A Real-Time NMR Method for Measurement of In Vitro Aggregation Kinetics Of Degarelix Drug Products

2021 ◽  
Vol 22 (2) ◽  
Author(s):  
Sharadrao M. Patil ◽  
Bin Qin ◽  
Yan Wang ◽  
Snober Ahmed ◽  
Huzeyfe Yilmaz ◽  
...  
Keyword(s):  
Real Time ◽  
Aggregation Kinetics ◽  
Drug Products ◽  
Nmr Method ◽  
Kinetics Of

scholarly journals Assessment of the Kinetics of Treponema pallidum Dissemination into Blood and Tissues in Experimental Syphilis by Real-Time Quantitative PCR

2007 ◽  
Vol 75 (6) ◽  
pp. 2954-2958 ◽  
Author(s):  
Juan C. Salazar ◽  
Asha Rathi ◽  
Nelson L. Michael ◽  
Justin D. Radolf ◽  
Linda L. Jagodzinski
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Whole Blood ◽  
Mononuclear Cells ◽  
Treponema Pallidum ◽  
Blood Components ◽  
Peripheral Blood Mononuclear ◽  
Real Time Quantitative Pcr ◽  
Kinetics Of

ABSTRACT Little is known about the size and kinetics of treponemal burdens in blood and tissues during acquired or experimental syphilitic infection. We used real-time quantitative PCR to measure Treponema pallidum DNA levels in rabbits infected intratesticularly with the prototype Nichols strain. At the outset, we performed a series of in vitro blood spiking experiments to determine the effect of blood processing procedures on the distribution of treponemes in various blood components. T. pallidum DNA levels in plasma and whole blood were approximately 10-fold higher than those in serum and more than 200-fold greater than those in peripheral blood mononuclear cells (PBMCs). Ten rabbits were inoculated intratesticularly with doses of treponemes ranging from 4 × 107 to 2 × 108 organisms. In five rabbits, T. pallidum DNA levels were measured sequentially in serum, plasma, whole blood, and PBMCs until sacrifice at peak orchitis, at which time brain, kidney, liver, spleen, and testicles were harvested; blood and organs were also harvested at orchitis from the other five rabbits. T. pallidum DNA was detected in plasma within 24 h postinfection. Treponeme levels in whole blood and blood components increased significantly with the development of peak orchitis. Overall, levels in serum and PBMCs were lower than those in plasma and whole blood; this disparity was particularly marked at early time points. Significantly greater numbers of spirochetes were found in the spleen than in liver, kidney, or brain tissue at the time of sacrifice. Our findings highlight the remarkable capacity of T. pallidum to disseminate from the site of infection to blood and tissues, and they identify the spleen as a prime target for treponemal invasion.

Nanomechanical Microcantilevers for In-Vitro Biomolecular Detection

2007 ◽  
Author(s):  
Kilho Eom ◽  
Tae Yun Kwon ◽  
Jinsung Park ◽  
Sungsoo Na ◽  
Dae Sung Yoon ◽  
...  
Keyword(s):  
Real Time ◽  
Biological Molecules ◽  
Label Free ◽  
Biomolecular Detection ◽  
Label Free Detection ◽  
Detection Of Biomolecules ◽  
Kinetics Of ◽  
Insight Into

Nanomechanical microcantilevers have enabled the sensitive label-free detection of chemical and/or biological molecules. In recent years, resonating microcantilevers have achieved the unprecedented sensitivity in detecting molecules. In this article, we review our current works on the label-free detection of biomolecules based on resonating microcantilevers. Our piezoelectric thick film microcantilevers exhibit the relatively high quality factor in a viscous liquid, indicating the potential of our cantilever to in situ biosensor applications for real-time detection of biomolecular interactions. It is shown that our microcantilevers allow the noise-free real-time monitoring of biomolecular recognitions, providing the insight into kinetics of biomolecular recognitions.

scholarly journals Sumoylation inhibits α-synuclein aggregation and toxicity

2011 ◽  
Vol 194 (1) ◽  
pp. 49-60 ◽  
Author(s):  
Petranka Krumova ◽  
Erik Meulmeester ◽  
Manuel Garrido ◽  
Marilyn Tirard ◽  
He-Hsuan Hsiao ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Dopaminergic Neurons ◽  
Double Mutant ◽  
Protein Solubility ◽  
Aggregation Kinetics ◽  
A Cell ◽  
Binding Interfaces ◽  
Kinetics Of

Posttranslational modification of proteins by attachment of small ubiquitin-related modifier (SUMO) contributes to numerous cellular phenomena. Sumoylation sometimes creates and abolishes binding interfaces, but increasing evidence points to another role for sumoylation in promoting the solubility of aggregation-prone proteins. Using purified α-synuclein, an aggregation-prone protein implicated in Parkinson’s disease that was previously reported to be sumoylated upon overexpression, we compared the aggregation kinetics of unmodified and modified α-synuclein. Whereas unmodified α-synuclein formed fibrils, modified α-synuclein remained soluble. The presence of as little as 10% sumoylated α-synuclein was sufficient to delay aggregation significantly in vitro. We mapped SUMO acceptor sites in α-synuclein and showed that simultaneous mutation of lysines 96 and 102 to arginine significantly impaired α-synuclein sumoylation in vitro and in cells. Importantly, this double mutant showed increased propensity for aggregation and cytotoxicity in a cell-based assay and increased cytotoxicity in dopaminergic neurons of the substantia nigra in vivo. These findings strongly support the model that sumoylation promotes protein solubility and suggest that defects in sumoylation may contribute to aggregation-induced diseases.

scholarly journals In Vitro Phosphorylation Does not Influence the Aggregation Kinetics of WT α-Synuclein in Contrast to Its Phosphorylation Mutants

2014 ◽  
Vol 15 (1) ◽  
pp. 1040-1067 ◽  
Author(s):  
Sarah Schreurs ◽  
Melanie Gerard ◽  
Rita Derua ◽  
Etienne Waelkens ◽  
Jean-Marc Taymans ◽  
...  
Keyword(s):  
Aggregation Kinetics ◽  
Kinetics Of

scholarly journals Impact of real-time shedding on binding kinetics of membrane-remaining L-selectin to PSGL-1

2019 ◽  
Vol 316 (5) ◽  
pp. C678-C689 ◽  
Author(s):  
Shuang Peng ◽  
Shen-Bao Chen ◽  
Lin-Da Li ◽  
Chun-Fang Tong ◽  
Ning Li ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Real Time ◽  
Jurkat Cells ◽  
Binding Kinetics ◽  
Time Dependent ◽  
Intact Cells ◽  
The Impact ◽  
Kinetics Of

L-selectin shedding induced by various cytokines is crucial in activating neutrophils (PMNs) in inflammatory cascade. While the real-time shedding in vivo lasts ~10 min after PMN activation, the impact of time-dependent shedding on binding kinetics of membrane-remaining L-selectins to its ligands is poorly understood at transient or steady state. Here, we developed an in vitro L-selectin shedding dynamics approach, together with competitive assays of cell adhesion, and proposed a theoretical model for quantifying the impact of real-time shedding on the binding kinetics of membrane-remaining L-selectins to P-selectin glycoprotein ligand-1 (PSGL-1). Our data indicated that the extent of L-selectin shedding on PMA activation is higher, but the terminating time is longer for Jurkat cells than those for human PMNs. Meanwhile, fMLF or IL-8 stimulation yields the longer terminating time than that on PMA stimulation but results in a similar shedding extent for PMNs. L-selectin shedding reduces L-selectin-PSGL-1-mediated cell adhesion in three ways: decreasing membrane-anchored L-selectins, increasing soluble L-selectins competitively binding to ligands, and presenting conformational alteration of membrane-remaining L-selectins themselves. Compared with those on intact cells, the binding affinities of membrane-remaining L-selectin-PSGL-1 pairs were all enhanced at initial and lowered at the late shedding phase for both PMN and Jurkat cells even with varied transition time points. The rolling velocities of both PMNs and Jurkat cells were increased following mechanically or biochemically induced shedding of L-selectin under shear flow. These findings help to further our understanding of the function of time-dependent L-selectin shedding during the inflammation cascade.

Diving under a Microscope—A New Simple and Versatile In Vitro Diving Device for Fluorescence and Confocal Microscopy Allowing the Controls of Hydrostatic Pressure, Gas Pressures, and Kinetics of Gas Saturation

2013 ◽  
Vol 19 (3) ◽  
pp. 608-616 ◽  
Author(s):  
Qiong Wang ◽  
Marc Belhomme ◽  
François Guerrero ◽  
Aleksandra Mazur ◽  
Kate Lambrechts ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Real Time ◽  
Decompression Sickness ◽  
Fluorescent Microscopy ◽  
Cell Activity ◽  
Real Time Monitoring ◽  
Isolated Cells ◽  
Gas Saturation ◽  
Kinetics Of

AbstractHow underwater diving effects the function of the arterial wall and the activities of endothelial cells is the focus of recent studies on decompression sickness. Here we describe an in vitro diving system constructed to achieve real-time monitoring of cell activity during simulated dives under fluorescent microscopy and confocal microscopy. A 1-mL chamber with sapphire windows on both sides and located on the stage of an inverted microscope was built to allow in vitro diving simulation of isolated cells or arteries in which activities during diving are monitored in real-time via fluorescent microscopy and confocal microscopy. Speed of compression and decompression can range from 20 to 2000 kPa/min, allowing systemic pressure to range up to 6500 kPa. Diving temperature is controlled at 37°C. During air dive simulation oxygen partial pressure is optically monitored. Perfusion speed can range from 0.05 to 10 mL/min. The system can support physiological viability of in vitro samples for real-time monitoring of cellular activity during diving. It allows regulations of pressure, speeds of compression and decompression, temperature, gas saturation, and perfusion speed. It will be a valuable tool for hyperbaric research.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

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