'BACKGROUND: Inhalation anthrax is characterized by a systemic spread of the challenge agent, Bacillus anthracis. It causes severe damage, including multiple hemorrhagic lesions, to host tissues and organs. It is widely believed that anthrax lethal toxin secreted by proliferating bacteria is a major cause of death, however, the pathology of ... More
Enzymatic microreactor-on-a-chip: protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices.
AuthorsPeterson DS, Rohr T, Svec F, Fréchet JM
JournalAnal Chem
PubMed ID12199578
'Enzymatic microreactors have been prepared in capillaries and on microfluidic chips by immobilizing trypsin on porous polymer monoliths consisting of 2-vinyl-4,4-dimethylazlactone, ethylene dimethacrylate, and acrylamide or 2-hydroxyethyl methacrylate. The azlactone functionalities react readily with amine and thiol groups of the enzyme to form stable covalent bonds. The optimized porous properties ... More
Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement.
AuthorsJones LJ, Upson RH, Haugland RP, Panchuk-Voloshina N, Zhou M, Haugland RP
JournalAnal Biochem
PubMed ID9299009
'We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon ... More
Functional screening of intracellular proteins in single cells and in patterned cell arrays using electroporation.
AuthorsNolkrantz K, Farre C, Hurtig KJ, Rylander P, Orwar O
JournalAnal Chem
PubMed ID12199607
'A tool for detection and characterization of intracellular enzyme-substrate and receptor-ligand interactions inside the cytoplasm of single targeted cells or small confined groups of cells is presented. Fluorogenic enzyme substrates and receptor ligands were rapidly delivered by electroosmosis and internalized by electroporation in cells using an electrolyte-filled capillary (EFC) biased ... More
Continuous assay of proteases using a microtiter plate fluorescence reader.
AuthorsMenges DA, Ternullo DL, Tan-Wilson AL, Gal S
JournalAnal Biochem
PubMed ID9398357
DNA shuffling of subgenomic sequences of subtilisin.
AuthorsNess JE, Welch M, Giver L, Bueno M, Cherry JR, Borchert TV, Stemmer WP, Minshull J
JournalNat Biotechnol
PubMed ID10471932
DNA family shuffling of 26 protease genes was used to create a library of chimeric proteases that was screened for four distinct enzymatic properties. Multiple clones were identified that were significantly improved over any of the parental enzymes for each individual property. Family shuffling, also known as molecular breeding, efficiently ... More
Analysis of proteins with caseinolytic activity in a human stratum corneum extract revealed a yet unidentified cysteine protease and identified the so-called
AuthorsBernard D, Méhul B, Thomas-Collignon A, Simonetti L, Remy V, Bernard MA, Schmidt R,
JournalJ Invest Dermatol
PubMed ID12648222
Desquamation is described as a protease-dependent phenomenon where serine proteases with a basic pH optimum play a key role. Recently proteases with an acidic pH optimum were identified in the stratumcorneum and associated with desquamation, e.g., cathepsin D and the stratum corneum thiol protease. The purpose of this study was ... More
Pluronic additives: a solution to sticky problems in digital microfluidics.
AuthorsLuk VN, Mo GCh, Wheeler AR,
JournalLangmuir
PubMed ID18481875
Digital microfluidics (DMF) is a promising technique for carrying out miniaturized, automated biochemical assays in which discrete droplets of reagents are actuated on the surface of an array of electrodes. A limitation for DMF is nonspecific protein adsorption to device surfaces, which interferes with assay fidelity and can cause droplets ... More
Upregulation of tPA/plasminogen proteolytic system in the periphery of amyloid deposits in the Tg2576 mouse model of Alzheimer's disease.
AuthorsLee JY, Kweon HS, Cho E, Lee JY, Byun HR, Kim DH, Kim YH, Han PL, Koh JY,
JournalNeurosci Lett
PubMed ID17662525
Although the tissue plasminogen activator (tPA)/plasminogen/plasmin proteolytic system is thought to modulate the catabolism of amyloid-beta (Abeta), in vivo evidence remains insufficient. In the brain of human amyloid precursor protein transgenic Tg2576 mice, we found co-accumulation of tPA and plasminogen at the periphery of compact amyloid deposits, mainly Abeta42-cored plaques, ... More
Extracellular antigen processing and presentation by immature dendritic cells.
In antigen presentation to CD4(+) T cells, proteins are degraded to peptide fragments and loaded onto class II MHC molecules in a process involving the peptide exchange factors H-2M (murine) or HLA-DM (human). In many antigen-presenting cells these processes occur in intracellular endosomal compartments, where peptides are generated and loaded ... More
Creation of an on-chip enzyme reactor by encapsulating trypsin in sol-gel on a plastic microchip.
AuthorsSakai-Kato K, Kato M, Toyo'oka T
JournalAnal Chem
PubMed ID12585462
Trypsin-encapsulated sol-gel was fabricated in situ onto a plastic microchip to form an on-chip bioreactor that integrates tryptic digestion, separation, and detection. Trypsin-encapsulated sol-gel, which is derived from alkoxysilane, was fabricated within a sample reservoir (SR) of the chip. Fluorescently labeled ArgOEt and bradykinin were digested within the SR followed ... More
Proteinase assay by capillary electrophoresis employing fluorescence-quenched protein-dye conjugates.
AuthorsWelder F, McCorquodale EM, Colyer CL
JournalElectrophoresis
PubMed ID12179975
Determination of proteinases--enzymes that catalyze the hydrolysis of peptide bonds--is often difficult due to the presence of interferences in complex biological media and limited sample size. Capillary electrophoresis (CE), with laser-induced fluorescence (LIF) can serve as a useful tool for such determinations. LIF detection offers the advantages of increased sensitivity ... More
Enzyme cytochemical techniques for metabolic mapping in living cells, with special reference to proteolysis.
AuthorsBoonacker E, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID11724895
Specific enzymes play key roles in many pathophysiological processes and therefore are targets for therapeutic strategies. The activity of most enzymes is largely determined by many factors at the post-translational level. Therefore, it is essential to study the activity of target enzymes in living cells and tissues in a quantitative ... More
The protease-activated receptor-2 upregulates keratinocyte phagocytosis.
AuthorsSharlow ER, Paine CS, Babiarz L, Eisinger M, Shapiro S, Seiberg M
JournalJ Cell Sci
PubMed ID10934047
The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 ... More
Matrix metalloproteinase-9 undergoes expression and activation during dendritic remodeling in adult hippocampus.
AuthorsSzklarczyk A, Lapinska J, Rylski M, McKay RD, Kaczmarek L
JournalJ Neurosci
PubMed ID11826121
Neurons of adult brain are able to remodel their synaptic connections in response to various stimuli. Modifications of the peridendritic environment, including the extracellular matrix, are likely to play a role during synapse remodeling. Proteolytic disassembly of ECM is a complex process using the regulated actions of specific extracellular proteinases. ... More
Secreted neutral metalloproteases of Bacillus anthracis as candidate pathogenic factors.
To evaluate the pathogenic potential of Bacillus anthracis-secreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1-, pXO2-). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. ... More
Evaluation of conditions for calpain inhibition in the rat spinal cord: effective postinjury inhibition with intraspinal MDL28170 microinjection.
AuthorsZhang SX, Bondada V, Geddes JW
JournalJ Neurotrauma
PubMed ID12614588
Calpains (calcium-activated cysteine proteases) are strongly implicated in the secondary damage that follows contusion injury to the spinal cord. Calpains are activated within a few minutes following injury and their elevated activity persists for 24 h, thereby providing a reasonable window of opportunity for postinjury inhibition. Previous studies demonstrated decreased ... More
A BODIPY fluorescent microplate assay for measuring activity of calpains and other proteases.
AuthorsThompson VF, Saldaña S, Cong J, Goll DE
JournalAnal Biochem
PubMed ID10706786
The use of 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) labeled casein in autoquenching assays of proteolytic activity has been recently described, and we have adapted this assay to measurement of calpain activity. BODIPY-FL coupled to casein at a ratio of 8 mol of BODIPY-FL/mol of casein or higher produces a BODIPY-FL-casein substrate ... More