60 research outputs found
A Theorem on Matroid Homomorphism
This note generalizes a result contained in a previous paper [ J. Sanders,
Circuit preserving edge maps II, J. Combin. Theory Ser. B 42 (1987), 146-155].Comment: 5 pages, 0 figure
Development of a DPAP1-specific HTS assay.
<p><b>A.</b> Continuous assay. The assay was carried out in 384-well plates using 1% of parasite lysates. Substrate turnover was continuously measured for 5 min. JCP410 (10 µM) was used as a positive inhibition control. Z’ factor, S/N, and % CV of the negative control are shown. <b>B.</b> End-point assay for HTS. The reaction described in A was quenched after 10 min by addition of 0.5 M acetic acid. The final concentration of rhodamine product was quantified by fluorescence.</p
Cat C-specific fluorogenic assay in rat liver lysates.
<p><b>A.</b> Labeling of Cat C with FY01. Rat liver extract extracts were treated with increasing concentrations of FY01 for 1 h and labeled proteins analyzed by SDS-PAGE followed by scanning of the gel using a flatbed laser scanner. The location of labeled Cat C is indicated. <b>B.</b> Inhibition of substrate turnover specifically correlates with Cat C labeling. The cleavage of (Pro-Arg)<sub>2</sub>-Rho substrate was measured prior to analysis of FY01 labeling shown in part A. Quantification of the indicated labeled proteins relative to DMSO control is shown. <b>C.</b> Cat C-specific HTS assay in rat liver extracts. Rat liver lysates were treated for 30 min with either DMSO or JCP410 (10 µM) followed by the addition of 10 µM of (Pro-Arg)<sub>2</sub>-Rho. The turnover rate was continuously measured for 5 min in a 384-well plate. Z’ factor, S/N, and % CV of the negative control are shown.</p
Use of an ABP to identify a DPAP1-selective substrate in parasite lysates.
<p><b>A.</b> Structure and reaction mechanism of the (Pro-Arg)<sub>2</sub>-Rho substrate. <b>B.</b> Measurement of (Pro-Arg)<sub>2</sub>-Rho apparent <i>K</i><sub>m</sub> in trophozoite lysates (circles) and with recombinant DPAP1 (triangle). Turnover rates at increasing concentrations of substrate were fitted to a Michaelis-Menten equation as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011985#s4" target="_blank">methods</a> section. <b>C.</b> Labeling of DPAP1 activity in parasite lysates with FY01. Trophozoite lysates were incubated for 1 h with increasing concentrations of FY01. Labeling was stopped by boiling the sample in SDS-PAGE loading buffer. DPAP1 activity was measured using a flatbed fluorescent scanner. <b>D.</b> DPAP1 labeling correlates with substrate turnover inhibition. An aliquot of the samples treated for 1 h with FY01 was diluted in assay buffer containing 10 µM of (Pro-Arg)<sub>2</sub>-Rho, and the initial turnover rate was measured in a 96-well plate (circles). This turnover rate is plotted with the labeling quantified in C.</p
Optimization of a Protease Activated Probe for Optical Surgical Navigation
Molecularly
targeted optical contrast agents have the potential
to enable surgeons to visualize specific molecular markers that can
help improve surgical precision and thus outcomes. Fluorescently quenched
substrates can be used to highlight tumor lesions by targeting proteases
that are highly abundant in the tumor microenvironment. However, the
majority of these and other molecularly targeted optical contrast
agents are labeled with reporter dyes that are not ideally matched
to the properties of clinical camera systems, which are typically
optimized for detection of indocyanine-green (ICG). While a wide range
of near-infrared (NIR) dyes are suitable for use with highly sensitive
and highly tunable research-focused small animal imaging systems,
most have not been evaluated for use with commonly used clinical imaging
systems. Here we report the optimization of a small molecule fluorescently
quenched protease substrate probe 6QC-ICG, which uses the indocyanine
green (ICG) dye as its optical reporter. We evaluated dosing and kinetic
parameters of this molecule in tumor-bearing mice and observed optimal
tumor over background signals in as little as 90 min with a dose of
2.3 mg/kg. Importantly, the fluorescence intensity of the probe signal
in tumors did not linearly scale with dose, suggesting the importance
of detailed dosing studies. Furthermore, when imaged using the FDA
approved <i>da Vinci</i> Si surgical system with Firefly
detection, signals were significantly higher for the ICG probe compared
to a corresponding probe containing a dye with similar quantum yield
but with a slightly shifted excitation and emission profile. The increased
signal intensity generated by the optimal dye and dose of the ICG
labeled probe enabled detection of small, flat lesions that were less
than 5 mm in diameter. Therefore, 6QC-ICG is a highly sensitive probe
that performs optimally with clinical imaging systems and has great
potential for applications in optical surgical navigation
Stage-Specific Expression of Falstatin
<p>Extracts from highly synchronized parasites were collected every 8 h, separated by SDS-PAGE, and evaluated by immunoblotting with anti-falstatin antibodies. Each sample of early-ring, late-ring, early-trophozoite, late-trophozite, early-schizont, or late-schizont extracts corresponded to 1.3 × 10<sup>7</sup> parasitized cells. The positions of molecular weight markers (kDa) are indicated. ER, early-ring; LR, late-ring; ET, early-trophozite; LT, late-trophozite; ES, early-schizont; LS, late-schizont.</p
Inhibition of Falstatin Function by Anti-Falstatin Antibodies
<p>Hydrolysis of the peptide substrate Z-Leu-Arg-AMC by falcipain-2 (FP2; 19.8 nM), falcipain-3 (FP3; 27.1 nM), or trophozoite extract (TE; corresponding to 5.5 × 10<sup>6</sup> parasites per reaction) was evaluated in the absence or presence of falstatin (31 nM) and the indicated quantities of anti-falstatin antibodies in 350 μl of 100 mM sodium acetate, 8 mM DTT (pH 6.0). Reaction components were incubated for 15 min before addition of substrate, and activity was measured as arbitrary fluorescence units over time (FU/min). Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p
Inhibitor Competition
<p>The indicated amounts of falstatin and anti-falstatin antibody were incubated with lysates from asynchronous parasite cultures before addition of [<sup>125</sup>I] DCG04, electrophoresis, and analysis by autoradiography. Results with increasing concentrations of falstatin (A), increasing concentrations of antibody (B), and increasing falstatin in the presence of antibody (C) are shown. Labels above the gels represent concentrations of falstatin and antibody (μg/ml). Proteins are labeled based on known migration patterns that were previously confirmed by mass spectrometry. FP, falcipain; DPAP1, dipeptidyl aminopeptidase1[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020117#ppat-0020117-b028" target="_blank">28</a>].</p
Activity of Falstatin against Different Classes of Proteases
<p>Equal amounts (4 μg) of proteases (FP2, falcipain-2; FP3, falcipain-3; trypsin; α-chymo, α-chymotrypsin; pepsin; renin; collagenase; MM-2, matrix-metalloprotease-2) were mixed with 350 μl of appropriate buffers containing falstatin (1.5 μg) for 15 min, FITC-casein (20 μg) was added, and hydrolysis of the substrate with and without falstatin was compared for each protease. Error bars represent the standard deviations of results from two different assays, each performed in duplicate.</p
Immunolocalization of Falstatin
<div><p>(A) Immunofluorescence microscopy. Erythrocytes infected with synchronized 3D7 or W2 parasites were collected every 8 h, stained with DAPI and anti-falstatin antibodies and FITC-second antibody, and then evaluated by immunofluorescence microscopy.</p><p>(B) Immunoelectron microscopy. Late-schizont stage parasites were incubated with anti-falstatin antibodies and gold-conjugated second antibody and then evaluated by electron microscopy. Labels show individual merozoites (M) and erythrocyte cytosol (EC).</p></div
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