34 research outputs found
Development and Application of a Simple Plaque Assay for the Human Malaria Parasite Plasmodium falciparum.
Malaria is caused by an obligate intracellular protozoan parasite that replicates within and destroys erythrocytes. Asexual blood stages of the causative agent of the most virulent form of human malaria, Plasmodium falciparum, can be cultivated indefinitely in vitro in human erythrocytes, facilitating experimental analysis of parasite cell biology, biochemistry and genetics. However, efforts to improve understanding of the basic biology of this important pathogen and to develop urgently required new antimalarial drugs and vaccines, suffer from a paucity of basic research tools. This includes a simple means of quantifying the effects of drugs, antibodies and gene modifications on parasite fitness and replication rates. Here we describe the development and validation of an extremely simple, robust plaque assay that can be used to visualise parasite replication and resulting host erythrocyte destruction at the level of clonal parasite populations. We demonstrate applications of the plaque assay by using it for the phenotypic characterisation of two P. falciparum conditional mutants displaying reduced fitness in vitro
Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts
Background: High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings: Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’.0.8) that are suitable for use in screening large collections of small molecules (i.e.300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions: We believe that this approach will have value for many enzymatic systems where access to large amounts o
Molecular Mechanisms of Bortezomib Resistant Adenocarcinoma Cells
Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response
Laboratory experiments on swelling due to crystal growth in sulphate argillaceous rocks
Crystal growth is a frequent cause of expansive deformations in natural soils, compacted soils, rocks and concrete. The sulphate crystallization in discontinuities and cracks due to evaporation of highly mineralized solutions can contribute in an important way to displacements and swelling pressures that usually appear in tunnels and deep foundations in argillaceous rocks. The present work is aimed to study the basic mechanisms that control the hydrated sulphate mineral growth in anhydritic-gypsiferous argillaceous rocks due to the evaporation of groundwater. The paper presents some laboratory works including
mineralogical and micro-structural analysis, as well as the design and development of new free swelling tests which were developed focusing on the capacity to discern the role carried out by the geochemical properties of the water, the main environmental variables and the degree of cracking on undisturbed sulphate argillaceous samples from different zones of the Lower Ebro Basin (Catalonia, Spain)
Laboratory experiments on swelling due to crystal growth in sulphate argillaceous rocks
Crystal growth is a frequent cause of expansive deformations in natural soils, compacted soils, rocks and concrete. The sulphate crystallization in discontinuities and cracks due to evaporation of highly mineralized solutions can contribute in an important way to displacements and swelling pressures that usually appear in tunnels and deep foundations in argillaceous rocks. The present work is aimed to study the basic mechanisms that control the hydrated sulphate mineral growth in anhydritic-gypsiferous argillaceous rocks due to the evaporation of groundwater. The paper presents some laboratory works including
mineralogical and micro-structural analysis, as well as the design and development of new free swelling tests which were developed focusing on the capacity to discern the role carried out by the geochemical properties of the water, the main environmental variables and the degree of cracking on undisturbed sulphate argillaceous samples from different zones of the Lower Ebro Basin (Catalonia, Spain).Postprint (published version
Laboratory experiments on swelling due to crystal growth in sulphate argillaceous rocks
Crystal growth is a frequent cause of expansive deformations in natural soils, compacted soils, rocks and concrete. The sulphate crystallization in discontinuities and cracks due to evaporation of highly mineralized solutions can contribute in an important way to displacements and swelling pressures that usually appear in tunnels and deep foundations in argillaceous rocks. The present work is aimed to study the basic mechanisms that control the hydrated sulphate mineral growth in anhydritic-gypsiferous argillaceous rocks due to the evaporation of groundwater. The paper presents some laboratory works including
mineralogical and micro-structural analysis, as well as the design and development of new free swelling tests which were developed focusing on the capacity to discern the role carried out by the geochemical properties of the water, the main environmental variables and the degree of cracking on undisturbed sulphate argillaceous samples from different zones of the Lower Ebro Basin (Catalonia, Spain)
Quantum mechanical calculations in vibrational relaxation in molecular collisions
SIGLEAvailable from British Library Document Supply Centre- DSC:DX59171 / BLDSC - British Library Document Supply CentreGBUnited Kingdo
A coupled protein and probe engineering approach for selective inhibition and activity-based probe labeling of the caspases
Caspases are cysteine proteases that play essential roles in apoptosis and inflammation. Unfortunately, their highly conserved active sites and overlapping substrate specificities make it difficult to use inhibitors or activity-based probes to study the function, activation, localization, and regulation of individual members of this family. Here we describe a strategy to engineer a caspase to contain a latent nucleophile that can be targeted by a probe containing a suitably placed electrophile, thereby allowing specific, irreversible inhibition and labeling of only the engineered protease. To accomplish this, we have identified a non-conserved residue on the small subunit of all caspases that is near the substrate-binding pocket and that can be mutated to a non-catalytic cysteine residue. We demonstrate that an active-site probe containing an irreversible binding acrylamide electrophile can specifically target this cysteine residue. Here we validate the approach using the apoptotic mediator, caspase-8, and the inflammasome effector, caspase-1. We show that the engineered enzymes are functionally identical to the wild-type enzymes and that the approach allows specific inhibition and direct imaging of the engineered targets in cells. Therefore, this method can be used to image localization and activation as well as the functional contributions of individual caspase proteases to the process of cell death or inflammation
Laboratory tests on swelling due to crystal growth in sulphate bearing rocks
Precipitation of sulphate crystals in discontinuities due to evaporation of sulphate solutions is an important contribution to the degradation and swelling behavior of sulphate bearing argillaceous rocks. Although the phenomenon has been studied in laboratory during decades linked to the expansive behavior in tunnels, tests do not adequately reproduce field conditions and all variables involved in the swelling mechanism. Size effect problems and the difficulty in correctly applying hydraulic boundary conditions are the main aspects behind these experimental difficulties. To this aim, a new experimental setup has been designed to allow applying relative humidity control on the top cap of the sample, while the bottom cap is in permanent contact with a saturated sulphate solution. This way, controlled solvent evaporation conditions are applied, which control the rate of sulphate precipitation on the sample. The crystals are deposited along preferential discontinuities of the sample, as well as on fissures generated by the hydraulically induced degradation of the material. A sulphate bearing claystone of the Lower Ebro Basin (Catalonia, Spain) has been tested. Selected swelling tests are presented at different relative humidity values. Microstructural and DRX analyses are also included to complement the information of the mineralogical phases deposited during these processes. © 2014 Taylor & Francis Group, London
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