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At least 1,243 records · Page 69Linked to original sources
PubMed2000

Localization and quantification of hydrophobicity: the molecular free energy density (MolFESD) concept and its application to sweetness recognition.

A method for the localization, the quantification, and the analysis of hydrophobicity of a molecule or a molecular fragment is presented. It is shown that the free energy of solvation for a molecule or the transfer free energy from one solvent to another can be represented by a surface integral of a scalar quantity, the molecular free energy surface density (MolFESD), over the solvent accessible surface of that molecule. This MolFESD concept is based on a model approach where the solvent molecules are considered to be small in comparison to the solute molecule, and the solvent can be represented by a continuous medium with a given dielectric constant. The transfer energy surface density for a 1-octanol/water system is empirically determined employing a set of atomic increment contributions and distance dependent membership functions measuring the contribution of the increments to the surface value of the MolFESD. The MolFESD concept can be well used for the quantification of the purely hydrophobic contribution to the binding constants of molecule-receptor complexes. This is demonstrated with the sweeteners sucrose and sucralose and various halogen derivatives. Therein the relative sweetness, which is assumed to be proportional to the binding constant, nicely correlates to the surface integral over the positive, hydrophobic part of the MolFESD, indicating that the sweetness receptor can be characterized by a highly flexible hydrophobic pocket instead of a localized binding site.

Models, Molecular↗
PubMed2000

Consideration of heteroduplexes and homoduplexes for the quantification by competitive PCR of human mitochondrial DNA deletions with ageing of tissues and cells.

The purpose of this work was first to construct two internal standards for human mitochondrial DNA mt DNA corresponding respectively to the fragment resulting from the 4,977 bp common deletion (H2del) and a fragment which was never reported to be deleted (H1). Secondly, we wished to consider the possible effect of annealing between the target and corresponding internal standard which forms heteroduplexes. These experiments show that the correction of the number of copies found by competitive PCR by considering the percentage of heteroduplexes allows a more accurate quantification of the number of target copies present in mt DNA samples. The design of internal standards specific to the fragment resulting from other deletions could also help a more accurate quantification of the frequency of other mt DNA deletions as well, and reconsideration of the role of mt DNA deletions in ageing.

Aging↗
PubMed2001

Improved quantification of angiogenesis in the rat aortic ring assay.

In vitro angiogenesis assays are essential for the identification of potential angiogenic agents and screening for pharmacological inhibitors. Among these assays, the rat aortic ring model developed by Nicosia bridges the gap between in vivo and in vitro models. The quantification of angiogenesis on this system must be applicable to characterise vascular networks of various states of complexity. We present here an improved computer-assisted image analysis which allows: (1) the determination of the aortic ring area and its factor shape; (2) the number of microvessels, the total number of branchings, the maximal microvessel length and the microvessel distribution; (3) the total number of isolated fibroblast-like cells and their distribution. We show that this method is suitable to quantify spontaneous angiogenesis as well as to analyse a complex microvascular network induced by various concentrations of vascular endothelial growth factor (VEGF). In addition, by evaluating a new parameter, the fibroblast-like cell distribution, our results show that: (1) during spontaneous angiogenic response, maximal fibroblast-like cell migration delimits microvascular outgrowth; and (2) the known angiogenic inhibitor Batimastat prevents endothelial cell sprouting without completely blocking fibroblast-like cell migration. Finally, this new method of quantification is of great interest to better understand angiogenesis and to test pro- or anti-angiogenic agents.

Animals↗
PubMed2002

Determination of left ventricular mass by three-dimensional echocardiography: in vitro validation of a novel quantification method using multiple equi-angular rotational planes for rapid measurements.

UNLABELLED: Measuring left ventricular mass by m-mode echocardiography or two-dimensional echocardiography is limited by the fact that calculations are based on assumptions, which describe left ventricular shape by simple geometric figures. The ability of three-dimensional echocardiography (3-DE) to accurately assess left ventricular mass has been shown previously, but 3-DE approaches to quantitative analysis of ventricular mass required multiple tomographic sectioning, manual tracing in various cut planes and were time consuming and laborious. We investigated the accuracy of a novel, rapid method of 3-DE mass quantification using multiple rotational planes in left ventricles in vitro. METHODS: Three-dimensional data sets of 10 fixed pig hearts were obtained using a TomTec 3-DE system. For 3-DE mass calculations, a rotational axis in the center of the ventricle (apical-basal orientation) was defined and 3, 6 and 12 equi-angular rotational planes were created. The endocardial and epicardial contour of the left ventricle was traced in each cut plane and the volume of the corresponding myocardial wedge was automatically calculated. Mass was calculated by multiplying the resulting myocardial volume by the specific weight of myocardial tissue. The measurements were performed by two investigators blinded to the anatomic true mass and were analyzed for interobserver and intraobserver variability. RESULTS: The anatomic left ventricular mass was measured 73-219 (168 +/- 50) g. 3-DE mass ranged from 88-247 (207 +/- 51) g (three planes), 84-250 (205 +/- 52) g (six planes) and 86-241 (202 +/- 50) g (12 planes) respectively. The correlation between 3-DE mass and anatomic LV mass measurements (r = 0.92) and between two observers (r = 0.97-0.98) was good. True mass was slightly overestimated by 3-DE measurement (SEE = 22-23 g). The intraobserver and interobserver variabilities were < or = 4 and < or = 7% respectively for all measurements. CONCLUSION: This new 3-DE method of left ventricular mass quantification with rotational approach provides accurate and reproducible measurements. In normal shaped left ventricles even three planes were sufficient to provide accurate mass measurements in vitro.

Animals↗
PubMed2002

Relative quantification of mRNA levels in Jurkat T cells with RT-real time-PCR (RT-rt-PCR): new possibilities for the screening of anti-inflammatory and cytotoxic compounds.

PURPOSE: Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential. METHOD: A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel. RESULTS: Cyclosporin A and capsaicin were potent inhibitors of PMA-induced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin down-regulated nearly all investigated mRNAs, resulting in a strong time-dependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability. CONCLUSIONS: The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real time-polymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.

Anthracenes↗
PubMed2003

Quantitative assessment of natural toxicity in sponges: toxicity bioassay versus compound quantification.

Microtox assay was used to assess the natural toxicity of two sponges, Dysidea avara and Ircinia variabilis. The activity of crude extracts and major metabolites were compared. Methanol extract of D. avara was more toxic than that of acetone and was as toxic as pure avarol, thus suggesting that the toxicity of the sponge was mainly due to this metabolite. We also quantified palinurin, the major metabolite of I. variabilis, in specimens from several habitats. With the same methanol extracts used for palinurin quantification, we ran the Microtox assay and found a positive significant regression between toxicity and concentration of this metabolite. Pure palinurin was tested at the same concentration present in the extract, and the toxicity recorded was higher than that of the methanol extract. As with avarol from D. avara, palinurin is the main secondary metabolite that confers toxicity to I. variabilis. The results confirm that the standardized Microtox assay is an accurate and reproducible tool for assessing the toxicity of crude extracts and pure metabolites of marine organisms. These results also suggest that methanol is more suitable than acetone for the detection of species toxicity by Microtox. The method is faster and easier to perform than chemical quantification even when the sponge chemistry is known, and is appropriate for studies on variation in natural toxicity over a range of environmental conditions.

Animals↗
PubMed2004

Model driven quantification of individual and collective cell migration.

While the control of cell migration by biochemical and biophysical factors is largely documented, a precise quantification of cell migration parameters in different experimental contexts is still questionable. Indeed, these phenomenological parameters can be evaluated from data obtained either at the cell population level or at the individual cell level. However, the range within which both characterizations of cell migration are equivalent remains unclear. We analyse here to which extent both sources of data could be integrated within a unified description of cell migration by considering the motility of the endothelial cell line EAhy926. Using time-lapse video-microscopy and associated analysis of digital image time series, we quantified EAhy926 random motility coefficient, migration speed and trajectory persistence time in two different migration assays: the in vitro wound healing assay, and the cell-populated agarose drop assay. In order to analyse the agreement between independent quantifications of cell motility based either on individual cell analysis or cell population dynamic analysis, a theoretical multi-agents cellular model was developed and discussed as a possible theoretical framework able to unify these multi-scale data. Model simulations especially reveal the potential bias induced by cell proliferation and cell-cell adhesion when cell migration parameters are estimated from the extensively used in vitro wound healing assay.

Cell Line↗
PubMedDate not supplied

Quantification and index of non-point source pollution in Taihu Lake region with GIS.

The contribution of phosphorus and nitrogen from non-point source pollution (NPS) in the Taihu Lake region was investigated through case study and surveying in the town of Xueyan, From experimental results coupled with survey and statistics in the studied area, the distribution of nitrogen and phosphorus input to the water body is achieved from four main sources: agricultural land, village, the town center and the poultry factory. The results showed that about 38% of total phosphorus (TP) and 48% of total nitrogen (TN) discharged is from agricultural land, 33% of TP and 40% TN from village residents, 25% of TP and 10% of TN from the town center and 4% of TP and 2% of TN from the poultry factory. The Agricultural Non-point Pollution Potential Index (APPI) system for identifying and ranking critical areas of NPS was established with a Geographic Information Systems (GIS)-based technology. Quantification of the key factors in non-point sources pollution was carried out utilizing the following: Sediment Production Index (SPI), Runoff Index (RI), People and Animal Loading Index (PALI) and Chemical Use Index (CUI). These are the core parts of the model, and the weighting factor of each index was evaluated according the results of quantification. The model was successfully applied for evaluating APPI in Xueyan. Results from the model showed that the critical area identified for NPS control in Xueyan. The model has several advantages including: requiring fewer parameters, easy acquirement of these parameters, friendly interface, and convenience of operation. In addition it is especially useful for identifying critical areas of NPS when the basic data are not fully accessible, which is the present situation in China.

China↗
PubMed1978

Quantification of three-dimensional structures.

A mathematical model, based on additive subcomponents of grouping, subitizing and adding, was derived to account for quantification latencies of three-dimensional block arrangements. Subitizing is the process that people use to directly quantify a small number of objects without counting. It was found that most people consistently subitized up to four blocks. With more than four blocks, people resorted to grouping and adding, and the model was able to account for these data. The structural variables of compactness, symmetry, linearity, and planarity were shown to have small effects on quantification latencies relative to the large effect of number of blocks. Of these structural variables, compactness had the largest effect, and in terms of the model, it is suggested that visual structure had its effect on the perceptual grouping subcomponent.

Adolescent↗
PubMed1993

Immunohistochemical distribution and quantification of crystal matrix protein.

The aim of this study was to determine the immunohistochemical distribution and quantification of crystal matrix protein (CMP). CMP, a 31 kDa glycoprotein, is the principal macromolecule found in calcium oxalate crystals generated in human urine, and is a potent inhibitor of crystal aggregation. A polyclonal rabbit anti-human CMP antibody was used to examine renal tissue by immunohistochemical techniques and light microscopy (N = 45). Twenty-five other human organs were similarly assessed. Quantification was performed using a visual analogue scale. CMP was visible as cytoplasmic staining in the epithelial cells of the TALH and the distal convoluted tubule including the macula densa in a subgroup of nephrons. CMP was not identified elsewhere in the urinary tract or in the extrarenal organs examined. Despite a trend indicating that the kidneys of normal men had more CMP than those of normal women, the difference failed to reach significance (P = 0.11). There was, however, more CMP in the stone formers group compared with either normal men (P < 0.01) or normal women (P < 0.01). This protein may be an important determinant of calcium oxalate kidney stone disease.

Aged↗
PubMed1996

Fractional direct dialysis quantification: a new approach for prescription and monitoring hemodialysis therapy.

We describe a new methodology, fractional direct dialysis quantification (FDDQ) utilizing the Fresinius Dialysate Sampling Module (DSM), for quantitating total solute removal during hemodialysis (HD). Our data demonstrate that this technique and Direct Dialysis Quantification (DDQ) yield virtually identical results. FDDQ, however, obviates the practical obstacles that have limited the applicability of DDQ. We discuss the theoretical and practical advantages of this methodology, as compared to urea kinetic modeling (UKM) with Kt/V, for prescribing and monitoring dialysis therapy. FDDQ provides reliable and accurate quantitative data of dialysis function and protein catabolic rate (PCR) independent of questionable theoretical assumptions and parameters required for UKM with Kt/V. It is simple to comprehend and apply. It permits easy comparison of standard and rapid high efficiency dialyses. It also facilitates the quantitative comparison of HD and continuous therapies (peritoneal dialysis and various types of continuous hemofiltration). FDDQ permits the use of other solutes, in place of or in addition to urea, for the quantitation of HD. Because of its simplicity and probable low cost, it can be used with each HD session. It will thus provide accurate data on delivered versus prescribed therapy. These features should permit more accurate monitoring and lead to a clearer understanding of the relationship of outcomes versus delivered dialysis dose, and consequently more effective adjustment of dialysis therapy.

Creatinine↗
PubMed2005

Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides.

Absolute quantification in proteomics usually involves simultaneous determination of representative proteolytic peptides and stable isotope-labeled analogs. The principal limitation to widespread implementation of this approach is the availability of standard signature peptides in accurately known amounts. We report the successful design and construction of an artificial gene encoding a concatenation of tryptic peptides (QCAT protein) from several chick (Gallus gallus) skeletal muscle proteins and features for quantification and purification.

Animals↗
PubMed2006

Digital quantification using amplified single-molecule detection.

We describe a scheme for biomolecule enumeration by converting nanometer-scale specific molecular recognition events mediated by rolling-circle amplification to fluorescent micrometer-sized DNA molecules amenable to discrete optical detection. Our amplified single-molecule detection (SMD) approach preserves the discrete nature of the molecular population, allowing multiplex detection and highly precise quantification of molecules over a dynamic range of seven orders of magnitude. We apply the method for sensitive detection and quantification of the bacterial pathogen Vibrio cholerae.

DNA, Bacterial↗
PubMed2006

First international consensus on the methodology of lymphangiogenesis quantification in solid human tumours.

The lymphatic system is the primary pathway of metastasis for most human cancers. Recent research efforts in studying lymphangiogenesis have suggested the existence of a relationship between lymphatic vessel density and patient survival. However, current methodology of lymphangiogenesis quantification is still characterised by high intra- and interobserver variability. For the amount of lymphatic vessels in a tumour to be a clinically useful parameter, a reliable quantification technique needs to be developed. With this consensus report, we therefore would like to initiate discussion on the standardisation of the immunohistochemical method for lymphangiogenesis assessment.

Biomarkers, Tumor↗
PubMed1999

Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection.

Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells.

Amelogenin↗
PubMed2004

Allele-specific quantification of HLA-DQB1 gene expression by real-time reverse transcriptase-polymerase chain reaction.

In addition to coding region polymorphism, allele-specific variation in the upstream regulatory region of the HLA-DQB1 gene has been detected. Reporter gene assays and transfection studies have indicated that HLA-DQB1 promoter polymorphism may be of functional significance. The aim of this study was to utilize real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for allele-specific quantification of HLA-DQB1 expression and to analyze cell-specific HLA-DQB1 expression in vivo. For the allele-specific quantification of DQB1 gene products, a real-time RT-PCR set of primer pairs (n=27) and probes (n=5) targeting exon 2 variability was established. The robustness and integrity of the assay system were confirmed by using recombinant DQB1 exon 2 plasmid clones as active exogenous controls. Sensitivity and reproducibility were assessed by serial dilution and allelic mixing analyses. In application to the study of allele-specific expression of DQB1 gene products during cytokine-driven maturation of monocyte-derived dendritic cells, differential patterns of allelic expression in heterozygous individuals were observed for DQB1*0301, compared to DQB1*0501 and DQB1*0602. At maximum, 1.9-fold (*0301/*0501) and 2.5-fold (*0301/*0602) higher induction was seen for DQB*0301. In conclusion, HLA-DQB1 expression can be analyzed by real-time RT-PCR suitable for cell- and allele-specific detection of HLA-DQB1 transcripts in homo- and heterozygous combinations.

Alleles↗
PubMedDate not supplied

Quantification of Stachybotrys chartarum conidia in indoor dust using real time, fluorescent probe-based detection of PCR products.

Analyses of fungal spores or conidia in indoor dust samples can be useful for determining the contamination status of building interiors and in signaling instances where potentially harmful exposures of building occupants to these organisms may exist. A recently developed method for the quantification of Stachybotrys chartarum conidia, using real-time, fluorescence probe--based detection of PCR products (TaqMan system) was employed to analyze indoor dust samples for this toxigenic fungal species. Dust samples ofup to 10 mg were found to be amenable to DNA extraction and analysis. Quantitative estimates of S. chartarum conidia in composite dust samples, containing a four-log range of these cells, were within 25 -- 104% of the expected quantities in 95% of analyses performed by the method. Calibrator samples containing known numbers of S. chartarum conidia were used as standards for quantification. Conidia of an arbitrarily selected strain of Geotrichum candidum were added in equal numbers to both dust and calibrator samples before DNA extraction. Partial corrections for reductions in overall DNA yields from the dust samples compared to the calibrator samples were obtained by comparative analyses of rDNA sequence yields from these reference conidia in the two types of samples. Dust samples from two contaminated homes were determined to contain greater than 10(3) S. chartarum conidia per milligram in collection areas near the sites of contamination and greater than 10(2) conidia per milligram in several areas removed from these sites in analyses performed by the method. These measurements were within the predicted range of agreement with results obtained by direct microscopic enumeration of presumptive Stachybotrys conidia in the same samples.

Air Pollution, Indoor↗
PubMed1998

Quantification of 17 beta-estradiol residues in bovine serum by liquid chromatography-tandem mass spectrometry with atmospheric pressure chemical ionization.

A method for the quantification of the natural hormone 17 beta-estradiol (17 beta-E2) in bovine serum by liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS-MS) was developed. Ethinylestradiol (EE2) was used as internal standard. Analytes were extracted from serum using acetate buffer, purified by C18 solid-phase extraction (SPE) and chromatographed on a polymeric reversed-phase (PLRP-S) LC column. They were ionized in a heated nebulizer (HN) interface operating in the negative ion mode, where only the intact deprotonated molecules, [M - H]-, were generated at m/z 271 and 295 for 17 beta-E2 and EE2, respectively. These served as precursor ions for collision-induced dissociation (CID) and diagnostic product ions were identified for the unambiguous hormone confirmation by selected reaction monitoring (SRM) LC-APCI-MS-MS. The method was validated on bovine serum and the limit of quantification (LOQ) was 30 pg ml-1 for 17 beta-E2. The inter-day precision (relative standard deviation, RSD) and accuracy (relative error, RE) derived from the analyses of validation samples at three concentrations ranged from 1.76 to 3.76 and from -4.18 to -2.01%, respectively. This method is currently being successfully applied to measure the bovine serum concentration of 17 beta-E2 in order to discriminate between the physiological concentrations of 17 beta/E2 and the hormone levels resulting from illegal administration.

Anabolic Agents↗