Hydrolysis of N-methyl-D-aspartate receptor-stimulated cAMP and cGMP by PDE4 and PDE2 phosphodiesterases in primary neuronal cultures of rat cerebral cortex and hippocampus.
AuthorsSuvarna NU, O'Donnell JM
JournalJ Pharmacol Exp Ther
PubMed ID12065724
'Stimulation of N-methyl-D-aspartate (NMDA) receptors on neurons activates both cAMP and cGMP signaling pathways. Experiments were carried out to determine which phosphodiesterase (PDE) families are involved in the hydrolysis of the cyclic nucleotides formed via this mechanism, using primary neuronal cultures prepared from rat cerebral cortex and hippocampus. The nonselective ... More
Leukocyte C5a receptor modulation during hemodialysis.
AuthorsLewis SL, Van Epps DE, Chenoweth DE
JournalKidney Int
PubMed ID2951551
'Exposure of blood to hemodialysis (HD) membranes results in the activation of the complement system. In this study, flow cytometry was used to analyze the binding of fluoresceinated chemotactic factors (C5a, f-Met-Leu-Phe-Lys [fMLPL], and casein) and aggregated IgG to PMN and monocytes isolated from normal whole blood following passage through ... More
Independent regulation of human neutrophil chemotactic receptors after activation.
AuthorsBender JG, Van Epps DE, Chenoweth DE
JournalJ Immunol
PubMed ID2822801
'The fluoresceinated chemotactic factors, C5a, formyl-methionyl-leucyl-phenylalanyl-lysine (FMLPL), and casein were used in conjunction with flow cytometry to examine chemotactic factor receptor expression on polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate (PMA), C5a, or formyl-methionyl-leucyl-phenylalanine. Activation with PMA resulted in a dose-dependent increase in binding of fluorescein-labeled (FL)-casein and (FL-FMLPL) ... More
C5a receptor modulation on neutrophils and monocytes from chronic hemodialysis and peritoneal dialysis patients.
AuthorsLewis SL, Van Epps DE, Chenoweth DE
JournalClin Nephrol
PubMed ID3488153
'Chronic renal failure patients have an increased risk for infection which may partially be due to altered chemotactic ability of their white blood cells. This study was designed to evaluate chemotactic factor and Fc receptor expression on neutrophils (PMN) and monocytes from chronic renal failure patients on hemodialysis (HD) or ... More
High throughput fluorescence polarization: a homogeneous alternative to radioligand binding for cell surface receptors.
AuthorsAllen M, Reeves J, Mellor G
JournalJ Biomol Screen
PubMed ID10803605
'High throughput fluorescence polarization (FP) assays are described that offer a nonradioactive, homogeneous, and low-cost alternative to radioligand binding assays for cell surface receptors (G protein-coupled receptors and ligand-gated ion channels). FP assays were shown to work across a range of both peptide (vasopressin V1a and delta-opioid) and nonpeptide (beta1-adrenoceptor, ... More
Analysis of density changes and chemotactic receptors of leukocytes from chronic hemodialysis and peritoneal dialysis patients.
AuthorsLewis SL, Van Epps DE, Chenoweth DE
JournalBlood Purif
PubMed ID3040039
'Analysis of standard Ficoll-Hypaque (density = 1.077 g/ml) separation profiles of peripheral white blood cells (WBC) from patients undergoing hemodialysis (HD) demonstrated that dialysis caused a marked decrease in the density of polymorphonuclear leukocytes (PMN) resulting in about 50% of these cells separating with the mononuclear cells. In vitro exposure ... More
Fluorescein isothiocyanate-labeled casein assay for proteolytic enzymes.
AuthorsTwining SS
JournalAnal Biochem
PubMed ID6442109
'A simple inexpensive sensitive protease assay was developed using soluble fluorescein isothiocyanate (FITC)-labeled casein. Casein was reacted with FITC to form the fluorescein thiocarbamoyl derivative. This substrate is cleaved by trypsin, chymotrypsin, elastase, subtilisin, and thermolysin in a linear time-dependent manner. These enzymes can be measured in the nanogram and ... More
A continuous fluorescent assay for measuring protease activity using natural protein substrate.
AuthorsFarmer WH, Yuan ZY
JournalAnal Biochem
PubMed ID1785689
'A continuous caseinolytic activity assay has been developed and characterized with trypsin, a serine protease, and transin, a metalloproteinase. Beta-casein labeled with both N-(7-dimethylamino-4-methylcoumarinyl)-maleimide (DACM) and fluorescein isothiocyanate (FITC) is used as the substrate in this assay. The effect of proteolysis of the substrate is a reduction of the intermolecular ... More
Proteolytic activity in infected and noninfected insect cells: degradation of HIV-1 Pr55gag particles.
'In this work the proteolytic activity in the supernatant and inside insect cells in culture was evaluated for different multiplicities of infection (MOI) and times of infection (TOI). Several methods to detect proteolytic activity in insect cells were tested and that using fluorescein thiocyanite-casein as a substrate was chosen. It ... More
Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum.
AuthorsAkopian TN, Kisselev AF, Goldberg AL
JournalJ Biol Chem
PubMed ID8999862
'Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its ... More
Fluorometric determination of bacterial protease activity using fluorescein isothiocyanate-labeled proteins as substrates.
AuthorsHomer KA, Beighton D
JournalAnal Biochem
PubMed ID2127659
'Intact fluorescein isothiocyanate-labeled proteins have relatively low background fluorescence at excitation and emission wavelengths of 495 and 525 nm, respectively. Degradation of these substrates leads to exposure of covalently linked fluorescein isothiocyanate molecules and to a concomitant increase in relative fluorescence at these wavelengths. The increase in relative fluorescence is ... More
Calpain zymography with casein or fluorescein isothiocyanate casein.
AuthorsArthur JS, Mykles DL
JournalMethods Mol Biol
PubMed ID10818754
Proteolytic activity of oral streptococci.
AuthorsHomer KA, Whiley RA, Beighton D
JournalFEMS Microbiol Lett
PubMed ID2182385
Streptococcus mutans and Streptococcus sobrinus were the least proteolytic of 8 species of oral streptococci while Streptococcus oralis and Streptococcus sanguis were the most proteolytic. Degradation of FITC-BSA was significantly correlated with the hydrolysis of synthetic endopeptidase substrates. As S. oralis strains proliferate in dental plaque in the absence of ... More
Flow cytometric analysis of entire microbial colonies.
AuthorsSahar E, Nir R, Lamed R
JournalCytometry
PubMed ID8187581
Much could be gained if the scope of flow cytometry could be broadened to the study of entire cell colonies, rather than to populations of single, separate cells. This can be achieved by encapsulating single microbial cells in small spheres in a way that allows each cell to multiply and ... More
Visualization and quantification of polymer distribution in microcapsules by confocal laser scanning microscopy (CLSM).
AuthorsLamprecht A, Schäfer UF, Lehr CM
JournalInt J Pharm
PubMed ID10699723
Confocal laser scanning microscopy (CLSM) was employed in order to characterize microcapsules. Microcapsules were prepared by complex coacervation: gelatin and arabic gum were labelled with fluorescent markers. In the capsule wall a homogeneous distribution for both gelatin and arabic gum throughout the capsule wall was depicted. By the use of ... More
Casein-mediated neutrophil chemotaxis: a parallel between surface binding and chemotaxis.
AuthorsVan Epps DE, Bankhurst AD, Williams RC
JournalInflammation
PubMed ID617803
This study was directed toward demonstrating an interaction between human polymorphonuclear leukocytes (PMNs) and the chemotactic factor,casein. Experiments conducted using fluorescein-labeled casein indicated that PMNs have a membrane receptor for this chemotactic factor. This receptor was specific for casein and was not found on lymphocytes. A direct parallel was shown ... More
A new protease activity assay using fluorescence polarization.
AuthorsBolger R, Checovich W,
JournalBiotechniques
PubMed ID7818914
Fluorescence polarization (FP) technology was used to develop an assay for protease activity that is more sensitive than other nonradioactive protease assays and requires no separations, precipitations or transfers of the reaction mixture. FP measures changes in the molecular volume of fluorescently labeled molecules. In the assay described in the ... More
A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.
AuthorsAbodeely M, DuBois KN, Hehl A, Stefanic S, Sajid M, DeSouza W, Attias M, Engel JC, Hsieh I, Fetter RD, McKerrow JH,
JournalEukaryot Cell
PubMed ID19749174
The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and ... More
Maturation factor for the activation process of an extracellular proteinase in Lactobacillus helveticus CP790.
AuthorsYamamoto N, Takano T
JournalJ Dairy Sci
PubMed ID9313133
A maturation factor that was needed for activation of an extracellular proteinase was partially purified from Lactobacillus helveticus CP790 by DEAE-Sepharose column chromatography, followed by gel filtration using HPLC. The protein had the ability to accelerate the conversion of proproteinase to active enzyme but had no proteolytic activity toward casein ... More
Demonstration of specific receptors for fluoresceinated casein on human neutrophils and monocytes using flow cytometry.
AuthorsLewis SL, Van Epps DE
JournalInflammation
PubMed ID6317559
Casein is chemotactic for human neutrophils (PMNs) and monocytes. The binding of fluorescein (FITC)-conjugated casein (mixture of alpha, beta, and kappa-casein) and purified alpha-casein to PMNs, monocytes, and lymphocytes was analyzed using flow cytometry. These studies demonstrate that 75-95% of PMNs and 46-85% of monocytes have membrane receptors for casein ... More
Evaluation of fluorometric and zymographic methods as activity assays for stromelysins and gelatinases.
AuthorsQuesada AR, Barbacid MM, Mira E, Fernández-Resa P, Márquez G, Aracil M
JournalClin Exp Metastasis
PubMed ID9009103
To measure matrix metalloproteinase (MMP) activity in a large number of samples it is advisable to use easily automated methods. We have evaluated and compared the activity of stromelysin-1 (MMP-3), matrilysin (MMP-7), 72 kDa gelatinase A (MMP-2) and 92 kDa gelatinase B (MMP-9) by zymogram analysis and fluorescent substrate degradation ... More
Effects of interleukin-1 and anti-inflammatory drugs on the degradation of human articular cartilage.
AuthorsShinmei M, Kikuchi T, Masuda K, Shimomura Y
JournalDrugs
PubMed ID2834169
It has been suggested that metalloproteases produced by chondrocytes play an important role in cartilage breakdown in joint diseases. The aim of this study was to investigate changes in enzyme activities in human rheumatoid and osteoarthritic articular cartilage. Cartilage fragments were incubated with various drugs for 48 hours. The concentrated ... More
Calpain in rat cornea.
AuthorsAzuma M, Anderson RS, Sameshima S, Matsumoto T, David LL, Shearer TR
JournalInvest Ophthalmol Vis Sci
PubMed ID1634345
This study was conducted to provide a description of calpain proteolytic enzyme (EC 3.4.22.17) in normal rat cornea and to document immunohistochemical changes in calpain distribution during maturation. Corneal soluble proteins were fractionated by diethylaminoethyl chromatography on high-performance liquid chromatography. Fractions were analyzed for calpain by enzyme-linked immunosorbent assay, immunoblotting, ... More
A rapid method for measuring protease activity in milk using radiolabeled casein.
AuthorsChristen GL
JournalJ Dairy Sci
PubMed ID3117854
A rapid means to detect the presence of protease activity in raw milk could be useful in predicting keeping ability of products made from that milk. A 30-min assay has been developed and compared with three other methods of detecting protease. Casein, [methyl-14C]-methylated-alpha was purchased from a radioisotope supplier. Concentrations ... More
Effect of monoclonal antibodies specific for the 28-kDa subunit on catalytic properties of the calpains.
AuthorsCong J, Thompson VF, Goll DE
JournalJ Biol Chem
PubMed ID7503986
Nine monoclonal antibodies (mAbs) specific for the 28-kDa subunit common to mu- and m-calpains have been assayed for their effects on mu- and m-calpains. All nine react with the COOH-terminal part (domain VI) of the 28-kDa subunit, and all nine affect the Ca2+ concentration required for autolysis of m-calpain, but ... More
Fluorescence imaging detection for capillary isoelectric focusing.
AuthorsWu XZ, Wu J, Pawliszyn J
JournalElectrophoresis
PubMed ID8529617
A simple laser-induced fluorescence (LIF) imaging detector and an ultrasensitive LIF imaging detector are described for capillary isoelectric focusing (CIEF). An argon ion laser beam of 496.5 nm is used as excitation source. In the simple LIF imaging detector, the excitation beam is directed into a capillary column by an ... More
The supplementation of culture medium with protease improves the hatching rate of mouse embryos.
AuthorsLee DR, Lee JE, Yoon HS, Lee HJ, Kim MK, Roh SI
JournalHum Reprod
PubMed ID9436692
Mammalian embryos are known to exhibit delayed development and have lower hatching rates in vitro than in vivo because of inadequate culture condition. These discrepancies may be due to a deficiency of the paracrine factors and proteolytic enzymes which exist in the oviduct and uterus. In order to evaluate the ... More
Structural determinants of the calpain inhibitory activity of calpastatin peptide B27-WT.
Calpastatin is the natural specific inhibitor of calpain. Recent research has linked uncontrolled calpain activation to tissue damage after neuronal and cardiac ischemias, traumatic spine and brain injuries, as well as Alzheimer's disease and cataract formation. An imbalance between the activities of calpain and calpastatin is believed to be responsible ... More
Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator.
Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These ... More
Structural and functional differentiation of follicular and oviductal mouse oocytes visualised with FITC-protein conjugates.
AuthorsKim H, Schuetz AW
JournalZygote
PubMed ID8081827
The fluorescence labelling characteristics of mouse oocytes were examined at various stages of periovulatory differentiation using FITC-protein conjugates. The zona pellucida, perivitelline space and plasma membrane underwent visible changes which were developmentally and environmentally related. Following exposure to fluorescein isothiocyanate (FITC)-casein conjugates, the zona pellucida (ZP) of germinal vesicle stage ... More
Oviductal protein produces fluorescence staining of the perivitelline space in mouse oocytes.
AuthorsKim H, Kim H, Kim SR, Kim MK, Schuetz AW
JournalJ Exp Zool
PubMed ID8636692
Mouse oocytes were previously observed to undergo structural changes involving the perivitelline space (PVS) within the oviduct following ovulation, as visualized by staining with fluorochrome-protein conjugates. In the present study, this phenomenon was investigated in detail to determine the role of the oviduct and oocyte. Mouse ovarian oocytes matured in ... More
Alterations in the visual cortex receptor pattern by operant conditioning in a reward paradigm.
AuthorsLöhrke B, Franz H, Brose R, Klein M
JournalBiol Chem Hoppe Seyler
PubMed ID7576247
The aim of the study was to investigate the impact of operant conditioning on the receptor pattern in the visual cortex of calves. A reward paradigm was used to induce conditioned preference for colours. Binding sites in visual cortex specimens from conditioned and naive animals were assayed in vitro on ... More
Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue.
AuthorsGalis ZS, Sukhova GK, Libby P
JournalFASEB J
PubMed ID7615167
Many physiological and pathological processes involve tissue remodeling due in part to degradation of extracellular matrix. Several factors limit current approaches used for detection of matrix-degrading enzymes in tissues. Matrix metalloproteinases (MMPs), enzymes specialized in catabolism of extracellular matrix constituents, require processing from inactive zymogen precursors to gain enzymatic function. ... More