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Results for “Functional assay”

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PubMedDate not supplied

[Heparin induced autoimmune thrombocytopenia: assessment of a rapid functional assay].

Heparin-induced thrombocytopenia (HIT) diagnosis is often difficult. Depending on the patients, the thrombocytopenia could be due to various causes. Despite their poor sensitivity and specificity, biological tests are necessary to clarify the diagnosis. In 1985, a new heparin-induced platelet aggregation assay was described that consists in determining the percentage of aggregated platelets by using an automated cell counter. Plasma samples from 18 patients with a definite HIT were tested with this quick easy-to-perform HIT diagnosis test. Positive results were obtained with 11 plasma (61%) when the test was performed with platelets from 3 different healthy volunteers (control platelets). As for the other functional tests, results are depending on control platelets and sensitivity seems to be increased when control platelets FcgammaRIIa-131 polymorphism was of His/His (but difference is not significant). In emergency situation, it is difficult to perform a functional test with control platelets from several healthy donors, and it is even more difficult to select volunteers on their FcgammaRIIa-131 polymorphism. In conclusion, in spite of its practicability, the test is not reliable to help in the rapid diagnosis of HIT. Indeed, 7 patients of 18 (39%) with definite HIT have been found negative with this test.

Anticoagulants↗

PubMed1996

Two functional assays of sperm responsiveness to progesterone and their predictive values in in-vitro fertilization.

We have recently reported, in a small cohort of subjects, that acrosome reaction (AR) and intracellular free calcium ([Ca2+]i) increase in response to progesterone were significantly correlated with in-vitro fertilization (IVF) rate. In the present study we extended these results to 90 subjects undergoing IVF. We confirm that both parameters were highly significantly correlated with the fertilization rate (P < 0.001). In particular, significantly lower responses to progesterone were detected in subjects with a fertilization rate < 50%, further enlightening the functional significance of sperm responsiveness to progesterone with respect to the process of fertilization. Moreover, we report here that both tests are highly discriminant of fertilization success, with positive predictive values > 90% for [Ca2+]i values which increase by > 1.2-fold and AR inducibility > 7% (cut-off values). Conversely, AR following challenge with the calcium ionophore A23187 was less significantly correlated with the percentage fertilization rate (P < 0.05), and showed lower predictive values than response to progesterone. All these tests ([Ca2+]i increase in response to progesterone, AR in response to progesterone and to A23187) appear highly sensitive and moderately specific. The positive predictive value may rise to > 95% when the combination of two tests ([Ca2+]i and inducibility of AR in response to progesterone) is considered. No correlation with fertilization rate has been found for spontaneous AR or basal [Ca2+]i. In conclusion, we propose that assessment of human sperm responsiveness to progesterone may be clinically useful in predicting fertilizing ability in vitro.

Acrosome↗

PubMed2005

In vitro functional assay of alleles and haplotypes of two COL1A1-promoter SNPs.

Osteoporosis is a common disease with a strong genetic component. We previously described two polymorphic sites in the COL1A1 gene promoter, -1997 G/T and -1663indelT, which have been associated with bone mineral density (BMD), a surrogate trait for osteoporosis. Here, we explore the molecular mechanisms underlying this association by performing transient transfections in MG-63 cells of constructs bearing different COL1A1 promoter regions, containing different alleles or haplotypes of the polymorphic sites. These promoter regions drove the transcription of a luciferase reporter gene. The main differences in transcriptional activity relied on an inhibitory region localized to the -1284 to -254 interval. Regarding the polymorphisms, reproducible differences were observed between the alleles of each of them: the G allele at -1997 showed a higher transcriptional activity than the T allele, as did the 7T allele of -1663 as compared with 8T. Accordingly, the T-8T haplotype was the weakest transcriber. A functional interaction was found between the -1997 and -1663 polymorphisms, in that the difference in transcriptional activity between the 7T and 8T alleles was dependent on the allele at -1997. This different transcriptional activity of the two -1663indelT alleles correlated with different binding capacities of the corresponding oligonucleotides to osteoblast nuclear proteins. Supershift assays allowed us to identify one of these proteins as the architectural transcription factor Nmp4/CIZ, a protein known to be an inhibitor of BMP/Smad signalling.

Alleles↗

PubMed1985

Preparation of interstitial lung cells by enzymatic digestion of tissue slices: preliminary characterization by morphology and performance in functional assays.

A technique is reported here for the quantitative extraction of live cells from the lung interstitium; it involves the incubation of slices of perfused lung in a mixture containing optimal concentrations of collagenase, DNAse, and fetal calf serum, followed by the simultaneous recovery and fractionation of cells released from the tissue matrix on a six-step discontinuous percoll gradient. Yields in the order of 10(8) viable cells per gram of lung were routinely achieved with tissues from rat, mouse and guinea-pig. Preliminary characterization of these cells has been performed in the rat by histological techniques (Giemsa staining, transmission electron microscopy), cytochemistry (acid phosphatase, esterase, peroxidase), by the capacity to bind monoclonal antibodies directed at lymphocyte surface markers, and by a range of functional tests. The cells comprised, on average, 32% macrophages, 44% lymphocytes (T and B cells and large granular lymphocytes), with small numbers of eosinophils, mast cells and epithelial cells. Transmission electron microscopy revealed minimal ultrastructural damage to extracted cells, with such functions as phagocytosis, FcR activity, mitogen responsiveness, antigen presentation, and NK-cell activity, being readily demonstrable. In addition, these activities segregated into defined areas of the six-step density gradient.

Animals↗

PubMed1984

Suppressor cells in end-stage renal disease. Functional assays and monoclonal antibody analysis.

Suppressor cell activity after concanavalin A induction was studied in peripheral blood mononuclear cells of patients undergoing long-term hemodialysis. Suppression both of the mixed lymphocyte reaction and of allogeneic cells stimulated with phytohemagglutinin was significantly higher with peripheral blood mononuclear cells from patients undergoing hemodialysis than with cells from control subjects. Expression of the Ia antigen on T lymphocytes (associated with immunologic activation) was studied by staining with monoclonal antibodies and two-color fluorescence analysis in a computer-linked cytofluorograph. In unstimulated cells, there was no significant difference between the patients and control subjects. After concanavalin A induction, the percentage of T4, and particularly of T8, cells expressing the Ia antigen was significantly higher in the group undergoing hemodialysis. The functional suppression seen after concanavalin A induction in the mixed lymphocyte reaction was significantly reduced by treatment with OKT8 monoclonal antibody and complement; in phytohemagglutinin cultures, both OKT8 and OKIa*1 antibodies were effective. The reduced in vitro response of uremic lymphocytes may thus be a consequence of increased suppressor activity associated with the T8-positive, Ia-positive subset of T cells.

Adult↗

PubMed1995

A sensitive platelet activation-based functional assay for the antileukemic agent bryostatin 1.

Bryostatin 1, a macrocyclic lactone activator of protein kinase C (PKC) currently in phase I evaluation, is a biologic response modifier which exhibits significant antitumor activity in several experimental systems. Clinical trials have been hampered by the absence of a sensitive assay for bryostatin 1 blood levels. The purpose of these studies was to exploit the exquisite sensitity of human platelets to bryostatin 1-induced aggregation in order to develop an assay capable of detecting plasma bryostatin 1 levels in the nanomolar range. Addition of bryostatin 1 (5-100 nM) to platelet-rich plasma resulted in complete platelet aggregation. A highly linear relationship was observed between low bryostatin 1 concentrations (i.e. 2-25 nM) and (i) reduction in the lag phase prior to aggregation and (ii) maximal rate of aggregation (R = 0.976). At higher bryostatin 1 concentrations (i.e. 10-100 nM), platelet aggregation was accompanied by detectable ATP release; both the extent and maximal rate of ATP secretion were highly linear functions of bryostatin 1 levels (R = 0.992). Bryostatin 1 concentrations in anticoagulated human blood samples could also be determined by mixing platelet poor plasma obtained from such samples with normal platelet-rich plasma. Notably, measurement of the delay in the aggregation lag phase permitted quantitation of bryostatin 1 concentrations of 5 nM or below. The capacity to detect bryostatin 1 plasma levels of 10 nM or lower should facilitate the conduct of pharmacokinetic and pharmacodynamic studies in conjunction with ongoing phase 1 trials.

Adenosine Triphosphate↗

PubMed1992

Isolation of maternal mononuclear cells from placentas for use in in vitro functional assays.

Interest in immunoregulatory mechanisms within uteroplacental tissues, particularly in malarial infection during pregnancy, prompted us to develop a technique to extract maternal mononuclear cells from human term placentas. This method is described. The phenotypes of isolated cells were characterised for expression of CD45, CD3, CD4, CD8, CD14, CD15, CD68, CD22, CAM 5.2 and class II MHC antigens and compared with those in situ in frozen sections of the same placentas. Isolated mononuclear cell preparations were examined for contamination by fetal trophoblasts. Fetal leukocyte contamination appeared unlikely since histological sections of placental tissue, after the extraction of maternal leukocytes, showed intact chorionic villi with no disruption of fetal stem vessels. This technique produces preparations of maternal placental mononuclear cells which are representative of cells in situ, show minimal fetal cell contamination and are suitable for functional studies.

Antibodies, Monoclonal↗

PubMed2001

Purification and functional assay of pluripotent hematopoietic stem cells.

Hematolymphopoietic stem cells (HSC) have the capacity for extensive self-renewal and pluripotent myelolymphoid differentiation. Recent studies have emphasized the heterogeneity of human HSC subsets in terms of proliferative and self-renewal capacity. In the NOD-SCID (nonobese diabetic-severe combined immunodeficient) mouse xenograft assay, most CD34+38- stem cell clones proliferate at early times, but then disappear, whereas only few clones persist: possibly, the latter ones consist of long-term engrafting CD34+38- HSC expressing the KDR receptor (i.e. the vascular endothelial growth factor receptor II). In this regard, isolation of the small KDR+ subset from the CD34+ hematopoietic progenitors (and possibly from the CD34-lin- population) may provide a novel and effective approach for the purification of long-term proliferating HSC. More importantly, KDR+ HSC isolation will pave the way to cellular/molecular characterization and improved functional manipulation of HSC/HSC subsets, as well as to innovative approaches for HSC clinical utilization, specifically transplantation, transfusion medicine and gene therapy.

Animals↗

PubMed1985

Tissue plasminogen activator inhibitor in human plasma: development of a functional assay system and demonstration of a correlating Mr = 50,000 antiactivator.

An assay system for determination of the "fast"-acting inhibitor (antiactivator, AA) of tissue-type plasminogen activator (tPA) in human plasma was developed. The system is based on incubation of plasma samples with various amounts of vessel wall-derived tPA for 8 minutes at 25 degrees C, followed by acidification and determination of the residual tPA activity by an indirect spectrophotometric assay. One unit of AA was defined as the amount inhibiting 1 U of tPA. AA levels in normal controls (n = 26) were 1.4 to 17.4 U/ml (median 3.0 U/ml) and 0.9 to 17.5 U/ml (median 3.0 U/ml) in patients with a history of deep venous thrombosis (n = 26). When plasma was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse fibrin autography, AA activity appeared as an inhibitory band corresponding to a relative molecular mass of 50,000. In six samples the inhibitory activity of this band was directly correlated to the functional AA activity of the plasma samples.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

[Consenting and declining patients for an intervention group after breast cancer surgery differ in terms of quality of life, coping and immunological functional assays].

OBJECTIVE: Psychosocial intervention during the course of cancer is still being controversially disputed in psychosomatic studies. Immunological and endocrinological parameters can serve as links in observing the influence of the interactive processes involved. MATERIAL AND METHODS: The approach to determine whether psychotherapeutic intervention can lead to an improved quality of life on the one hand, and to a change in immune defenses in breast cancer patients as well, employed using the EORTC QLQ-C30 and optimistic self-efficacy (OKE) as well as evaluating lymphocyte subpopulations, lymphocyte proliferation and NK cell activity. Fifty-three breast cancer patients were offered the chance to participate in a psychosocial intervention group. Twenty-three accepted the offer. Randomized sampling of the group participants was determined twice by using the 30 EORTC QLQ-C30 parameters. In addition, immune function was measured before beginning (T1) and after completing the group intervention sessions (T2). This data was compared with that of the declining group. RESULTS: The analysis of the individual EORTC parameters in the pre- and post-comparison showed a decrease in "emotional strain" and an increase in "all-around quality of life" at the T2 test period. NK cell activity was increased at T2 as well in the participating group and was higher than that of the declining group both at T1 and T2 testing times. CONCLUSION: Participation in the psychosocial intervention group resulted in a marked decrease in the emotional strain and an increase in the quality of life. The increased NK-cell activity with lowered T-cell activation in comparison to the decliners hints at a coherence between coping behaviour and changes of the immunological cell function. The results of this study will be investigated further in a greater sample.

Adaptation, Psychological↗

PubMed1995

Tobacco mitotic cyclins: cloning, characterization, gene expression and functional assay.

Three cyclin cDNA clones (Ntcyc25, Ntcyc27, Ntcyc29) have been isolated from tobacco (Nicotiana tabacum) using the PCR cloning method. The encoded Ntcyc cyclins were highly homologous to mitotic cyclins. In synchronized tobacco suspension cultured cells, the mRNA levels of Ntcyc25 and Ntcyc27 were detectable through S, G2 and M phases, while the Ntcyc29 mRNA was detected from G2 to M phase. These expression patterns classified the Ntcyc25 and Ntcyc27 into A-type cyclin and Ntcyc29 into B-type cyclin. The three genes were expressed in growing tobacco cultured cells but ceased to be expressed when cells entered the stationary phase, indicating that the expression of these cyclin genes was well correlated with cell growth. The N-terminal truncated Ntcyc25 and Ntcyc29 cDNAs were able to rescue G1 cyclin mutant of Saccharomyces cerevisiae, while the full-length of Ntcyc27 protein could also partially perform G1 cyclin functions.

Amino Acid Sequence↗

PubMed2002

Fluorescence-based functional assay for Wnt/beta-catenin signaling activity.

Aberrant activation of beta-catenin signaling has been implicated in the development of human cancers. As a Wnt signal transducer, beta-catenin forms a complex with the lymphocyte enhancer-binding factor/T cell factor transcription factor and activates downstream targets that promote cell proliferation. Here we developed a Wnt-dependent beta-catenin-mediated heterologous transactivation system, which consisted of a chimeric transcription factor constructed by fusing the GAL4 DNA-binding domain with the full-length beta-catenin, and a GAL4-responsive reporter expressing GFP. The chimeric transcription factor was highly unstable and exerted no detectable transactivating effect on the GAL4-responsive reporter. However, lithium and Wnt1 significantly stabilized this chimeric transactivator, indicating that this transactivation system is regulated by beta-catenin in a Wnt-responsive fashion. Thus, this transactivation system could be used as a functional reporter to identify potential upstream factors that deregulate beta-catenin signaling during tumorigenesis, as well as to screen for potential anti-cancer agents that specifically inhibit beta-catenin signaling in human tumors.

Adenocarcinoma↗

PubMed1996

Expression of the three alternative forms of the sphingolipid activator protein precursor in baby hamster kidney cells and functional assays in a cell culture system.

Sphingolipid activator proteins (SAPs) are non-enzymatic glycoproteins required for lysosomal degradation of various sphingolipids with short oligosaccharide chains by their respective exohydrolases. Four of these (SAP-A to SAP-D or saposins A to D) are derived from a common precursor by proteolytic processing. Alternative splicing of the SAP-precursor gene results in insertion of additional 6 or 9 bases of exon 8' or 8, respectively, into the SAP-B coding region of the transcribed mRNAs. To examine the features of the three different SAP-precursor proteins (prosaposins), the respective cDNAs were stably expressed in baby hamster kidney cells. Pulse-chase experiments with transfected cells and endocytosis studies on human fibroblasts showed that synthesis, transport, and maturation of all SAP-precursor led to formation of the four mature SAPs (SAP-A to SAP-D). In order to determine the biological function of the three different SAP-B isoforms, SAP-precursor-deficient human fibroblasts were loaded with recombinant SAP-precursor proteins with or without 2- and 3-amino acid insertions, respectively, purified from the medium of the baby hamster kidney cells. They were found to stimulate at nanomolar concentrations the turnover of biosynthetically labeled ceramide, glucosylceramide, and lactosylceramide. Since the physiological function of SAP-B is to stimulate the degradation of sulfatide by arylsulfatase A (EC 3.1.6.1) and globotriaosylceramide by beta-galactosidase (EC 3.2.1.23) loading studies with the respective exogenously labeled lipids on SAP-precursor-deficient fibroblasts were performed. Addition of different purified SAP-precursors to the medium of the lipid-loaded fibroblasts showed positive stimulation of the lipid degradation by all three SAP-B isoforms derived from the SAP-precursors. These findings establish that all three forms of the SAP-B can function as sulfatide/globotriaosylceramide activator.

Alternative Splicing↗

PubMed2005

Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist.

The human Neuropeptide Y (NPY) receptors 1 (hY1), 2 (hY2), 4 (hY4), and the mouse type 5 (mY5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPgammaS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gqo5, Gqi5 and Gqi9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY1, hY2 and hY4 receptors coupled to Gqo5, Gqi5 and Gqi9, the mY5 receptor stimulated transient calcium release only when co-expressed with Gqi9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY5 receptor with high affinity (Ki=32.1+/-1.8 nM), competitively antagonized NPY-mediated GTPgammaS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.

Animals↗

PubMed1988

Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action.

The effects of inhibitors of protein kinase C on the activities of the intact enzyme, the proteolytically-generated catalytic domain, and [3H]phorbol 12,13-dibutyrate (PDBu) binding were compared in an effort to evaluate this approach for clarifying mechanisms of inhibitor action. Staurosporine, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], and quercetin inhibited the catalytic fragment with similar potencies as for the intact enzyme while having little or no effect on binding, consistent with reports that they are competitive with ATP. Adriamycin, trifluoperazine, and tamoxifen, suggested to disrupt hydrophobic interactions between the regulatory domain of protein kinase C and phospholipid, were all most effective on the intact enzyme. They appear to possess a mixed mechanism, however, inhibiting activity of the catalytic domain with approximately 3-fold lower potencies. Gossypol inhibited intact enzyme, catalytic fragment, and PDBu binding with similar potencies. In light of multiple apparent sites of action for such protein kinase C inhibitors, comparison of their activities on the individual functional domains of the kinase may provide a useful complement to studies with the intact enzyme.

Animals↗

PubMed1982

The suitability of various plasmin preparations for the functional assay of alpha 2-antiplasmin in plasma.

Various plasmin preparations were tested for their suitability for use in the assay of alpha 2-antiplasmin in blood plasma by the immediate plasmin inhibition test. Activation of plasminogen, viz., 1-Glu-plasminogen and/or 77-Lys-plasminogen, by immobilized urokinase results in plasmin preparations suitable for this alpha 2-antiplasmin test. Plasmins obtained by "spontaneous" activation procedures in glycerol containing solutions, however, appeared not to be suitable. In this second group, the behaviour of the plasmins resembles that of 442-Val-plasmin (miniplasmin), which is known to show a low inactivation rate with alpha 2-antiplasmin due to the absence of lysine-binding sites in the plasmin molecule. Evidence is presented that, in the nonsuitable plasmins, the lysine-binding sites, although not completely absent, have at least partly lost their functional integrity.

Blood Coagulation Tests↗

PubMed2005

Analysis of allosterism in functional assays.

The theoretical basis for analyzing the effects of an allosteric modulator on the response to an agonist is described. The effects of an allosteric modulator on the concentration-response curve to an agonist can be attributed to changes in the observed dissociation constant and intrinsic efficacy of the agonist-receptor complex. Each of these two changes can be represented by a coefficient or factor. It is possible to estimate the ratio of the coefficient of change in agonist efficacy divided by that for the agonist dissociation constant. This ratio is designated as the relative activity (RA) of the agonist in the presence of the allosteric modulator. The RA value can be estimated for each concentration of allosteric modulator by nonlinear regression analysis, regardless of the shape of the concentration-response curve. Regression analysis of the RA values against the concentration of allosteric modulator yields estimates of the dissociation constant (K(A)) of the allosteric modulator and the maximal RA value. If the concentration-response curve to the agonist obeys a logistic function and the allosteric modulator influences the maximal response, it is possible to distinguish between the maximal change in affinity from that of efficacy. If the agonist concentration-response curve obeys a logistic equation with a Hill slope of 1, the RA values can be estimated easily from the agonist EC(50) and E(max) values. This analysis illustrates the utility of the RA value in quantifying allosteric effects.

Algorithms↗

PubMed2000

Sperm aster formation and pronuclear decondensation during rabbit fertilization and development of a functional assay for human sperm.

Microtubule organization and chromatin configurations in rabbit eggs after in vivo rabbit fertilization and after intracytoplasmic injection with human sperm were characterized. In unfertilized eggs, an anastral barrel-shaped meiotic spindle, oriented radially to the cortex, was observed. After rabbit sperm incorporation, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei. The microtubules extending from the decondensed sperm head participated in pronuclear migration, and organization around the female pronucleus may also be important for pronuclear centration. Support for these observations was found in parthenogenetically activated eggs, in which microtubule arrays were organized around the single female pronucleus that formed after artificial activation. These observations support a biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern suggested from previous studies. In rabbit eggs that received injected human donor sperm, an astral array of microtubules radiated from the sperm neck and enlarged as the sperm head underwent pronuclear decondensation. gamma-Tubulin was observed in the center of the sperm aster. We conclude that the rabbit egg exhibits a blended centrosomal contribution necessary for completion of fertilization and that the rabbit egg may be a novel animal model for assessing centrosomal function in human sperm and spermatogenic cells following intracytoplasmic injection.

Animals↗