Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “quantitative complementation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,333 records · Page 74Linked to original sources
PubMed1987

The fifth component of complement (C5) in the mouse. Analysis of the molecular basis for deficiency.

C5-deficient mice differed from C5-sufficient mice both quantitatively and qualitatively in C5 protein, C5 mRNA, and the C5 gene. C5-deficient protein was present as decreased amounts of an unprocessed, single-chain precursor. C5-deficient mRNA was decreased in amount and present in two forms, the smaller of which was the same as the single form in normal cells. Nuclei from both normal and deficient cells contained the larger form of C5 mRNA, and C5-deficient DNA demonstrated differences from the normal pattern on Southern analysis for two restriction enzymes. These data suggest that the primary transcript of the C5-deficient gene is abnormal, retarding the processing of the C5 mRNA, and that the C5-deficient mRNA codes for an abnormal protein.

Animals↗
PubMed1993

Temporal sequence of changes in tear film composition during sleep.

Overnight eye closure induces a shift in the nature and composition of the tear film, from a dynamic reflex tear-rich to a stagnant secretory IgA-rich layer. This is accompanied by the induction of a state of sub-clinical inflammation, as evidenced by increases in albumin levels, plasminogen activation, conversion of complement C3 to C3c, and the recruitment of polymorphonuclear (PMN) cells into the tear film. To determine the time course and functional relationship between these potentially interdependent processes, tear samples were collected from ten non-contact lens wearers after 1, 2, 3 and 5 hours of sleep. A subgroup of 6 subjects also self-collected tear samples after 8 hours of sleep. Tear samples were analysed for albumin by quantitative immunofixation assay, secretory IgA (sIgA) by radial immunodiffusion assay, plasmin-like activity using a chromogenic substrate, and complement C3 to C3c conversion by immunoblot assay. Epithelial and PMN cells in the precorneal tear film were recovered from corneal washings from the same subjects after 1, 3, 5 and 8 hours of sleep, and quantified. Results revealed that, unlike epithelial cells which exhibited a slow progressive accumulation as a function of the period of sleep, PMN cell concentration exhibited a lag phase, with recruitment occurring after between 3 and 5 hours of eye closure. This was preceded by plasminogen activation, increases in albumin and sIgA levels, and complement C3 to C3c conversion, all of which occurred within 1 to 3 hours after eye closure. Plasmin-like activity appeared to plateau after 3 hours and then decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1986

Functional characterization of toposomes from sea urchin blastula embryos by a morphogenetic cell aggregation assay.

This paper documents the evidence that the large oligomeric glycoprotein complexes of unknown function first isolated as 22S particles from sea urchin embryos are the sole agents responsible for the adhesive integrity of sea urchin blastula embryos. The conclusion rests on the demonstration that polyclonal IgG (as serum or monovalent Fab) against whole membranes or butanol-solubilized components of membranes, as well as against the purified particle itself, completely blocks reaggregation of dissociated blastula cells and that this inhibition is reversed by neutralization of the inhibitory antibodies with purified 22S antigen. An essential aspect of the evidence is the combination of quantitative endpoint titrations in microtiter wells with the qualitative parameters of morphogenesis. The new data complement previous evidence that morphogenesis is mediated by a general class of particles, toposomes, responsible for mechanical linkage between cells and their positional guidance in embryogenesis.

Animals↗
PubMed1988

The use of tonometry in the assessment of upper extremity lymphoedema.

The use of a specially designed tissue tonometer in 17 lymphoedema patients, who were treated with multiple lymphaticovenous anastomoses, with a follow-up period of up to 18 months, gives a quantitative measurement of "softness" and correlates well with their subjective feeling. It would appear to complement limb volume and circumferential limb measurements.

Anastomosis, Surgical↗
PubMed2005

Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase proteins in winter flounder (Pleuronectes americanus): comparison to the effects of Saint John's Wort.

4-Nonylphenol (4-NP), a major by-product of alkylphenol ethoxylates, is used in several industries and as a consequence is quite common in rivers, estuaries and other aquatic environments that receive sewage discharges or are near offshore oil platforms. 4-NP is an environmental estrogen that also binds human and rodent Pregnane X-receptor (PXR), the orphan nuclear receptor that controls the expression of several detoxication genes in mammals, including several CYP3A and CYP2B family members. These P450s preferentially hydroxylate testosterone in the 6beta- and 16beta-positions, respectively. In this study, the effects of 4-NP on testosterone metabolism and hepatic CYP3A induction were compared to the effects of St. John's Wort (SJW), a well established mammalian PXR agonist, in winter flounder. Male winter flounder (Pleuronectes americanus) were injected with 100 mg/kg/day 4-NP or 500 mg/kg/day SJW or both (S and N) every 24 h. Forty-eight hours after the initial injections, flounder were euthanized. Western blots and testosterone 6beta-hydroxylation indicated that CYP3A was increased 50% by 4-NP, but was not affected by SJW. Testosterone 16beta-hydroxylase activity was also significantly increased in flounder treated with 4-NP (2.8 x), but not with SJW. This is not consistent with our hypothesis that both SJW and 4-NP would induce CYP3A. Subtractive hybridization was performed between control and 4-NP treated hepatic mRNA samples to isolate differentially expressed genes. Subtractive hybridization indicated that several acute phase proteins were altered by 4-NP. Quantitative real-time PCR (Q-PCR) confirmed 4-NP altered the expression of complement components C8b, cathepsin L, C-type lectin domain, FK506 binding protein 2 precursor (FKBP2) and an EST (expressed sequence tag). SJW and 4-NP treated flounder demonstrated similar induction profiles for the EST, cathepsin L and FKBP2, suggesting that SJW was at a sufficient dose to alter gene expression but not induce P450s. In conclusion, testosterone hydroxylase activity and Western blots indicate that SJW did not activate detoxication pathways in a similar manner to 4-NP.

Acute-Phase Proteins↗
PubMed2006

Structural and functional analysis of a salt stress inducible gene encoding voltage dependent anion channel (VDAC) from pearl millet (Pennisetum glaucum).

We have cloned and characterized a gene encoding voltage-dependent anion channel from Pennisetum glaucum (PgVDAC). PgVDAC was identified while isolating genes that were differentially up-regulated following salt stress. The genomic organization of PgVDAC clone was well conserved compared to other plant VDAC genes in terms of number of introns, their position and phasing, however, the primary amino acid sequence of voltage dependent anion channel (VDAC) proteins did not show much conservation with other plant VDACs but their secondary and tertiary structures are well conserved as predicted by in silico structural and CD spectra analyses and results show it to be a typical membrane-spanning beta-barrel leading to the formation of pore in the membrane. The heterologous expression of PgVDAC protein in yeast strain lacking the endogenous mitochondrial VDAC gene could not functionally complement it as was also previously observed for the potato VDAC. Using real-time quantitative PCR analysis it was found that transcript expression profile of PgVDAC was quantitatively and kinetically up-regulated in response to salinity, desiccation, cold and exogenous application of salicylic acid (SA); however, there was no effect of exogenous application of abscisic acid (ABA) on its expression. Constitutive over-expression of PgVDAC appears to be deleterious in transgenic rice plant; however, low level of up-regulation imparted salinity stress adaptive response. A search for a more suitable inducible transgene system is currently under way to understand PgVDAC expression levels in plant development and its role in stress adaptation.

Amino Acid Sequence↗
PubMed1997

A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines.

The well-established methods of generating stably transfected cell lines, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotoxicity assays. In these assay systems, the detection of released enzymes was used to quantitate the leakage of intracellular proteins after membrane disintegration. Target cell lines were transfected with a luciferase reporter gene under the control of a strong eucaryotic promoter. Release of the intracellular expressed enzyme into the culture supernatant occurred after membrane perforation and was measured as an indicator of cellular death. The quantitation of released enzyme was a reliable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially established with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intracellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial beta-galactosidase to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than luciferase. In a direct comparison between the standard 51Cr release and beta-galactosidase release, the enzyme release showed a much higher signal-to-noise ratio, i.e., low background and high induced release if spontaneous release and detergent induced maximal lysis were measured. Since a wide range of human and murine cell lines can be stably transfected and several reporter genes are available, the system should provide an alternative for conventional cytotoxicity assays. The detection of released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of luminometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular death using transgenic animals feasible.

Animals↗
PubMed2002

Proteomics goes quantitative: measuring protein abundance.

Protein-based methodologies are catching up with established DNA-based methods at an astonishing speed. Recent developments in mass spectrometry enable high-throughput automated identification of proteins as is already the case with DNA sequencing methods. Furthermore, methods for the quantitation of relative protein abundance at the protein level are getting more advanced, which should complement gene expression monitoring at the mRNA level.

Isotope Labeling↗
PubMed2002

Unbiased stereological estimates of neuron number in subcortical auditory nuclei of the rat.

The mammalian auditory system consists of a large number of cell groups, each containing its own complement of neuronal cell types. In recent years, much effort has been devoted to the quantitation of auditory neurons with common morphological, connectional, pharmacological or functional features. However, it is difficult to place these data into the proper quantitative perspective due to our lack of knowledge of the number of neurons contained within each auditory nucleus. To this end, we have employed unbiased stereological methods to estimate neuron number in the cochlear nuclei, superior olivary complex, lateral lemniscus, inferior colliculus and medial geniculate body. Additionally, we generated a three-dimensional model of the superior olivary complex. The utility of unbiased stereological estimates of auditory nuclei is discussed in the context of various experimental paradigms.

Animals↗
PubMedDate not supplied

Treatment of endothelial cells with serum from women with preeclampsia: effect on neutrophil adhesion.

OBJECTIVE: The purpose of our study was to determine whether the previously reported neutrophil activation that occurs in the maternal circulation of women with preeclampsia is due to a factor (or factors) in serum that increases neutrophil adhesion to endothelial cells. METHODS: The extent of neutrophil adhesion to endothelial cells incubated with serum from women with preeclampsia (n = 12) was compared with serum from normal, pregnant women matched for maternal age and gestational age at blood sampling (n = 12). Preeclampsia was defined as persistent diastolic blood pressure above 90 mmHg, with proteinuria greater than 0.3 g per 24 hours, in patients who were normotensive before 20 weeks' gestation. The ability of serum (with and without heat inactivation of the complement system) from both groups of patients to stimulate neutrophil adhesion to endothelial monolayers was tested in a 15-minute quantitative assay using fluorescence-labeled neutrophils. The extent of neutrophil adhesion was quantified indirectly from fluorescence counts. RESULTS: No significant differences were found regarding neutrophil-endothelial cell adhesion in response to media alone, serum from women with preeclampsia, and serum from normal, pregnant women. This was also the case when the serum was heat inactivated to destroy the complement system. However, heat inactivated serum produced a significantly greater extent of adhesion compared with serum containing an intact complement system, regardless of whether the patient had preeclampsia. CONCLUSION: This study found no evidence of a factor in serum from women with preeclampsia that could alter neutrophil-endothelial cell adhesion via a direct effect on the endothelium. However, our data suggest that adhesion may be regulated in an inhibitory manner by the complement system.

Adult↗
PubMed2006

Sepsis plasma protein profiling with immunodepletion, three-dimensional liquid chromatography tandem mass spectrometry, and spectrum counting.

Sepsis is a systemic, often fatal inflammatory response whose biochemical pathways are not fully understood and with no single biomarker capable of its reliable prediction. Increased interest in protein profiling to reveal fundamental biochemical events as well as disease diagnosis has grown considerably, largely due to advances in mass spectrometry and related front-end technologies. In this study, patients with sepsis and systemic inflammatory response syndrome (SIRS) were examined using plasma protein profiling following immunodepletion treatment to remove the most abundant proteins, serum albumin, transferrin, haptoglobin, anti-trypsin, IgG, and IgA. These proteins cause significant signal suppression, and their removal allows for lower abundance proteins to be examined through improved ion signal. Analyses after immunodepletion were performed using 3-dimensional reverse phase/strong cation exchange/reverse phase liquid chromatography with electrospray ion trap mass spectrometry (3D LC-MS/MS) and spectrum counting for comparative quantitation. The results revealed a major theme in immune system activity, including activation of the complement and coagulation pathways. Additionally, lipid transport may prove to be important in distinguishing sepsis from SIRS. Specifically, significant multi-fold changes were observed in 10 proteins and are now being investigated for the early diagnosis of sepsis.

Biomarkers↗
PubMed1985

Thyrotrophin receptors, tumour radioiodine concentration and thyroglobulin secretion in differentiated thyroid cancers.

Tumour radioiodine concentration has been compared with serum thyroglobulin (Tg) and, in a few cases, with tumour complement of thyrotrophin receptors in patients with differentiated thyroid carcinoma. All tumours examined possessed TSH receptors. In most the complement was similar to that of normal thyroid tissue although all but one of the tumours had no detectable 131I concentration in vivo even with excess TSH stimulation. Elevated serum Tg (patient taking T4 in suppressive dose) was generally associated with tumours which had 131I concentrating function when stimulated by excess TSH. Some patients, however, had high serum Tg concentration but only low or indetectable tumour 131I uptake. We conclude that (a) measurement of tumour TSH receptor complement is unlikely to be useful in clinical management as tumours which do not significantly concentrate 131I in vivo may have a normal TSH receptor complement and (b) the capacity to secrete Tg is usually associated with 131I concentration but quantitatively the relationship varies considerably between tumours.

Adult↗
PubMed2003

Dopa decarboxylase (Ddc) affects variation in Drosophila longevity.

Mutational analyses in model organisms have shown that genes affecting metabolism and stress resistance regulate life span, but the genes responsible for variation in longevity in natural populations are largely unidentified. Previously, we mapped quantitative trait loci (QTLs) affecting variation in longevity between two Drosophila melanogaster strains. Here, we show that the longevity QTL in the 36E;38B cytogenetic interval on chromosome 2 contains multiple closely linked QTLs, including the Dopa decarboxylase (Ddc) locus. Complementation tests to mutations show that Ddc is a positional candidate gene for life span in these strains. Linkage disequilibrium (LD) mapping in a sample of 173 alleles from a single population shows that three common molecular polymorphisms in Ddc account for 15.5% of the genetic contribution to variance in life span from chromosome 2. The polymorphisms are in strong LD, and the effects of the haplotypes on longevity suggest that the polymorphisms are maintained by balancing selection. DDC catalyzes the final step in the synthesis of the neurotransmitters, dopamine and serotonin. Thus, these data implicate variation in the synthesis of bioamines as a factor contributing to natural variation in individual life span.

Animals↗
PubMed2002

Variant XRCC3 implicated in cancer is functional in homology-directed repair of double-strand breaks.

Polymorphisms in DNA repair genes, including double-strand break (DSB) repair genes, are postulated to confer increased cancer risk. A variant of the XRCC3 gene, which is involved in DSB repair, has been associated with increased risk of malignant skin melanoma and bladder cancer. We tested the hypothesis that this variant, Thr241Met, may affect cancer risk by disrupting a critical function of XRCC3, i.e., promoting homology-directed repair (HDR) of chromosomal DSBs. Using a quantitative fluorescence assay, we find that the variant XRCC3 protein is functionally active for HDR, complementing the HDR defects of an XRCC3 mutant cell line as well as the wild-type protein. We also examined cells expressing this variant for sensitivity to the interstrand cross-linking agent, mitomycin C (MMC), as HDR mutant cell lines, including the XRCC3 mutant, have been found to be hypersensitive to this DNA damaging agent. Cells expressing the variant protein were found to be no more sensitive than cells expressing the wild-type protein. These results suggest that the increased cancer risk associated with this variant may not be due to an intrinsic HDR defect.

Amino Acid Sequence↗
PubMed1990

Binding of monoclonal antibodies and T cell effector function in vivo.

The capacity of adoptively transferred CD8+ effector T cells to induce meningitis in immunosuppressed, or unsuppressed, recipients infected with lymphocytic choriomeningitis virus (LCMV) may be diminished by prior incubation of the lymphocytes with IgM monoclonal antibodies (MAbs) specific for CD8 or Thy1.2. The same is true, though to a lesser extent, for the further proliferation of donor T cells in the spleens of the immunosuppressed mice. This inhibition of cell mediated immunity can be overcome, at least for the unsuppressed recipients, by increasing the numbers of cells that are transferred, even though exposure to Mab+ complement abrogates all cytotoxic T cell activity in vitro. The LCM model thus provides a quantitative system for assessing the consequences of MAb binding for T cell trafficking and effector function in vivo.

Animals↗
PubMed2006

Dyspnea and asthma.

PURPOSE OF REVIEW: Dyspnea--the perception of respiratory discomfort--is a primary symptom of asthma. This review examines possible ways to link mechanisms, measurement and treatment that will increase our understanding of this condition. RECENT FINDINGS: Functional neuroimaging methods have proven to be powerful tools that serve as advanced models of sensor motor brain function. Studies examining functional neuroimaging methods have revealed activation of distinct brain areas associated with increased dyspnea. Pulmonary hyperinflation has been proposed to influence the perception of dyspnea. The association of hyperinflation with minor levels of bronchoconstriction reflects the partition of the sensory effect of airway narrowing per se from that of the attendant elastic loading of the inspiratory muscles. There is evidence to suggest, however, that hyperinflation does not play an important role in the pathogenesis of exercise dyspnea as it does during induced bronchoconstriction. Decreased levels of perception of airway obstruction may be a risk factor associated with life-threatening asthma. A poor perceiver may be vulnerable to further hypoxia-induced suppression of respiratory sensation. Monitoring the response to bronchodilator therapy with formoterol and salbutamol in patients with acute or chronic asthma has resulted in significantly faster improvement in dyspnea, within 2 min. SUMMARY: Regardless of the factors involved, much variability in dyspnea scores remains unexplained. Quantitative and qualitative assessment of the perception of dyspnea, symptom measurement and quality of life complement physiological measurements and contribute to our understanding of dyspnea in asthma.

Airway Resistance↗
PubMed1976

Cell mediated lympholysis in man: an attempt to type with cytotoxic lymphocytes.

From approximately 3,000 CML combinations, originally established in order to evaluate the qualitative and quantitative influence of the serologically defined HLA-A, B, and C antigens on cellular, complement independent cytolysis, 12 combinations were selected yielding reproducible positive cytolysis on allogenic target cells, although no HLA-antigenic sharing could be demonstrated between stimulator and target lymphocytes. These 12 CytoToxic Lymphocytes (CTL's) have been tested in parallell as "CML typing combinations" against lymphocytes from a random population sample of 100 unrelated Danes. Based on a pairwise analysis 11 of these CTL's could be classified into two groups of significantly correlated CTL's. These two groups do not define monospecific traits of allelic genetic origin as judged by a mutually positive correlation and a poor fit to Hardy-Weinberg equilibrium. The traits defined by these groups may be either partially identical or governed by closely linked loci. The same groups were identified and the same conclusions reached after exclusion of those individuals in the population sample where HLA-A, B, C, or D antigens may be targets for destruction. Thus, this study gives direct evidence that known HLA antigens are not sole target determinants in CML or that cytotoxic lymphocytes recognize HLA molecules in a different way than lymphocytotoxic antibodies. The studies underline the immunogenetic complexity of CML although this reaction is most probably governed by genes in the HLA region. It is suggested that cytotoxic lymphocytes may recognize "backbone structures" of the HLA molecules.

Cytotoxicity Tests, Immunologic↗
PubMed1997

Prevalence and biological effects of anti-trophoblast and anti-endothelial cell antibodies in patients with recurrent spontaneous abortions.

PROBLEM: Trophoblasts and endothelial cells represent a potential target for antibodies in women with recurrent spontaneous abortions. These antibodies have been shown to be associated with anti-phospholipid antibodies. Are they also present in women with unexplained pregnancy losses in the absence of anti-phospholipid antibodies? METHOD OF STUDY: The anti-trophoblast antibodies were tested by an immunofluorescence assay on cells purified from pooled first-trimester placentae, whereas the anti-endothelial cell antibodies were measured by enzyme-linked immunoadsorbent assay (ELISA) on cells isolated from the umbilical vein and were cultured to confluence. The cytotoxicity of trophoblasts was evaluated in a homologous system. The expression of adhesion molecules on endothelial cells was quantitated by ELISA using specific monoclonal antibodies, and the expression of tissue factor was quantitated by a chromogenic assay measuring the formation of factor Xa. RESULTS AND CONCLUSIONS: Complement-fixing antibodies to trophoblast represent a better marker to discriminate patients with recurrent spontaneous abortions from controls and are cytotoxic for the target cells. Anti-endothelial antibodies are also present in these patients and exhibit pro-inflammatory and pro-coagulant activities.

Abortion, Habitual↗