scholarly journals Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 673
Author(s):  
Yan Liu ◽  
Yali Shen ◽  
Yang Song ◽  
Lei Xu ◽  
J. Jefferson P. P. Perry ◽  
...  
Keyword(s):  
Real Time ◽  
Catalytic Efficiency ◽  
Protein Activity ◽  
Kinetic Determination ◽  
Pioneer Effort ◽  
Living Organisms ◽  
E2 Enzymes ◽  
Isopeptidase Activity ◽  
Kinetics Of

Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases.

scholarly journals Kinetics of Recombinant Adenovirus Type 5, Vaccinia Virus, Modified Vaccinia Ankara Virus, and DNA Antigen Expression In Vivo and the Induction of Memory T-Lymphocyte Responses

2008 ◽  
Vol 15 (4) ◽  
pp. 691-696 ◽  
Author(s):  
Ralf Geiben-Lynn ◽  
John R. Greenland ◽  
Kwesi Frimpong-Boateng ◽  
Norman L. Letvin
Keyword(s):  
Immune Responses ◽  
Real Time ◽  
T Lymphocyte ◽  
Recombinant Adenovirus ◽  
Antigen Expression ◽  
Vaccine Antigen ◽  
Photon Imaging ◽  
Lymphocyte Responses ◽  
Kinetics Of

ABSTRACT While a new generation of vaccine vectors has been developed for eliciting cellular immune responses, little is known about the optimal routes for their administration or about the ramifications of the kinetics of in vivo vaccine antigen expression for immunogenicity. We evaluated the kinetics of vaccine antigen expression by real-time in vivo photon imaging and showed dramatic differences in these kinetics using different vectors and different routes of administration. Further, using a gamma interferon enzyme-linked immunospot assay to measure T-lymphocyte immune responses, we observed an association between the kinetics of vaccine antigen expression in vivo and the magnitude of vaccine-elicited memory T-lymphocyte responses. These results highlight the utility of the real-time in vivo photon-imaging technology in evaluating novel immunization strategies and suggest an association between the kinetics of vaccine antigen clearance and the magnitude of vaccine-elicited T-lymphocyte memory immune responses.

scholarly journals Real-time nanodiamond thermometry probing in vivo thermogenic responses

2020 ◽  
Vol 6 (37) ◽  
pp. eaba9636 ◽  
Author(s):  
Masazumi Fujiwara ◽  
Simo Sun ◽  
Alexander Dohms ◽  
Yushi Nishimura ◽  
Ken Suto ◽  
...  
Keyword(s):  
Real Time ◽  
Thermal Imaging ◽  
Biological Activities ◽  
Temperature Monitoring ◽  
Potential Applications ◽  
Sample Chamber ◽  
Chemical Stimuli ◽  
Living Organisms ◽  
Mitochondrial Uncouplers

Real-time temperature monitoring inside living organisms provides a direct measure of their biological activities. However, it is challenging to reduce the size of biocompatible thermometers down to submicrometers, despite their potential applications for the thermal imaging of subtissue structures with single-cell resolution. Here, using quantum nanothermometers based on optically accessible electron spins in nanodiamonds, we demonstrate in vivo real-time temperature monitoring inside Caenorhabditis elegans worms. We developed a microscope system that integrates a quick-docking sample chamber, particle tracking, and an error correction filter for temperature monitoring of mobile nanodiamonds inside live adult worms with a precision of ±0.22°C. With this system, we determined temperature increases based on the worms’ thermogenic responses during the chemical stimuli of mitochondrial uncouplers. Our technique demonstrates the submicrometer localization of temperature information in living animals and direct identification of their pharmacological thermogenesis, which may allow for quantification of their biological activities based on temperature.

scholarly journals Enhanced enzyme kinetics of reverse transcriptase variants cloned from animals infected with SIVmac239 lacking viral protein X

2020 ◽  
Vol 295 (50) ◽  
pp. 16975-16986
Author(s):  
Si'Ana A. Coggins ◽  
Dong-Hyun Kim ◽  
Raymond F. Schinazi ◽  
Ronald C. Desrosier ◽  
Baek Kim
Keyword(s):  
Steady State ◽  
Viral Protein ◽  
Catalytic Efficiency ◽  
Myeloid Cell ◽  
Immunodeficiency Virus ◽  
Protein X ◽  
Kinetics Of ◽  
Hiv 1

HIV Type 1 (HIV-1) and simian immunodeficiency virus (SIV) display differential replication kinetics in macrophages. This is because high expression levels of the active host deoxynucleotide triphosphohydrolase sterile α motif domain and histidine-aspartate domain–containing protein 1 (SAMHD1) deplete intracellular dNTPs, which restrict HIV-1 reverse transcription, and result in a restrictive infection in this myeloid cell type. Some SIVs overcome SAMHD1 restriction using viral protein X (Vpx), a viral accessory protein that induces proteasomal degradation of SAMHD1, increasing cellular dNTP concentrations and enabling efficient proviral DNA synthesis. We previously reported that SAMHD1-noncounteracting lentiviruses may have evolved to harbor RT proteins that efficiently polymerize DNA, even at low dNTP concentrations, to circumvent SAMHD1 restriction. Here we investigated whether RTs from SIVmac239 virus lacking a Vpx protein evolve during in vivo infection to more efficiently synthesize DNA at the low dNTP concentrations found in macrophages. Sequence analysis of RTs cloned from Vpx (+) and Vpx (−) SIVmac239–infected animals revealed that Vpx (−) RTs contained more extensive mutations than Vpx (+) RTs. Although the amino acid substitutions were dispersed indiscriminately across the protein, steady-state and pre-steady-state analysis demonstrated that selected SIVmac239 Vpx (−) RTs are characterized by higher catalytic efficiency and incorporation efficiency values than RTs cloned from SIVmac239 Vpx (+) infections. Overall, this study supports the possibility that the loss of Vpx may generate in vivo SIVmac239 RT variants that can counteract the limited availability of dNTP substrate in macrophages.

Real-time in vivo kinetics of protoporphyrin IX after administration of 5-aminolevulinic acid in meningiomas and comparative analyses with glioblastomas

2020 ◽  
Vol 162 (9) ◽  
pp. 2197-2202 ◽  
Author(s):  
Sadahiro Kaneko ◽  
Benjamin Brokinkel ◽  
Eric Suero Molina ◽  
Nils Warneke ◽  
Markus Holling ◽  
...  
Keyword(s):  
Real Time ◽  
Aminolevulinic Acid ◽  
Protoporphyrin Ix ◽  
Comparative Analyses ◽  
In Vivo 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.

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.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Hepatobiliary Kinetics of 113mIn-Phenolphthalexon

1981 ◽  
Vol 20 (02) ◽  
pp. 90-93
Author(s):  
P.B. Parab ◽  
U.R. Raikar ◽  
R.D. Ganatra ◽  
M. C. Patel
Keyword(s):  
Biliary Excretion ◽  
Iminodiacetic Acid ◽  
Organ Distribution ◽  
Liver Uptake ◽  
On Line ◽  
Rapid Clearance ◽  
Chemical Purity ◽  
Kinetics Of ◽  
High Liver

Phenolphthalexon, a compound with iminodiacetic acid as a functional group, has been labelled with 113mIn to high chemical purity and its usefulness in studies of biliary excretion patency has been studied. Organ distribution of 113mIn-phenolphthalexon in mice was characterized by high liver uptake (50.8% of the administered dose after 5 min) and rapid clearance through the gall bladder. An animal model for studying obstruction of biliary excretion has been developed. Data on the kinetics of the radiopharmaceutical were obtained by collecting in-vivo data through an on-line computer.

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