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 Near-Infrared Fluorophore (NIRF)-Labeled Activity-Based Probes for <i>in Vivo</i> Imaging of Legumain

Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease

Development of Activity-Based Probes for Cathepsin X

Cathepsin X is a lysosomal cysteine protease that functions as a carboxypeptidase with broad substrate specificity. Cathepsin X was discovered only recently, and its physiological roles are still not well understood. A number of studies suggest that cathepsin X may be involved in a variety of biological processes, including cancer, aging and degenerative conditions of the brain, inflammation, and cellular communication. Here we present the synthesis and characterization of several activity-based probes (ABPs) that target active cathepsin X. These ABPs were used to label cathepsin X in complex lysates, whole cells, and in vivo. Furthermore, we have developed a method for selectively labeling and visualizing active cathepsin X in vitro and in vivo. Overall, the probes developed in this study are valuable tools for the study of cathepsin X function

A General Solid Phase Method for the Preparation of Diverse Azapeptide Probes Directed Against Cysteine Proteases

A solid phase approach is presented for the synthesis of azapeptide inhibitors and activity based probes (ABPs) for cysteine proteases. This synthetic method allows the incorporation of diverse reactive warheads linked to different peptide recognition elements. Application of this method to the synthesis of a series of caspase probes is described

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

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)₂-Rho substrate. <b>B.</b> Measurement of (Pro-Arg)₂-Rho apparent <i>K</i>_m 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)₂-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

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)₂-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)₂-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

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 × 10⁷ 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

LC-MS/MS spectral counts of functionally interesting proteins identified in sporocyst/sporozoite fractions of <i>Toxoplasma gondii</i> oocysts – SRS family proteins.

<p>LC-MS/MS spectral counts of functionally interesting proteins identified in sporocyst/sporozoite fractions of <i>Toxoplasma gondii</i> oocysts – SRS family proteins.</p