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At least 217 records · Page 12Linked to original sources
PubMed2001

Novel functional assay for proteases and modulators. Application in beta-secretase studies.

Proteases play a critical role in many cellular functions and have been an attractive therapeutic target due to their involvement in a number of disease processes. One prominent example is the secretases responsible for the generation of amyloid beta peptide, which is believed to be central for the development of Alzheimer's disease. It is therefore desirable to identify and characterize these proteases. We have developed a novel functional approach for identification of proteases and modulators by coupling the protease activity to caspase-mediated apoptosis. Here we show the proof of principle for this approach using beta-secretase as an example. We provide data showing that 1. A modified caspase-3 containing beta-secretase cleavage site induces apoptosis in 293T cells. 2. The modified caspase-3 induced apoptosis is correlated with the susceptibility of beta-secretase recognition sequence to beta-secretase. 3. In vivo beta-secretase competitors BACE2 and BACE2(D110A) prevent the modified caspase-3 induced cell death. Therefore, this approach can be a useful tool in studies of proteolytic cleavage provided only that the protease recognition sequence is known.

Alzheimer Disease↗
PubMed1997

Characterization of functional assays of multidrug resistance P-glycoprotein transport activity.

P-glycoprotein-mediated multidrug resistance has emerged as one of the most attractive targets to improve anticancer therapy. The P-glycoprotein functions as an energy-dependent, membrane transport pump capable of decreasing the intracellular concentration of a broad range of chemotherapeutic agents. Pharmaceuticals which inhibit P-glycoprotein transport activity are currently being evaluated in clinical trials. Characterization of P-glycoprotein functional activity is critical in determining if these multidrug resistance reversal agents improve therapeutic responses of tumors expressing P-glycoprotein. In this report, we directly compare and characterize assays using rhodamine 123, dimethyloxadicarbocyanine iodide (DiOC2), [3H]daunorubicin and hexakis(2-methoxyisobutyl isonitrile)technetium(I) ([(99m)Tc]Sestamibi) as P-glycoprotein transport probes to quantitate functional activity. The accumulation of certain substrates is concentration dependent and the parameters which determine probe accumulation are impacted by the level of P-glycoprotein expression. In addition, higher concentrations of reversal agents are required to inhibit multidrug resistance in cell lines expressing higher levels of P-glycoprotein. Furthermore, the concentration of reversal agents required to inhibit completely P-glycoprotein transport activity is higher than generally recognized. Thus, the level of P-glycoprotein expression may confound intersample comparisons unless sensitive probes are used in combination with saturating concentrations of potent reversal agents. These results highlight the importance of carefully characterizing assay systems under uniform conditions to quantitate P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Evaluation of a series of novel CCKB antagonists using a functional assay in the rat central nervous system.

1. Electrophysiological recordings from rat ventromedial hypothalamus (VMH) in vitro have been used to compare the effects of novel chemical entities on CCKB receptor activation in the rat central nervous system. 2. Twenty compounds from three different chemical series were evaluated for their ability to reduce pentagastrin-induced increases in action potential firing rate. 3. All twenty compounds studies were found to be CCKB antagonists, with equilibrium constants spanning a concentration-range of several orders of magnitude. The rank order for their ability to block pentagastrin responses correlated well with values obtained for their relative affinities for the mouse cortex CCKB binding site. 4. It is concluded that the VMH preparation provides a good functional correlate to binding assays in the rodent central nervous system for a structurally diverse series of CCKB antagonists.

Action Potentials↗
PubMedDate not supplied

Comparison of a new automated kinetically determined fibrinogen assay with the 3 most used fibrinogen assays (functional, derived and nephelometric) in Austrian laboratories in several clinical populations and healthy controls.

A new automated kinetically determined fibrinogen assay was measured in plasmas of healthy subjects and three clinical cohorts (acute-phase reaction, liver cirrhosis and fibrinolytic therapy) that were expected to show normal, high and low levels of fibrinogen. The results were compared with the results of fibrinogen measurement using the derived method, the method according to Clauss and an immunological-nephelometric method. Altogether, the best correlation was achieved between the kinetic and the derived method. However, results from the derived method were generally higher than values obtained through the kinetic method. This was particularly true for high concentration levels above 400 mg/dl (patients with acute phase reaction) as well as for plasmas containing fibrin(ogen) degradation products and low concentrations of fibrinogen (below 150 mg/dl). Fibrinogen determinations in several commercial plasma pools with declared fibrinogen levels show remarkable heterogeneity when different methods were applied. To improve the discernment of fibrinogen determinations we suggest adjustment of standard preparations to international reference materials and the specification of the method used. Furthermore the attending physician is asked to cast a critical eye on fibrinogen values with regard to the used method of determination.

Acute-Phase Reaction↗
PubMed2003

Molecular cloning of p53 cDNA of Mongolian gerbil and establishment of yeast p53 functional assay system.

BACKGROUND: Epidemiological studies have shown a correlation between Helicobacter pylori infection and human gastric carcinogenesis. A Mongolian gerbil model has demonstrated that H. pylori infection induced gastric carcinoma. However, the disadvantage of this animal model is a lack of information regarding the cellular genes involved in oncogenesis. Mutation of the p53 gene is one of the most common steps in gastric carcinogenesis. In this study, we aimed to clone the p53 gene of the Mongolian gerbil and detect the functional mutations in H. pylori-infected animals. MATERIALS AND METHODS: The p53 complementary DNA (cDNA) of Mongolian gerbil was cloned by the methods of reverse-transcribed polymerase chain reaction and rapid amplification of cDNA ends. RESULTS: The p53 cDNA of Mongolian gerbil has a 78.8% homology to that of humans. A novel yeast p53 assay system was established and enabled to detect the functional mutations of the p53 gene in the stomach of the Mongolian gerbil. CONCLUSIONS: This is the first report of the complete sequence of wild-type p53 cDNA of the Mongolian gerbil. This genetic information and an assay system designed to detect the functional mutations of the p53 gene are useful for further investigations of gastric oncogenesis in this animal model.

Animals↗
PubMed1980

Functional assay of alpha 1-antitrypsin in obstructive lung disease.

A decreased concentration of alpha 1-antitrypsin is associated with a high risk for obstructive lung disease. We measured the elastase inhibitory capacity, the most important biologic measure of alpha 1-AT function, using a natural substrate. The gel plate assay that we developed uses a commercial gelatin film and is more sensitive, faster, and cheaper than similar elastin-elastase methods. In serum samples from 76 patients with emphysema, there was a high correlation between the immunologically measured alpha 1-AT and the elastase inhibitory capacity. There was no evidence for a functionally deficient alpha 1-AT in any of these patients.

Humans↗
PubMed2025

Using the Tether Function Assay to Identify Potential Regulators of mRNA Translation and mRNA Decay.

RNA binding proteins (RBPs) and their associated partners are key factors of posttranscriptional control of gene expression. To study and manipulate the functional consequences of binding of these regulators to their targets, several tethering assays have been developed, in which a protein of interest is brought to a reporter mRNA through heterologous RNA-protein interaction motifs. The effect of such constrained binding is then monitored by measuring the accumulation of the reporter protein and mRNA. This chapter describes a protocol for the λN-BoxB tether system in transiently transfected mammalian cells. Combining the luciferase reporter technology to quantify protein amounts by light measurement and RNA amounts by RT-qPCR, this assay provides a simple and robust way to analyze the consequences of any protein binding in a controlled and defined manner.

RNA, Messenger↗
PubMed2005

Functional assay of effectors of ADP ribosylation factor 6 during clathrin/AP-2 coat recruitment to membranes.

Arf proteins play pivotal roles in membrane traffic, cell signaling, and actin cytoskeletal rearrangements. We describe here methods to functionally analyze interacting partner proteins of recombinantly produced N-myristoylated Arf6. Combined evidence from affinity purification and chemical crosslinking experiments, in vitro recruitment assays, and the analysis of lipid kinase activities indicates that Arf6-GTP facilitates clathrin/AP-2 recruitment to synaptic membranes by direct binding and activation of the brain-specific phosphatidylinositol 4-phosphate 5-kinase type Igamma (PIPK Igamma). These methods shall help to mechanistically dissect the role of Arf6 in regulating exo-endocytic vesicle cycling at synapses and in related membrane trafficking events.

ADP-Ribosylation Factor 1↗
PubMed2003

Organochlorine contaminant exposure and associations with hematological and humoral immune functional assays with dam age as a factor in free-ranging northern fur seal pups (Callorhinus ursinus).

Potential effects of organochlorine contaminant (OC) exposure on humoral immune function and health of free-ranging northern fur seals were investigated. Forty-two "neonates" were captured for blood sample collection and re-sampled as "pups" 29-51 days later. OCs were extracted from whole blood to identify polychlorinated biphenyl congeners and chlorinated pesticides by high performance liquid chromatography. Humoral immune function was assessed by antibody responses to vaccination and immunoglobulin concentrations. Additional health status indicators included leukocyte counts and haptoglobin concentrations. Mean OC concentrations were higher in neonates than at recapture. Neonates of young dams had higher mean OC concentrations than neonates of older dams. A higher proportion of old dam's pups developed a twofold or greater increase in antigen-specific antibodies compared to young dam's pups. Higher OC exposure and poor immune responses in first-born pups may indicate a higher risk of secondary morbidity and mortality than pups of multiparous dams.

Age Factors↗
PubMed1997

Identification and functional assay of an extracellular calcium-sensing receptor in Necturus gastric mucosa.

In mammals and amphibians, increases in extracellular Ca2+ can activate bicarbonate secretion and other protective functions of gastric mucosa. We hypothesized that the recently cloned extracellular Ca(2+)-sensing receptor (CaR) is functioning in the gastric mucosa. In Necturus maculosus gastric mucosa, reverse transcription-polymerase chain reaction using primers based on previously cloned CaR sequences amplified a 326-bp DNA fragment that had 84% nucleotide sequence identity with the rat kidney CaR. Immunohistochemical localization of the CaR using specific anti-CaR antiserum revealed its presence on the basal aspect of gastric epithelial cells. In microelectrode studies of Necturus antral mucosa, exposure to elevated Ca2+ (4.8 mM) and the CaR agonists NPS-467 and neomycin sulfate resulted in significant hyperpolarizations of basal membrane electrical potentials and increases in apical-to-basal membrane resistance ratios. Circuit analysis revealed that these changes reflected specific decreases in basolateral membrane resistance. Inhibition of prostaglandin synthesis using indomethacin significantly attenuated these effects. We conclude that the CaR is present and functioning in Necturus gastric antrum.

Amiloride↗
PubMed1997

The clinical application of a new specific functional assay to detect the factor V(Leiden) mutation associated with activated protein C resistance.

Resistance to activated protein C (APC) is the most common defect found in patients who have venous thromboembolism. The molecular basis of APC resistance is a single-point mutation (arginine506-glutamine) in the gene that encodes for coagulation factor V. This mutation results in a factor V molecule (factor V(Leiden)) that is less effectively downregulated by APC than is normal factor V. The gold standard for the detection of this defect is DNA analysis. Several functional tests, which are based on activated partial thromboplastin time clotting assays, are also commercially available for the detection of APC resistance. These tests, however, have not been satisfactory. Compared with the results of DNA analysis, the results of these tests are frequently discordant. Further, in some patients (eg, those who have lupus anticoagulant or have been receiving heparin), these tests cannot be performed at all. DNA analysis is therefore required in most patients to distinguish congenital APC resistance (factor V(Leiden)) from other causes of abnormal response in functional APC-resistance tests. The purpose of this study was to investigate the clinical use of a new chromogenic APC-resistance assay that is based on direct measurement of the effect of APC on factor Va cofactor activity in highly diluted, thrombin-activated plasma specimens. All individuals who provided plasma samples for the study underwent DNA analysis to detect the presence of the factor V mutation. In all patient subgroups, including patients who had lupus anticoagulant and those who were receiving unfractionated heparin or coumarin derivatives, the chromogenic test showed excellent discrimination between normal individuals and those who were heterozygous or homozygous for the factor V(Leiden) mutation. No discordant results with DNA analysis were found in 150 cases. The new test easily can be incorporated in any laboratory that has an automated coagulation apparatus with an option for chromogenic measurements. All reagents are commercially available at low cost, and the test is easy to perform and is not time-consuming. This new, sensitive, and specific test allows large-scale screening for the factor V(Leiden) mutation without the need for DNA analysis.

Blood Coagulation Disorders↗
PubMed2001

Identification of glucose transporter-1 and it's functional assay in mouse glomerular mesangial cells cultured in vitro.

OBJECTIVE: To evaluate the role of glucose transporter-1 (GLUT1) in the glucose uptake of glomerular mesangial cells. METHODS: Cultured C57/SJL mouse mesangial cells were used in the study. The expression of GLUT1 mRNA was detected by RT-PCR. The expression of GLUT1 protein was detected by immunofluorescence and flow cytometry. The uptake of glucose and its kinetics were determined by 2-deoxy-[3H] -D-glucose uptake. RESULTS: Both GLUT1 mRNA and protein were found in mouse glomerular mesangial cells. 2-deoxy-D-glucose uptake and kinetics assay showed that this glucose transporter had high affinity for glucose and the glucose uptake specificity was further confirmed by phloretin. CONCLUSION: Functional GLUT1 did present in mouse mesangial cells cultured in vitro and it might be the predominant transporter mediated the uptake of glucose into mesangial cells.

Animals↗
PubMed2005

Non-destructive micromethod for MRP1 functional assay in human lung tumor cells.

Defense against toxic endo- and xenobiotics is a major concern of all living species and ABC transporters play a vital role in this defense system. Multidrug resistance associated proteins 1 (MRP1) is a cellular detoxifying factor supposed to transport a wide range of compounds across cell membranes either as GSH conjugates or as co-transport accompanying glutathione transposition. The cellular localization of MRP1 is a determining factor whether the transport function can take place. In this study we have undertaken experiments on the transport activity of MRP1 in cultured human lung tumor cells in order to check whether MRP1 is expressed as a functionally active protein. For this purpose we have adapted a quantitative fluorescence imaging assay to conditions where a small number of attached cells should be repeatedly measured by a non-destructive method. In cultured A549, H358 and H322 cells MRP1 is located in the cell membrane as observed by immunocytochemistry. Efflux of 5,6-carboxy-2'-7'-dichloro-fluorescein (CDF) from lung cells was sensitive toward the MRP1 inhibitor MK571 while verapamil had no effect. On the other hand, efflux of Rhodamin 123, a Pgp-glycoprotein substrate, from lung cells reacted to inhibition by verapamil, while MK571 had no effect. Modulation of glutathion content of lung cells by N-acetyl cystein and buthionine sulfoximine shifted CDF efflux toward higher or lower rates, respectively. These experiments confirm that MRP1 function can be followed in the attached cells in vitro under non-toxic concentrations of the substrates without the need to harvest and destroy the cells.

ATP-Binding Cassette Transporters↗
PubMed1992

Characterization of myocardial extracellular ATP receptors by photoaffinity labelling and functional assays.

Extracellular ATP receptors in rat ventricular myocytes were investigated through intact cell photolabelling followed by protein isolation. 8-Azido-ATP (8Az-ATP) was used for labelling under specific conditions determined by parallel functional studies. In those studies ATP-induced cytosolic Ca2+ transients were irreversibly and specifically inhibited by UV-photolyzed 8Az-ATP, but not by 2-azido-ATP (2Az-ATP), even in the presence of high concentrations of phosphonucleotides not affecting myocardial ATP receptors. Under those conditions background labelling is minimized and radioactive 8Az-ATP specifically labels a band of 45-48 kDa on a SDS gel. Labelling under the above conditions in the presence of ATP gamma S or 2-methylthio-ATP (2-meSATP), which are distinct for two functionally different cardiac ATP receptors, shows two different proteins within the same band consistent with the possible labelling of these two receptors.

Adenosine Diphosphate↗
PubMed2006

Functional assays for replication protein A (RPA).

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding protein. RPA is conserved in all eukaryotes and is essential for DNA replication, DNA repair, and recombination. RPA also plays a role in coordinating DNA metabolism and the cellular response to DNA damage. Assays have been established for many of these reactions. This chapter provides an overview of the methods used for analyzing RPA-DNA interactions, RPA-protein interactions, and functional activities of RPA. Methods are also discussed for visualizing RPA in the cell and analyzing the effects of RPA function on cell cycle progression in mammalian cells.

Chromatography, Affinity↗
PubMed2000

A comparative study of functional assays for tissue factor pathway inhibitor using normal plasma and clinical samples.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type inhibitor that regulates the initiation of tissue factor-mediated coagulation. Recent reports in the literature have described variable results using different methodologies for TFPI measurement. In this study, we used one clotting and two amidolytic methodologies to assess TFPI functional levels. The study groups included normal healthy donors as well as patients with acute hepatitis, diabetes, coronary artery bypass graft operations, deep vein thrombosis, and prior to and during heparin therapy. The aims were to compare the results obtained in normal plasma using different assay systems, to compare TFPI levels in a range of clinical samples, including those previously not determined using a clotting methodology, and to report TFPI levels in patient groups previously not investigated. The results clearly demonstrate poor correlation between functional TFPI values using the different methodologies, highlighting the requirement for standardization.

Biological Assay↗
PubMed2002

Intra- and intermolecular interactions in sucrose transporters at the plasma membrane detected by the split-ubiquitin system and functional assays.

Interaction of two separately expressed halves of sucrose transporter SUT1 was detected by an optimized split-ubiquitin system. The halves reconstitute sucrose transport activity at the plasma membrane with affinities similar to the intact protein. The halves do not function independently, and an intact central loop is not required for membrane insertion, plasma membrane targeting, and transport. Under native conditions, the halves associate into higher molecular mass complexes. Furthermore, the N-terminal half of the low-affinity SUT2 interacts functionally with the C-terminal half of SUT1. Since the N terminus of SUT2 determines affinity for sucrose, the reconstituted chimera has lower affinity than SUT1. The split-ubiquitin system efficiently detects intramolecular interactions in membrane proteins, and can be used to dissect transporter structure.

Amino Acid Sequence↗