Production of Neuraminidase in Cell Monolayers infected with Influenza Virus
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Cell‐in‐cell structures represent live cell events in which one cell internalizes another. Because formation of cell‐in‐cell structures is a rare event in most cell types and the event is associated with cell death, there has been limited clarification of this phenomenon, and its physiological role and molecular mechanism are yet to be precisely elucidated. In this study, we established a mutagenized cell line that exhibited cell‐in‐cell structures at a more than 10‐fold higher frequency as compared to the parent cells. Interestingly, both engulfment and invasion were increased in the mutagenized cell line as compared with that in the parent cell line in the suspension culture condition. This finding indicates that this mutagenized cell line showed an interchangeable status in terms of its ability to form cell‐in‐cell structures, and the system described here could be useful for elucidation of the mechanisms regulating the formation of cell‐in‐cell structures, including engulfment and invasion, in a given cellular environment. Further studies using this cell line are warranted to understand the mechanism of formation and biological significance of the cell‐in‐cell formation.
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This chapter contains sections titled: Introduction Influenza Neuraminidase as a Drug Target Neuraminidase Active Site and Inhibitor Binding Small-Molecule Inhibitors of Influenza Neuraminidase Mechanism of Resistance Influenza Neuraminidase Inhibitors Based on Other Scaffolds Clinical Use of Neuraminidase Inhibitors Concluding Remarks References
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The wild type influenza strain A/Aichi/2/68 (H 3 N 2 ), when disrupted with SDS and electrophoresed on cellulose acetate paper, yielded two separate neuraminidases, NA(H+) and NA(H−). These enzymes after extraction were biologically active and possessed different specific activities. Enzyme NA(H+) possessed neuraminidase as well as hemagglutinin activities whereas enzyme NA(H−) demonstrated only neuraminidase activity. Similar results were obtained when the Aichi strain was treated with Tween–ether and the two enzymes were separated by affinity chromatography. Techniques used failed to separate the hemagglutinin activity from neuraminidase NA(H+). These results suggest that the dual activity present in enzyme NA(H+) may be characteristic of this protein. Both enzymes are antigenically different and are apparently present as distinct entities in the Aichi strain. Experiments showed that only enzyme NA(H−) of the Aichi strain was incorporated into the hybrid X-32 virus during genetic recombination.
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Objective:To determine the inhibitory effect of compounds L20051117 and AMMS607 on influenza virus neuraminidase at molecular level.Methods:The anti-influenza virus neuraminidase activity of L20051117 and AMMS607 was examined by the fluorimetric assay.Results:The inhibitory effect of L20051117 on influenza virus neuraminidase was higher than that of AMMS607,the inhibition of influenza A virus neuraminidase by L20051117 and AMMS607 was higher than that of influenza B virus neuraminidase.Conclusion:L20051117 is a potent inhibitor of the neuraminidase activity of both influenza A and B virus.Its in vitro activity is higher than AMMS607.
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본 연구에서는 인플루엔자 바이러스 표면에 존재하는 neuraminidase 효소의 활성을 평가 할 수있는 종이칩 기반의 분석 시스템을 구축하였다. 종이칩의 장점을 살려 분석 전문가와 장비 없이 현장 진단(Point-of-care)이 가능하도록 X-Neu5Ac 기질을 이용한 비색분석법을 통해 시료 내 neuraminidase 효소의 존재를 정량적으로 확인 할 수 있도록 설계 및 제작하였다. Neuraminidase 효소의 활성을 확인할 수 있는 종이칩 센서(Paper-based neuraminidase assay sensor; PNAS) 성능 실험 결과 neuraminidase를 0.004 U/mL 농도부터 검출 가능하였으며, 인간 혈청에 각기 다른 농도로 존재하는 neuraminidase 효소의 양을 활성 평가를 통해 정량적으로 검출할 수 있음을 입증하였다($R^2$ > 0.99). 또한, 보관기간에 따른 종이칩의 안정성 평가 결과 빛이 차단 된 $4^{\circ}C$ 환경에서 보관 시 70일까지 초기 성능이 안정하게 유지됨을 확인하였다. 마지막으로, PNAS 상에서 효소 반응의 신뢰성 평가를 위해 미카엘리스-멘텐 동역학 (Michaelis-Menten kinetics)을 적용하여 X-Neu5Ac 기질에 대한 neuraminidase의 동역학 분석 결과 $K_m$ 값은 $8.327{\times}10^{-3}M$ 으로 확인되었으며, 이 값은 용액상에서의 효소 반응 속도 계산으로부터 산출된 값과($K_m=8.327{\times}10^{-3}M$ ) 근사한 수치임을 확인하였다. 본 연구로부터 개발된 종이칩 기반의 neuraminidase 효소 활성 평가 시스템은 인플루엔자 바이러스의 신속하고 안전한 검출에 다양하게 응용 될 수 있을 것으로 생각된다. In this study, we described a paper-based neuraminidase assay sensor (PNAS) which can be applied to detect the infection by influenza viruses. The PNAS was designed and manufactured to quantitatively identify the levels of neuraminidase in the sample, which is based on colorimetric analysis using the X-Neu5Ac substrate. The limit of detection of the PNAS was determined as 0.004 U/mL of neuraminidase. According to the amount of neuraminidase in human serum, the PNAS could monitor the enzyme activity with a good linearity ($R^2$ > 0.99). In addition, the initial performance of the PNAS has been maintained up to 70 days in the $4^{\circ}C$ . Finally, we demonstrated whether the Michaelis-Menten kinetics is applied to the PNAS, which can show the reliability of the enzyme reactions. The kinetic studies indicated that the PNAS provides the good condition for enzyme reactions ($K_m=8.327{\times}10^{-3}M$ ), but they were performed on paper chip nonetheless. The paper-based neuraminidase assay sensor may be useful in a wide range of rapid and safe detection of influenza virus.
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We have developed a temperature-responsive culture dish grafted with a poly(N-isopropylacrylamide) (PIPAAm). Various types of cells adhere, spread, and proliferate on the grafted dishes in the presence of serum at 37°C. By reducing only temperature, these cells can be harvested noninvasively from the dishes according to rapid hydration of the grafted polymer. Because the harvest does not need enzymatic digestion, differentiated cell phenotypes are retained. In the present study, a renal epithelial cell line, Madin-Darby canine kidney (MDCK) cell, was cultured on the dishes, and cell behavior was examined. MDCK cells showed differentiated phenotypes such as dome formation during long-term culture, similar to on ungrafted dishes. After 1-week culture at 37°C, trypsin digestion disrupted cell–cell junctions but failed to liberate cells from both ungrafted and grafted dishes. However, short-term incubation at 20°C released confluent MDCK cells as a single contiguous cell sheet only from the polymer-grafted dishes because of selective disruption of the cell-surface binding. Immunocytochemistry with anti-β-catenin antibody revealed that functional cell–cell junctions were organized even in the recovered cell sheets. Intriguingly, incubation time at 20°C required for cell sheet detachment gradually shortened during long-term culture before reducing temperature. The acceleration of cell detachment was correlated to the decrease of a single cell area by means of cell contractile force. These findings suggest that cell sheet detachment from PIPAAm-grafted dishes should be accomplished by both PIPAAm hydration and cellular metabolic activity such as cell contraction. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 51, 216–223, 2000.
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