury causes mechanical allodynia and structural imbalance of neurotransmission, which were typically associated with calcium overload. Storeoperated calcium entry (SOCE) is considered crucial elements-mediating intracellular calcium homeostasis, ion channel activity, and synaptic plasticity. However, the underlying mechanism of SOCE in mediating neuronal transmitter release and synaptic transmission remains ambiguous in neuropathic pain. Neuropathic rats were operated by spinal nerve ligations. Neurotransmissions were assessed by whole-cell recording in substantia gelatinosa. Immunofluorescence staining of STIM1 with neuronal and glial biomarkers in the spinal dorsal horn. The endoplasmic reticulum stress level was estimated from qRT-PCR. Intrathecal injection of SOCE antagonist SKF96365 dose-dependently alleviated mechanical allodynia in ipsilateral hind paws of neuropathic rats with ED50 of 18 ㎍. Immunofluorescence staining demonstrated that STIM1 was specifically and significantly expressed in neurons but not astrocytes and microglia in the spinal dorsal horn. Bath application of SKF96365 inhibited enhanced miniature excitatory postsynaptic currents in a dosage-dependent manner without affecting miniature inhibitory postsynaptic currents. Mal-adaption of SOCE was commonly related to endoplasmic reticulum (ER) stress in the central nervous system. SKF96365 markedly suppressed ER stress levels by alleviating mRNA expression of C/ EBP homologous protein and heat shock protein 70 in neuropathic rats. Our findings suggested that nerve injury might promote SOCE-mediated calcium levels, resulting in long-term imbalance of spinal synaptic transmission and behavioral sensitization, SKF96365 produces antinociception by alleviating glutamatergic transmission and ER stress. This work demonstrated the involvement of SOCE in neuropathic pain, implying that SOCE might be a potential target for pain management.

Integrating the Theory of Planned Behavior and Social Ecological Model to Explain Exercise Intention (운동의도 설명을 위한 계획된 행동이론과 사회생태학적 모델의 통합)

  • Lee, Yoon-Gu;Suh, Kwang-Bong
    • 한국체육학회지인문사회과학편
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    • v.55 no.5
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    • pp.117-125
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    • 2016
  • Most of health behavioral research were conducted with only individual psychological variables. The purpose of this study was to integrate the theory of planned behavior and social ecological model to explain exercise intention through multilateral approaches. 50s male 252 exercise participants were used in the final data analysis. Reliability analysis, confirmatory factor analysis, correlation analysis, structural equation modeling were used to analyze. The results indicated that physical environment has a positive influence on the subjective norm. Attitude, subjective norm and perceived behavioral control have positive influence on intention. Lastly, Social environment has a positive influence on the attitude. According to the results of this study, it will be able to contribute to integration of theoretical model for various health behavior and also help to develop intervention for physical activity promotion.

Wheat phytase potentially protects HT-29 cells from inflammatory nucleotides-induced cytotoxicity

  • Jeongmin An;Jaiesoon Cho
    • Animal Bioscience
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    • v.36 no.10
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    • pp.1604-1611
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    • 2023
  • Objective: The aim of this study was to investigate the protective effect of wheat phytase as a structural decomposer of inflammatory nucleotides, extracellular adenosine triphosphate (ATP), and uridine diphosphate (UDP) on HT-29 cells. Methods: Phosphatase activities of wheat phytase against ATP and UDP was investigated in the presence or absence of inhibitors such as L-phenylalanine and L-homoarginine using a Pi Color Lock gold phosphate detection kit. Viability of HT-29 cells exposed to intact- or dephosphorylated-nucleotides was analyzed with an EZ-CYTOX kit. Secretion levels of pro-inflammatory cytokines (IL-6 and IL-8) in HT-29 cells exposed to substrate treated with or without wheat phytase were measured with enzyme-linked immunosorbent assay kits. Activation of caspase-3 in HT-29 cells treated with intact ATP or dephosphorylated-ATP was investigated using a colorimetric assay kit. Results: Wheat phytase dephosphorylated both nucleotides, ATP and UDP, in a dose-dependent manner. Regardless of the presence or absence of enzyme inhibitors (L-phenylalanine and L-homoarginine), wheat phytase dephosphorylated UDP. Only L-phenylalanine inhibited the dephosphorylation of ATP by wheat phytase. However, the level of inhibition was less than 10%. Wheat phytase significantly enhanced the viability of HT-29 cells against ATP- and UDP-induced cytotoxicity. Interleukin (IL)-8 released from HT-29 cells with nucleotides dephosphorylated by wheat phytase was higher than that released from HT-29 cells with intact nucleotides. Moreover, the release of IL-6 was strongly induced from HT-29 cells with UDP dephosphorylated by wheat phytase. HT-29 cells with ATP degraded by wheat phytase showed significantly (13%) lower activity of caspase-3 than HT-29 cells with intact ATP. Conclusion: Wheat phytase can be a candidate for veterinary medicine to prevent cell death in animals. In this context, wheat phytase beyond its nutritional aspects might be a novel and promising tool for promoting growth and function of intestinal epithelial cells under luminal ATP and UDP surge in the gut.

A Study on the Effect of Internal and External Pressures on ESG Activities and Business Performance (내외부 압력이 ESG 활동과 경영성과에 미치는 영향에 관한 연구)

  • TaeYang Park;Jong Dae Kim
    • Journal of Korean Society of Industrial and Systems Engineering
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    • v.46 no.1
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    • pp.1-14
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    • 2023
  • This study is a leading case of empirical analysis of whether, when corporate stakeholders (government, investors, customers, managers, employees) put pressure on companies for ESG management, it affects the introduction and implementation of ESG activities (environmental, social, governance) and affects business performance. As for the research method, a sustainability report was published, and a web survey of Korea Research Inc. was conducted from May 10 to May 20, 2022 targeting ESG management managers of 192 companies, and analyzed through the PLS structural equation model. As a result of the study, it was found that the introduction and execution of ESG is closely influenced by the pressure from the government, investors, managers, and employees, and in particular, the internal pressure of current managers and executives and employees has a great impact on the introduction and implementation of environmental, social, and governance activities. In particular, although external pressure also has some influence, it is practical to suggest that strong internal pressure is necessary for continuous activities and performance. And, methodologically, the main activity indicators of the GRI Reporting Guidelines, which are the most representative ESG management indicators, were developed as a questionnaire, and reliability, validity, and model fit were secured through comparison with indicators of multiple systems and expert reviews. The limitations of this study are that more in-depth analysis by industry or size is possible when ESG management is mature and sufficient samples are secured, and complex ESG pressure factor modeling is possible when more diverse stakeholders are added.

Inhibitory Effects of Collagen Coated Coffee Bean Intake on Skin Aging

  • Lee, In-Ah;Ha, Mi-Ae;Shin, Yong-Wook
    • Journal of People, Plants, and Environment
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    • v.22 no.1
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    • pp.39-52
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    • 2019
  • To evaluate the protective effect of collagen peptide-coated coffee extract on skin aging, cell viability was measured with a MTT assay using cultured CCD-986sk fibroblasts, and its effect on wrinkles in the skin of hairless mice induced by UVB-irradiation was examined. In addition, its effect on procollagen synthesis and anti-oxidative, and its inhibitory activity against collagenase, elastase, tyrosinase and MMP-1 were analysed. After the 30-minute topical treatment, the animals were exposed to UVB irradiation (60-100 mJ/cm2) for 4 weeks and its intensity increased during the period. Under the experimental conditions set in this study, the skin thickness of hairless mice significantly decreased (11.8-21.3%) compared to the control group. Based on these results, the prolonged oral intake of a collagen peptide mixture with coffee is expected to significantly increase the synthesis of procollagen in dermal fibroblasts, thereby contributing to the alleviation of wrinkling and lowered elasticity due to structural damage to the dermal layer caused by UV. The oral intake of collagen-coated coffee contributes to increasing collagen biosynthesis in a dose-dependent manner and alleviates the symptoms of thickened keratin caused by UV irradiation. However, it did not inhibit the enzymes involved in skin aging, whitening, wrinkle improvement, and antioxidation. Based on the these results, it can be concluded that the intake of collagen peptide-coated coffee extract can be utilized as an alternative material for the prevention or treatment of diseases associated with photoaging.

Seismic behavior of steel and sisal fiber reinforced beam-column joint under cyclic loading

  • S.M. Kavitha;G. Venkatesan;Siva Avudaiappan;Chunwei Zhang
    • Structural Engineering and Mechanics
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    • v.88 no.5
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    • pp.481-492
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    • 2023
  • The past earthquakes revealed the importance of the design of moment-resisting reinforced concrete framed structures with ductile behavior. Due to seismic activity, failures in framed structures are widespread in beam-column joints. Hence, the joints must be designed to possess sufficient strength and stiffness. This paper investigates the effects of fibers on the ductility of hybrid fiber reinforced self-compacting concrete (HFRSCC) when subjected to seismic actions; overcoming bottlenecks at the beam-column joints has been studied by adding low modulus sisal fiber and high modulus steel fiber. For this, the optimized dose of hooked end steel fiber content (1.5%) was kept constant, and the sisal fiber content was varied at the rate of 0.1%, up to 0.3%. The seismic performance parameters, such as load-displacement behavior, ductility, energy absorption capacity, stiffness degradation, and energy dissipation capacity, were studied. The ductility factor and the cumulative energy dissipation capacity of the hybrid fiber (steel fiber, 1.5% and sisal fiber, 0.2%) added beam-column joint specimen is 100% and 121% greater than the control specimen, respectively. And also the stiffness of the hybrid fiber reinforced specimen is 100% higher than the control specimen. Thus, the test results showed that adding hybrid fibers instead of mono fibers could significantly enhance the seismic performance parameters. Therefore, the hybrid fiber reinforced concrete with 1.5% steel and 0.2% sisal fiber can be effectively used to design structures in seismic-prone areas.

Molybdenum-Based Electrocatalysts for Direct Alcohol Fuel Cells: A Critical Review

  • Gaurav Kumar Yogesh;Rungsima Yeetsorn;Waritnan Wanchan;Michael Fowler;Kamlesh Yadav;Pankaj Koinkar
    • Journal of Electrochemical Science and Technology
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    • v.15 no.1
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    • pp.67-95
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    • 2024
  • Direct alcohol fuel cells (DAFCs) have gained much attention as promising energy conversion devices due to their ability to utilize alcohol as a fuel source. In this regard, Molybdenum-based electrocatalysts (Mo-ECs) have emerged as a substitution for expensive Pt and Ru-based co-catalyst electrode materials in DAFCs, owing to their unique electrochemical properties useful for alcohol oxidation. The catalytic activity of Mo-ECs displays an increase in alcohol oxidation current density by several folds to 1000-2000 mA mgPt-1, compared to commercial Pt and PtRu catalysts of 10-100 mA mgPt-1. In addition, the methanol oxidation peak and onset potential have been significantly reduced by 100-200 mV and 0.5-0.6 V, respectively. The performance of Mo-ECs in both acidic and alkaline media has shown the potential to significantly reduce the Pt loading. This review aims to provide a comprehensive overview of the bifunctional mechanism involved in the oxidation of alcohols and factors affecting the electrocatalytic oxidation of alcohol, such as synthesis method, structural properties, and catalytic support materials. Furthermore, the challenges and prospects of Mo-ECs for DAFCs anode materials are discussed. This in-depth review serves as valuable insight toward enhancing the performance and efficiency of DAFC by employing Mo-ECs.

A comparative study on the hepatoprotective effect of selenium-nanoparticles and dates flesh extract on carbon tetrachloride induced liver damage in albino rats

  • Ghada Nady Ouais;Doaa Mohamad Hassan
    • Anatomy and Cell Biology
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    • v.56 no.4
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    • pp.538-551
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    • 2023
  • Exposure to environmental pollutants such as carbon tetrachloride (CCL4) causes liver damage. This study aimed to compare the ameliorative activity of the dates flesh extract (DFE) and selenium-nanoparticles (SeNPs) on CCL4-induced hepatotoxicity and if DFE could be a useful alternative supplement. Twenty-four male albino rats were enrolled and randomly divided into four equal groups (6 rats in each group): control group received only basal diet with no medications. Group II received CCL4 in a dose of 0.5 mg/kg intraperitoneal injection twice weekly for four weeks. Group III rats were pretreated with SeNPs in a dose of 2.5 mg/kg once a day orally three times/wk for four weeks alone then combined with the previously described dose of CCL4 for another four weeks. Group IV rats were pretreated with DFE in a dose of 8 ml of the aqueous extract/kg/d orally for four weeks alone then combined with the previously described dose of CCL4 for another four weeks. The liver damage was assessed by estimation of plasma concentration of albumin and enzymes activities of alanine aminotransferase and tissue genes expression. Liver oxidation levels were assessed by measuring the tissue concentration of the malondialdehyde, superoxide dismutase, and the total glutathione. Additionally, inflammatory mediators tumour necrosis factor--α and interleukin-6 were estimated. Detecting the liver's cellular structural damage was done by histopathological and immunohistochemical examination. This study suggests that CCL4-induced liver damage in rats can be protected by administration whether the costly SeNPs or the economical DFE.