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Satyajeet Das Sagar Batra Pramodkumar P. Gupta Mukesh Kumar Vijay Kumar Srivastava Anupam Jyoti Nagendra Singh Sanket Kaushik 《Journal of molecular recognition : JMR》2019,32(11)
Enterococcus faecalis is a gram‐positive, rod‐shape bacteria responsible for around 65% to 80% of all enterococcal nosocomial infections. It is multidrug resistant (MDR) bacterium resistant to most of the first‐line antibiotics. Due to the emergence of MDR strains, there is an urgent need to find novel targets to develop new antibacterial drugs against E. faecalis. In this regard, we have identified naphthoate synthase (1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase, EC: 4.1.3.36; DHNS) as an anti‐E. faecalis target, as it is an essential enzyme for menaquinone (vitamin K2) synthetic pathway in the bacterium. Thus, inhibiting naphtholate synthase may consequently inhibit the bacteria's growth. In this regard, we report here cloning, expression, purification, and preliminary structural studies of naphthoate synthase along with in silico modeling, molecular dynamic simulation of the model and docking studies of naphthoate synthase with quercetin, a plant alkaloid. Biochemical studies have indicated quercetin, a plant flavonoid as the potential lead compound to inhibit catalytic activity of EfDHNS. Quercetin binding has also been validated by spectrofluorimetric studies in order to confirm the bindings of the ligand compound with EfDHNS at ultralow concentrations. Reported studies may provide a base for structure‐based drug development of antimicrobial compounds against E. faecalis. 相似文献
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Singhal P Gaur A Behl V Gautam A Varshney B Paliwal J Batra V 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,852(1-2):293-299
A simple, sensitive and rapid liquid chromatography/tandem mass spectrometric (LC-MS/MS) method was developed and validated for quantification of chloroquine, an antimalarial drug, in plasma using its structural analogue, piperazine bis chloroquinoline as internal standard (IS). The method is based on simple protein precipitation with methanol followed by a rapid isocratic elution with 10 mM ammonium acetate buffer/methanol (25/75, v/v, pH 4.6) on Chromolith SpeedROD RP-18e reversed phase chromatographic column and subsequent analysis by mass spectrometry in the multiple reaction monitoring mode (MRM). The precursor to product ion transitions of m/z 320.3-->247.2 and m/z 409.1-->205.2 were used to measure the analyte and the IS, respectively. The assay exhibited a linear dynamic range of 2.0-489.1 ng/mL for chloroquine in dog plasma. The limit of detection (LOD) and lower limit of quantification (LLOQ) were 0.4 and 2.0 ng/mL, respectively in 0.05 mL plasma. Acceptable precision and accuracy were obtained for concentrations over the standard curve range of 2.0-489.1 ng/mL. A run time of 2.0 min for a sample made it possible to achieve a throughput of more than 400 plasma samples analyzed per day. The validated method was successfully used to analyze samples of dog plasma during non-clinical study of chloroquine. 相似文献
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The ability to sense and respond to the environment is a hallmark of living systems. These processes occur at the levels of the organism, cells and individual molecules. Sensing of extracellular changes could result in a structural or chemical alteration in a molecule, which could in turn trigger a cascade of intracellular signals or regulated trafficking of molecules at the cell surface. These and other such processes allow cells to sense and respond to environmental changes. Often, these changes and the responses to them are spatially and/or temporally localized, and visualization of such events necessitates the use of high-resolution imaging approaches. Here we discuss optical imaging approaches and tools for imaging individual events at the cell surface with improved speed and resolution. 相似文献
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Shreyasi Asthana Zaved Hazarika Parth Sarathi Nayak Jyoti Roy Anupam Nath Jha Bibekanand Mallick Suman Jha 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):153-166
Background
Injection localized amyloidosis is one of the most prevalent disorders in type II diabetes mellitus (TIIDM) patients relying on insulin injections. Previous studies have reported that nanoparticles can play a role in the amyloidogenic process of proteins. Hence, the present study deals with the effect of zinc oxide nanoparticles (ZnONP) on the amyloidogenicity and cytotoxicity of insulin.Methods
ZnONP is synthesised and characterized using XRD, Zeta Sizer, UV-Visible spectroscope and TEM. The characterization is followed by ZnONP interaction with insulin, which is studied employing fluorescence spectroscopes, isothermal titration calorimetry and molecular dynamics simulations. The interaction leads insulin conformational rearrangement into amyloid-like fibril, which is studied using thioflavin T dye binding assay, circular dichroism spectroscopy and TEM, followed by cytotoxicity propensity using Alamar Blue dye reduction assay.Results
Insulin has very weak interaction with ZnONP interface. Insulin at studied concentration forms amorphous aggregates at physiological pH, whereas in presence of ZnONP interface amyloid-like fibrils are formed. While the amyloid-like fibrils are cytotoxic to MIN6 and THP-1 cell lines, insulin and ZnONP individual solutions and their fresh mixtures enhance the cells proliferation.Conclusions
The presence of ZnONP interface enhances insulin fibrillation at physiological pH by providing a favourable template for the nucleation and growth of insulin amyloids.General significance
The studied protein-nanoparticle system from protein conformational dynamics point of view throws caution over nanoparticle use in biological applications, especially in vivo applications, considering the amyloidosis a very slow but non-curable degenerative disease. 相似文献60.
Coonrod SA Naaby-Hansen S Shetty J Shibahara H Chen M White JM Herr JC 《Developmental biology》1999,207(2):334-349
The effect of phosphatidyinositol-specific phospholipase C (PI-PLC) on mouse sperm-egg interaction was investigated in this study to determine if glycosyl-phosphatidylinositol (GPI)-anchored proteins are involved in mammalian fertilization. When both sperm and zona-intact oocytes were pretreated with a highly purified preparation of PI-PLC and coincubated, there was no significant effect on sperm-zona pellucida binding; however, fertilization was reduced from 59.6% (control group) to 2.8% (treatment group). A similar reduction in fertilization rates was found when zona-intact oocytes were treated with PI-PLC and washed prior to incubation with untreated sperm. The effect of PI-PLC on sperm binding and fusion with zona-free oocytes was then investigated. Treatment of sperm with PI-PLC had no significant effect on sperm-egg binding or fusion. However, treatment of eggs with PI-PLC significantly reduced sperm-egg binding and fusion from 6.2 bound and 2.1 fused sperm per egg in the control group to 2.1 bound and 0.02 fused sperm per egg in the treatment group. This decrease in sperm-egg binding and fusion depended on the dose of PI-PLC employed, with a maximal inhibitory effect on binding and fusion at 5 and 1 U/ml, respectively. PI-PLC-treated oocytes could be artificially activated by calcium ionophore, demonstrating that the oocytes were functionally viable following treatment. Furthermore, treatment of oocytes with PI-PLC did not reduce the immunoreactivity of the non-GPI-anchored egg surface integrin, alpha6beta1. Taken together, these observations support the hypothesis that PI-PLC affects fertilization by specifically releasing GPI-anchored proteins from the oolemma. In order to identify the oolemmal GPI-anchored proteins involved in fertilization, egg surface proteins were labeled with sulfo-NHS biotin, treated with PI-PLC, and analyzed by two-dimensional gel electrophoresis followed by avidin blotting. A prominent high-molecular-weight protein cluster (approximately 70 kDa, pI 5) and a lower molecular weight (approximately 35-45 kDa, pI 5.5) protein cluster were released from the oolemmal surface as a result of PI-PLC treatment. It is likely that these GPI-anchored egg surface proteins are required for sperm-egg binding and fusion. 相似文献