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PubMed1997

The quantitative analysis of multiple mRNA species using oligonucleotide probes in an S1 nuclease protection assay.

The quantitative measurement of steady-state mRNA levels is fundamental to the analysis of gene expression. A variety of techniques are widely used to achieve this including Northern blotting, RNase protection, and S1 nuclease protection. We describe here in detail a relatively recent extension of the S1 nuclease protection technique (1) in which radiolabeled oligonucleotides are used as probes in a solution hybridization assay (2). The principle advantage of this technique is that it allows, in a single RNA sample, the simultaneous measurement of the relative levels of at least six mRNA species, including that of a control mRNA. Further, a large number of RNA samples can be analyzed at one time.

Nucleic Acid Hybridization↗
PubMed1995

Quantitative analysis of the plain radiographic appearance of eosinophilic granuloma.

RATIONALE AND OBJECTIVES: To quantitate features that distinguish the plain radiographic appearance of eosinophilic granuloma (EG) from other solitary lesions of bone. MATERIALS AND METHODS: Seven hundred nine focal bone lesions, including 26 EGs, were analyzed according to demographic, anatomic, and plain radiographic features. Vector analysis of groups of features was performed to determine those that are most sensitive and specific for the appearance of EG in contrast to other lesions in the data base. RESULTS: The radiographic appearance of EGs was most consistently that of a medullary based lytic lesion (100%) with geographic destruction (76.9%), lobular contour (73.1%), and well-defined edges (73.1%). Long bone lesions showed these features more frequently than EGs in other locations. Periosteal reaction was visible in all long bone lesions but in only one nonlong bone lesion (5.9%). Characterization of EG in long bones as a lytic, medullary-based metaphyseal or diaphyseal lesion with geographic destruction, lobular contours, periosteal reaction, no matrix, and no subarticular extension showed a sensitivity of 55.6% of EG and prevalence of 22.7%. The vector analysis-generated differential diagnoses include unicameral bone cyst, aneurysmal bone cyst, giant cell tumor, Brodie's abscess, enchondroma, chondrosarcoma, and malignant fibrous histiocytoma. CONCLUSIONS: The radiographic appearance of EG is relatively nonspecific but, using vector analysis, can be better elucidated than in current textbook descriptions.

Adolescent↗
PubMed1997

Quantitative analysis of dopamine receptor messages in the mouse cochlea.

Dopamine receptor isoforms were examined in the cochlea of the CBA(J) mouse by RT-PCR analysis and nucleotide sequencing, utilizing primers specific for known dopamine receptor isoforms. Cochlear cDNA sequences corresponding to dopamine D2(long) and D3 receptors were amplified, whereas those representing D1A, D1B, D2(short), and D4 were not detected. Utilizing quantitative competitive PCR analysis, relative levels of dopamine receptor transcripts were found to be 0.002, 0.014, 0.016, and 1.000 for D2(long) cochlea, D3 cochlea, D3 brain, and D2(long) brain, respectively. In the context of previously published findings, the current work provides key quantitative evidence necessary to establish that dopamine is a neurotransmitter in the auditory inner ear.

Animals↗
PubMed1995

Quantitative analysis of the plain radiographic appearance of nonossifying fibroma.

RATIONAL AND OBJECTIVES: To quantitate radiographic features that distinguish the plain radiographic appearance of nonossifying fibroma (NOF) from other solitary lesions of bone. MATERIALS AND METHODS: Seven hundred nine cases of focal bone lesions, including 34 NOFs, were analyzed according to demographic, anatomic, and plain radiographic features. Vector analysis of groups of features was performed to determine those that are most sensitive and specific for the appearance of NOF in contrast to other lesions in the data base. RESULTS: The radiographic appearance of NOFs was most consistently a medullary based (97%), lytic lesion (100%) with geographic bone destruction (100%), marginal sclerosis (97%), and well-defined edges (94%). A statistically significant number of lesions were located in the distal aspect of long bones. Unicameral bone cyst shared the most radiographic features with the NOF. Vector analysis showed a large degree of overlap between NOF and other lesions such as aneurysmal bone cyst, chondromyxoid fibroma, and eosinophilic granuloma. The description that optimized sensitivity and prevalence for detection of NOF is a medullary based, ovoid lesion in the distal or proximal portions of a long bone with well-defined edges, a partial or complete rind of sclerosis, and absence of fallen fragment, periosteal reaction, and cortical disruption. CONCLUSION: The radiographic appearance of NOF is relatively nonspecific but, using vector analysis, can be better elucidated over current textbook descriptions.

Adolescent↗
PubMed1989

HPLC quantitative analysis of plantaginin in Shazenso (Plantago asiatica L.) extracts and isolation of plantamajoside.

Quantitative analytical method by means of high-performance liquid chromatography (HPLC) has been developed for the quality control of Shazenso (Plantago asiatica L.) extracts. Plantaginin (I) which is one of the known constituents of Shazenso, was selected as a standard compound of the analysis. A chemically labile component has been isolated from the methanol extract and identified to be plantamajoside (II). It was found that II was converted into III even in hot water.

Chromatography, High Pressure Liquid↗
PubMed1992

[Quantitative analysis of electroencephalographic (EEG) activity during sevoflurane anesthesia].

The change in electroencephalographic (EEG) activity during sevoflurane anesthesia was quantitatively evaluated by using zero-crossing analysis in 10 adult patients. The deceleration of EEG activity was significant and dose-dependent. Such change was not significantly different among the regions. Burst suppression appeared at 2-2.5 MAC in all patients. The significant decrease in both alpha and beta activities and the significant increase in delta activity disappeared with 1% or less of sevoflurane. Toward the emergence from anesthesia, EEG activity accelerated and there was no significant difference in its activity between the emergence period and control period. The results demonstrated that the effect of sevoflurane on EEG activity is similar to that of other inhalation anesthetics, but the difference among anesthetics should be clarified in a further study.

Adult↗
PubMed1998

Quantitative analysis of lipid deposits from Schnyder's corneal dystrophy.

AIM: To report the quantitation of the lipid composition of a corneal button from a Japanese woman in her 60s with clinically and histopathologically proved Schnyder's corneal dystrophy. METHODS: Total lipids extracted from the corneal button of the patient were analysed by the method of thin layer chromatography flame ionisation detection. Two different solvent systems were used for neutral lipid analysis and phospholipid analysis. Results were compared with three age matched corneal buttons obtained from cadaveric eyes. RESULTS: The lipids that accumulated in the cornea in Schnyder's dystrophy consisted mainly of unesterified cholesterol and phospholipids. The analysis of phospholipids showed sphingomyelin to be the predominant phospholipid in the patient's cornea. CONCLUSION: Findings suggest that this disorder involves a disturbance of the metabolism of cholesterol and/or sphingomyelin metabolism that is limited to the cornea.

Cholesterol↗
PubMedDate not supplied

Quantitative analysis of human heart valves: does anorexigen exposure produce a distinctive morphological lesion?

The need for more detail regarding the clinical and morphological features of human heart valves has become evident due to recent controversy regarding anorexigen-associated valvular dysfunction. In the present study, we used quantitative digital image analysis of geometric and compositional features to compare the histopathology of cardiac valves excised from patients treated with anorexigens as compared to normal, floppy, rheumatic and carcinoid valves. Anorexigen-exposed valves had the greatest number of onlays/valve (P<.0001), while rheumatic valves showed the greatest average onlay size and thickness of the comparison groups studied (P=.01). The valve onlays from anorexigen-exposed, carcinoid and floppy valves contained a greater percentage of glycosaminoglycans (GAGs) as compared to normal and rheumatic valves (P=.01). The anorexigen-exposed valve propers contained more GAGs than any other comparison group (P=.02). Vessels were prominent in both onlay and valve proper regions of carcinoid valves, in the anorexigen-exposed valve onlays and in rheumatic valve propers. Thus, the number of onlays, their size, the degree of GAG deposition, and the presence and location of vessels and leukocytes were important features distinguishing anorexigen-exposed valves from normal valves. Discriminant analyses, based on geometry, color composition or color composition, and vessel and leukocyte counts combined, were able to separate the valves into distinguishable groups. Our findings demonstrate that specific microscopic features can be used to separate anorexigen-associated heart valve lesions from normal valves and valve lesions associated with other pathologies, and suggest that a distinctive pathological process may exist in many anorexigen-exposed valves.

Adolescent↗
PubMedDate not supplied

Qualitative and quantitative analysis of hydrochlorothiazide in equine plasma and urine by high-performance liquid chromatography.

A sensitive, quantitative method has been developed for the determination of hydrochlorothiazide in equine plasma and urine. Thin-layer chromatography is used to screen for the presence of the drug in unknown samples. The TLC screening methods described provide minimum detection limits of 50 ng/mL in plasma and 25 ng/mL in urine. A silica micro chromatography column is used to clean up ethyl acetate extracts for HPLC analysis and mass spectral confirmation. An internal standard, trichloromethiazide, is used to derive quantitative data at concentrations as low as 25 ng/mL for plasma disappearance curves and urinary excretion rates.

Animals↗
PubMed2004

Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra.

To take advantage of the potential quantitative benefits offered by tandem mass spectrometry, we have modified the method in which tandem mass spectrum data are acquired in 'shotgun' proteomic analyses. The proposed method is not data dependent and is based on the sequential isolation and fragmentation of precursor windows (of 10 m/z) within the ion trap until a desired mass range has been covered. We compared the quantitative figures of merit for this method to those for existing strategies by performing an analysis of the soluble fraction of whole-cell lysates from yeast metabolically labeled in vivo with (15)N. To automate this analysis, we modified software (RelEx) previously written in the Yates lab to generate chromatograms directly from tandem mass spectra. These chromatograms showed improvements in signal-to-noise ratio of approximately three- to fivefold over corresponding chromatograms generated from mass spectrometry scans. In addition, to demonstrate the utility of the data-independent acquisition strategy coupled with chromatogram reconstruction from tandem mass spectra, we measured protein expression levels in two developmental stages of Caenorhabditis elegans.

Algorithms↗
PubMed1978

Quantitative analysis of the amino-terminal residues of spectrin by use of the transamination reaction.

The metal ion-catalysed transamination reaction has been examined as a means of quantitative amino-terminal analysis of proteins. Application of this method to the erythrocyte membrane protein, spectrin, showed that this protein contained a single amino-terminal residue per 240,000 daltons. This value supports the hypothesis that spectrin is comprised of two polypeptide chains of approx. 220,000 and 250,000 daltons, respectively.

Amination↗
PubMed2001

Quantitative analysis of the stabilization by substrate of Staphylococcus aureus PC1 beta-lactamase.

BACKGROUND: The stabilization of enzymes in the presence of substrates has been recognized for a long time. Quantitative information regarding this phenomenon is, however, rather scarce since the enzyme destroys the potential stabilizing agent during the course of the experiments. In this work, enzyme unfolding was followed by monitoring the progressive decrease of the rate of substrate utilization by the Staphylococcus aureus PC1 beta-lactamase, at temperatures above the melting point of the enzyme. RESULTS: Enzyme inactivation was directly followed by spectrophotometric measurements. In the presence of substrate concentrations above the K(m) values, significant stabilization was observed with all tested compounds. A combination of unfolding kinetic measurements and enzymatic studies, both under steady-state and non-steady-state regimes, allowed most of the parameters characteristic of the two concurrent phenomena (i.e. substrate hydrolysis and enzyme denaturation) to be evaluated. In addition, molecular modelling studies show a good correlation between the extent of stabilization, and the magnitude of the energies of interaction with the enzyme. CONCLUSIONS: Our analysis indicates that the enzyme is substantially stabilized towards heat-induced denaturation, independently of the relative proportions of non-covalent Henri-Michaelis complex (ES) and acyl-enzyme adduct (ES*). Thus, for those substrates with which the two catalytic intermediates are expected to be significantly populated, both species (ES and ES*) appear to be similarly stabilized. This analysis contributes a new quantitative approach to the problem.

Acylation↗
PubMed2006

A new method of competitive reverse transcription polymerase chain reaction with SYBR Gold staining for quantitative analysis of mRNA.

There are several methods available to analyze the mRNA concentration quantitatively. Among them, the competitive reverse transcription (RT-)PCR method is very useful. For this method, Cy5-labeled primers were used, and after gel electrophoresis in 7 M urea, the Cy5-labeled single-strand DNA was measured by a fluorescence detector. However, as the equipment to measure the Cy5-labeled fluorescence is expensive, we developed a new method using SYBR Gold staining. After gel electrophoresis in 7 M urea, the single-strand PCR product DNA was stained with SYBR Gold, and photographed with a standard UV-transilluminator and a standard digital camera with a specific filter for SYBR Gold staining. The photographic image was digitized by an imaging software. We measured beta-actin and plasma glutathione peroxidase (Gpx3) mRNA concentrations of HepG2 cell cultured at 5 and 20% oxygen tension. The Gpx3 expression was increased by hypoxia. The result was equivalent to the data obtained by the real-time PCR analysis.

Actins↗
PubMed2001

[Quantitative analysis of sparfloxacin injection by high performance liquid chromatography].

Analytical method of the quantitative determination of sparfloxacin injection by HPLC is described. The analytical conditions were as follows. A Waters Symmetry C18(5 microns, 150 mm x 3.9 mm i.d.) column was used as the analytical column. The detection wavelength was UV-298.8 nm. The column temperature was 30 degrees C. The mobile phase was 0.2% KH2PO4 buffer (pH 3.2)-CH3CN-CH3OH (80:15:5, volume ratio) and the flow-rate was 1.0 mL/min. The injection volume was 10 microL. The linear range (the peak area vs. the mass concentration of sparfloxacin) was from 39.94 mg/L to 199.68 mg/L, and the correlation coefficient was 0.9999. The average recovery of sparfloxacin was 100.1% (n = 5), and its RSD was 0.72%. The RSDs of continuous injections, within day injections per 2 hours and between day injections in three days were 0.19%, 0.14% and 0.13% respectively. The above analytical results show that this method has good precision and stability. It is a rapid, sensitive and accurate method for the analysis of sparfloxacin.

Antitubercular Agents↗
PubMed1992

Quantitative analysis of immunoglobulin G subclass responses to Pseudomonas aeruginosa antigens in cystic fibrosis.

The four subclasses of IgG have different structures, functions and implications in the antibody response. IgG subclass reactions to individual Pseudomonas aeruginosa structural antigens in 22 adolescents and young adults with cystic fibrosis (CF) were studied qualitatively and quantitatively by densitometric analysis of Western blot assays. These patients had been infected by P. aeruginosa for 7 years or longer and were divided into two groups according to their pulmonary status: Group 1 comprised 11 patients with relatively good pulmonary status; Group 2 consisted of 11 patients with poor pulmonary status. There was a relative decrease of IgG1 and a relative increase of IgG2 and, especially, of IgG3 and IgG4 antibodies against P. aeruginosa antigens in the CF patients. Comparison of the two CF patient groups showed a significant increase in the proportion of IgG3 in the Group 2 patients. This could be a potential cause or effect in the deterioration of their pulmonary function. Densitometric analysis of Western blots revealed more than 24 P. aeruginosa antigens and indicated those that were the targets of the isotype antibody response(s) that were apparently most harmful. Thus, there was a significant increase of IgG2 or IgG3 reactivity (or of both) against proteins F, H (H1 and H2), and I in the Group 2 patients. One other striking observation of this study was the high reactivity of IgG4 antibodies to protein H. IgG4 was the major antibody to this protein in seven of the 11 Group 1 patients compared to two of the 11 in Group 2. We hypothesise that IgG4 antibodies may antagonise IgG2 antibodies, helping to preserve stable pulmonary function.

Adolescent↗
PubMed1983

A quantitative analysis of cell surface glycosphingolipid with a fluorescence activated cell sorter.

The fluorescence activated cell sorter (FACS) was used with an indirect membrane immunofluorescence technique to detect antibody against the Forssman antigen, a glycosphingolipid. Sheep erythrocytes, which contain Forssman antigen as a major membrane glycosphingolipid, were used as the target antigen. Detection of the anti-Forssman antibody on the sheep erythrocytes was done with specific fluorescein-conjugated second antibody and analyzed on a FACS. Compared to other available methods, analysis with the FACS was simple, sensitive, reproducible and quantitative. More than 250 pg of antibody could be detected. In addition, as little as 1 ng of Forssman antigen could be estimated by a binding inhibition experiment.

Animals↗
PubMed2004

The use of targeted microbeads for quantitative analysis of the phagocytic properties of human monocyte-derived dendritic cells.

The relationship between phagocytic capacity and morphology of dendritic cells (DCs) has not been investigated previously. Therefore, in order to approach this question, we have developed a novel assay, which is described here. The model of dendritic cells (DCs) used was based upon cytokine-induced differentiation of peripheral blood mononuclear cells, followed by culture on a fibronectin substratum. Under these conditions, standard current methods of quantifying phagocytosis are not applicable, as they rely upon flow cytometric analysis of fluid phase cells; and for adherent cells, quantitative efficiency of uptake is very difficult to measure. Furthermore, for both fluid phase and adherent cells, it is difficult to discriminate between internal and externally bound probe, and degradation of internalised probes can lead to artefacts. Therefore, in this study, these technical issues have been overcome by a simple and flexible assay. Phycoerythrin (PE)-conjugated antibodies are used to target microbeads to the DCs. Following an appropriate incubation period, secondary staining with fluorescein isothiocyanate (FITC)-conjugated antibody allows discrimination between internal and externally bound beads. Microscopic visualisation allows individual beads to be studied easily and thus phagocytosis quantified, whilst permitting parallel examination of morphological parameters. In particular, the relationship between bead uptake and the nature and distribution of the dendritic processes can be evaluated.

Antibodies↗
PubMed2006

Quantitative analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography tandem mass spectrometry.

Buprenorphine is an opioid analgesic drug that is used as an alternative to methadone to treat heroin addiction. Established methods for the analysis of buprenorphine and its metabolites in urine such as gas chromatography-mass spectrometry (GC-MS) involve complicated sample extraction procedures. The aim of the present study was to develop a sensitive yet straightforward method for the simultaneous analysis of buprenorphine and norbuprenorphine in urine using liquid chromatography-MS-MS. The method comprised an enzymatic hydrolysis using Patella vulgata b-glucuronidase, followed by centrifugation and direct analysis of the supernatant. The limits of detection and quantitation were < 1 microg/L for buprenorphine and < 1 and 4 microg/L, respectively, for norbuprenorphine. Assay coefficients of variation (CVs) were < 15%, with the exception of concentrations close to the limit of quantitation, where CVs were below 20%. In direct comparison with an established GC-MS protocol, the method showed minimal negative bias (8.7% for buprenorphine and 1.8% for norbuprenorphine) and was less susceptible to sample carryover. The extent of conjugation in unhydrolyzed urine was investigated and found to be highly variable, with proportions of unconjugated buprenorphine and norbuprenorphine of 6.4% [range 0% to 67%; standard deviation (SD) 9.7%] and 34% (range 0% to 100%; SD 23.8%), respectively.

Analgesics, Opioid↗