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At least 235 records · Page 13Linked to original sources
PubMed2006

Functional assay of Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general method for characterization of outer membrane proteins.

The immunodominant trimeric beta-barrel outer membrane protein OmpC from Salmonella typhi, the causative agent of typhoid, has been functionally characterized here. The activity in the vesicle environment was studied in vitro using OmpC reconstituted into proteoliposomes. Passage of polysaccharides and polyethyleneglycols through OmpC has been examined to determine the permeability properties. The relative rate of neutral solute flux yields a radius of 1.1 nm for the S. typhi OmpC pore. This is almost double the pore size of Escherichia coli. This provides an example of large pore size present in the porins that form trimers as in the general bacterial porin family. The method used in this study provides a good membrane model for functional studies of porins.

Bacterial Outer Membrane Proteins↗
PubMed2004

A rapid platelet function assay used to regulate platelet transfusion prophylaxis following cardiopulmonary bypass surgery.

Subsequent to cardiac surgery with cardiopulmonary bypass (CPB), patients are at risk of postoperative bleeding caused by acquired defects associated with the procedure. As such, many patients receive prophylactic blood product transfusion. The effectiveness of measuring platelet function using a near-patient platelet function analyzer for the purpose of regulating platelet transfusion and potentially modulating other blood products including fresh frozen plasma (FFP) and cryoprecipitate (CRYO) transfusion was evaluated prospectively in patients undergoing CPB. A 6-month prospective study was designed that encompassed all CPB patients at Aultman Hospital (Canton, OH) during 2001. Each patient was assessed postoperatively for platelet count and function using a point-of-care analyzer. Data were used to help determine whether a platelet transfusion would be administered. Transfusion of CRYO and FFP was also recorded. The data were then compared to the retrospective same 6-month period in the preceding year for the purpose of determining the value of this system in guiding transfusion triage. There were 1770 platelet packs transfused during the study period, representing a 34% reduction in platelet usage. Platelet transfusions were reduced by 51% (5.75 in 2001 vs. 11.75 in 2000) per patient. CRYO/FFP usage was also substantially reduced. During the study period, the patient caseload had increased by 72 (310 vs. 238). Monitoring platelet count and function as a result of CPB and administering an appropriate transfusion protocol can positively impact transfusion outcome.

Autoanalysis↗
PubMed1989

In vitro assembly of yeast U6 snRNP: a functional assay.

U6 small nuclear RNA (snRNA) is the most highly conserved spliceosomal RNA, and it has been postulated to have a fundamental role in pre-mRNA splicing. To elucidate this role, we developed an in vitro system for reconstituting the functional U6 small ribonucleoprotein (snRNP). Treating splicing extracts with an oligonucleotide complementary to the central domain of U6 snRNA leads to both RNase H cleavage of the endogenous U6 snRNA and loss of splicing activity. Yeast U6 RNA, synthesized in vitro using T7 RNA polymerase, is then added to the oligonucleotide-treated extract, and restoration of splicing activity is monitored by the subsequent addition of substrate pre-mRNA. Addition of full-length, unmodified T7U6 snRNA (113 nucleotides) to oligonucleotide-treated extracts restores splicing activity efficiently. Using U6 RNA transcripts truncated at their 3' ends, we show that large deletions (39 nucleotides) produce molecules that are unable to restore splicing activity in vitro and cannot interact with the endogenous U4 snRNA or form a mature spliceosome. Finally, we show that substitution of the invariant G81 with C within the T7U6 RNA abolishes its ability of restoring splicing activity. Although the U4/U6 snRNP forms correctly, mature spliceosomes do not assemble.

Base Sequence↗
PubMed1997

Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe.

Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170). Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123. With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye. Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form. Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm. JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence. In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence. The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels. The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells. Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells. The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1983

Reconstitution of a functional beta-adrenergic receptor using cholate and a novel method for its functional assay.

Solubilization of purified turkey erythrocyte membranes at increasing cholate to protein ratios and in the presence of salt, extracts up to 20% of the beta-adrenergic receptor together with the GTP stimulatory protein (Ns) of adenylate cyclase. Upon removal of the cholate, by active absorption on Bio-beads, the functional interaction between the beta-receptor and the GTP regulatory protein Ns is quantitatively restored. The receptor (R) in the presence of l-isoproterenol and p[NH]ppG is able to catalyze the activation of Ns to its permanently active state, N's p[NH]ppG, with a rate constant (kon) identical to that of the native membrane. Reconstitution of the R/Ns mixture using poly(ethyleneglycol)-6000 restores the receptor binding properties as effectively as SM-2 Bio-beads. Unlike SM-2 Bio-beads, however, poly(ethyleneglycol) is not as efficient in restoring the R to Ns functional coupling. In this communication we also report on the ability to monitor quantitatively N's . p[NH]ppG, using native turkey erythrocyte membranes in the presence of Lubrol-PX as the source of the catalytic unit (c) of adenylate cyclase. The latter method is as efficient as using S49 AC- lymphoma cell membranes but much less expensive. Using this technique, we also demonstrate that when the Ns to C interaction is nullified, employing treatment with N-ethylmaleimide, the parameters which characterize R to Ns coupling remain unchanged.

Adenylyl Cyclases↗
PubMed2005

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay.

AIM: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. METHODS: Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. RESULTS: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor. CONCLUSION: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

Animals↗
PubMed1986

A simplified functional assay for protein C in plasma samples.

The important role of protein C (PC) in the regulation of hemostasis has been appreciated since the description of patients who were deficient in PC and presented with severe thromboembolic events. The potentially fatal complications associated with PC-deficiency require an early and reliable identification of those patients affected with this inherited disorder. The present study introduces a test procedure for the functional assessment of PC in plasma samples. The test utilizes the thrombin/thrombomodulin complex to achieve complete and rapid formation of activated PC whose proteolytic capacity is subsequently determined with a chromogenic substrate. Homogenate obtained from rabbit lung effectively substituted the purified component thrombomodulin in the assay system. This new approach simplifies the test procedure without losing specificity and accuracy. Proteases, such as plasmin, streptokinase and urokinase did not influence the assay and the inhibitory effect of heparin on the PC-activation could easily be overcome by the addition of protamine sulphate. The PC-activity in a group of unselected patients (n = 50), who did not reveal any abnormalities in global coagulation tests, amounted to 100 +/- 12% (mean +/- SD) with a range from 54 to 143% when analyzed in comparison to a plasma pool constituted from healthy volunteers. Since the synthesis of PC depends on the availability of vitamin K, patients receiving phenprocoumon have also been analyzed. These patients (n = 103) presented 40 +/- 11% residual PC-activity accompanied by a concomitant decrease in PC-antigen levels to 43 +/- 10% (mean +/- SD). The test described is specific, sensitive, less time-consuming and can be performed on a routine basis.

Adult↗
PubMed1995

Comparison of immunological and functional assays for measurement of soluble fibrin.

Various assays have been developed for quantitation of soluble fibrin or fibrin monomer in clinical plasma samples, since this parameter directly reflects in vivo thrombin action on fibrinogen. Using plasma samples from healthy blood donors, patients with cerebral ischemic insult, patients with septicemia, and patients with venous thrombosis, we compared two immunologic tests using monoclonal antibodies against fibrin-specific neo-epitopes, and two functional tests based on the cofactor activity of soluble fibrin complexes in tPA-induced plasminogen activation. Test A (Enzymun-Test FM) showed the best discriminating power among normal range and pathological samples. Test B (Fibrinostika soluble fibrin) clearly separated normal range from pathological samples, but failed to discriminate among samples from patients with low grade coagulation activation in septicemia, and massive activation in venous thrombosis. Functional test C (Fibrin monomer test Behring) displayed good discriminating power between normal and pathological range samples, and correlated with test A (r = 0.61), whereas assay D (Coa-Set Fibrin monomer) showed little discriminating power at values below 10 micrograms/ml and little correlation with other assays. Standardization of assays will require further characterization of analytes detected.

Antibodies, Monoclonal↗
PubMed2000

A flow cytometric immune function assay for human peripheral blood dendritic cells.

CD11c+ and CD11c- (CD123+) dendritic cells (DCs) have been described in blood. Both cell types express high levels of HLA-DR and lack the lineage markers CD3, CD14, CD19, CD20, CD16, and CD56. These immunophenotypic properties were used along with analysis of activation-related surface antigens and intracellular staining of cytokines to characterize functional responses of these DC subsets to stimuli in whole human blood (WB). Samples from healthy donors were activated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). The only distinct response in CD11c- DCs was the expression of CD25 upon PMA+I activation. CD11c+ cells responded to LPS stimulation by producing high levels of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), and lower levels of IL-6, IL-1Ra, and IL-8 and an increased expression of accessory molecules (CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ). PMA+I activation of CD11c+ cells resulted in high levels of IL-1beta and lower levels of IL-8, IL-1Ra, and TNF-alpha and up-regulation of CD80, CD86, HLA-DR, and HLA-DQ. Our data support prior observations of functional differences between peripheral blood DC subsets and demonstrate the power of multiparameter flow cytometry to characterize the pleiotropic responses of these cells to various stimuli.

Blood Cells↗
PubMed1997

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system.

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP3), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca2+ channel, the IP3 receptor (IP3R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf (S. frugiperda) cell system that can be used to look at IP3R function. Agonist-evoked changes in intracellular Ca2+ levels [Ca2+]i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vm1AchR). Furthermore, we have constructed a recombinant BV (vIP3R), with the core of the IP3R ligand-binding domain from the Drosophila IP3R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vm1AchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP3R construct and vm1AchR have been used to assay the modulation of IP3R-mediated Ca2+ release, by the ligand sink.

Animals↗
PubMed2005

Functional assays for the investigation of the role of Rab GTPase effectors in dense core granule release.

Rab3 and Rab27 GTPases control late events in the secretory pathway of mammalian cells including docking and fusion of secretory vesicles with the plasma membrane. The action of Rab3 and Rab27 on the exocytotic process is exerted through the activation of specific effectors. Several proteins with the capacity to interact in a GTP-dependent manner with Rab3 and Rab27 have been identified. However, for most of these potential Rab effectors a precise function in the secretory process has not yet been attributed. In this chapter we describe a series of approaches that can be applied to assess the properties of Rab3 and Rab27 effectors and their potential role in the regulation of dense core granule release.

Animals↗