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At least 757 records · Page 42Linked to original sources
PubMed1997

Clonal X-inactivation analysis of human tumours using the human androgen receptor gene (HUMARA) polymorphism: a non-radioactive and semiquantitative strategy applicable to fresh and archival tissue.

Assessment of clonality of cellular proliferations is important in experimental and clinical cancer research. X-chromosome inactivation studies are widely used to assess clonality, but most assays require relatively large amounts of high molecular weight DNA. Two PCR-based strategies, the phosphoglycerate kinase (PGK) and the human androgen receptor (HUMARA) clonality assays allow studies of small tissue samples. The HUMARA assay was adapted to non-radioactive analysis taking advantage of an automated sequencer providing high resolution of alleles and immediate quantitation. This assay was validated by comparison with X-inactivation patterns obtained by Southern analysis with the probes M27 beta and PGK. Fifteen gastrointestinal carcinomas, 25 benign goiter nodules and normal peripheral leukocytes of 27 individuals (12 who were under 15 years and 15 over 80 years) were analysed. Furthermore, DNA extracted from formalin-fixed paraffin-embedded tissue (FPT) was analysed with the two PCR-based methods and compared with X-inactivation patterns determined by Southern analysis of high molecular weight (HMW) DNA. This modified HUMARA assay is reliable in most patients; as with other clonality assays, constitutive skewing in normal tissue precludes clonal analysis in some individuals. Extremely skewed X-inactivation patterns were found in normal peripheral leukocytes of 7 out of 15 old females (over 80 years) and in 1 of 12 of the young females tested (under 15 years). Comparison of results obtained with HMW and FPT DNA yielded consistent results for the HUMARA assay whereas the PGK PCR assay was much less reliable. The HUMARA assay thus permits studies of selected areas of tissue sections without significant stromal components, allowing correlation of histological and genotype findings in fresh and archival specimens.

Adolescent↗
PubMed1983

Electron microscopic localization of choline acetyl transferase activity in the electric organ of Torpedo marmorata.

The light microscopic method for demonstration of choline acetyltransferase (CAT) activity based on the formation of a lead mercaptide of free SH-acetyl Coenzyme A was adapted for electron microscopy. In samples of electric organ of Torpedo marmorata CAT activity was found to be restricted to synaptic vesicles and cysternae. The precipitate formed was mostly fine grained and distributed more or less evenly throughout the vesicles. Generally, the reaction product seemed not to adhere to the inner side of the vesicle membrane. CAT activity was found only in the presynaptic region of the synapse, neither the synaptic cleft nor the postsynaptic region reacted positively. CAT activity was found also within synaptic vesicles in nerve endings prepared from electric organ. Samples of Torpedo brain reacted positively too. Complete suppression of CAT activity with inhibitors, judged on the basis of lead mercaptide deposited, was rather difficult to achieve. From a group of 10 presumed enzyme inhibitors, only 2 compounds reacted satisfactorily, namely trans-1,2-dihydro-2-imino-4-(1-naphthylvinyl)-1-pyridine-ethanol hydrobromide and 5,5-dithio-bis-(2-nitrobenzoic acid) (3,3'-6). On the whole, the results obtained show the viability of the method used and furthermore it offers also some new insight into the turnover of acetylcholine, since it may be deduced from the results that under certain circumstances acetylcholine may be synthesized in synaptic vesicles.

Animals↗
PubMed1998

Relative renal uptake and transit time measurements using functional factor images and fuzzy regions of interest.

The aim of the study was a quantitative comparison of relative renal uptake and both the whole-kidney and the parenchymal transit time derived from factor analysis of image sequences and provided by standard clinical procedues. In order to extract the stable, well-interpretable factors, factor analysis was performed locally in the problem-specific time and spatial windows and the resulting factor images either evaluated directly as functional images or used as fuzzy regions of interest (ROIs) for the subsequent extraction of time-activity curves from the analysed data. The values of relative renal uptake of the left kidney measured in the functional factor images, which demonstrate the initial accumulation of activity in renal parenchyma (mean 51.0%), did not differ significantly from the values obtained by a standard method (mean 51.5%, r = 0.98, P<0.001). Whole-kidney transit time calculated using fuzzy ROI curves correlated well with the reference values (r = 0.84, P<0.001); however, both its mean value (336.5 s) and the standard deviation (151.5 s) were substantially greater than those of the values provided by a standard procedure (262.8+/-86.9 s). Parenchymal transit time calculated using ROI curves correlated better with the transit time through a wider corticomedullary region rather than through a narrow cortical region, which is decisive in a differential diagnosis of renal disorders. In general, values of transit times provided by factor analysis correlated well with those provided by reference methods but with a shift towards the higher numerical values. This may have been a consequence of a greater extent of the automatically extracted fuzzy ROIs, or of occasionally delayed accumulation in the upper calyces. Results of the study provide quantitative evidence that the factor analysis of dynamic data, even without the introduction of prior physiological information, may yield clinically relevant information. However, some basic requirements, such as sufficiently high sampling frequency and count rate, adaption of the method to a specific clinical task, and proper selection of time and spatial windows for locally performed analysis, have to be fullfilled if the method is to be successfully applied clinically.

Algorithms↗
PubMed1984

Nylon shavings enzyme reactor for batch determination of urea.

The design and performance of an enzyme reactor (enzyme electrode) which features (i) incorporating nylon shavings onto which an enzyme is covalently bonded, (ii) a flat-surface combination pH electrode for proton monitoring, and (iii) a body providing an injection port for sample injection and washing and stirring capabilities is described. The reactor configuration described here offers good diffusional and partition characteristics which result in relatively fast response, good stability, simplicity of operation, low sample and reagent consumption, and adaptability to flow systems. Application to the determination of urea in standards and physiological salt solutions is demonstrated by use of immobilized urease (EC 3.5.1.5).

Chemical Phenomena↗
PubMed1988

Separation and o-phthaldehyde fluorometric determination of 5-hydroxy- and 5-methoxyindoleamines by thin-layer chromatography: selected applications.

Individual 5-hydroxyindoles and 5-methoxyindoles were simultaneously isolated and quantified, after o-phthaldehyde condensation in different concentrations of HCl, followed by separation on silica gel plates. The 5-hydroxyindoleamines and 5-methoxyindoleamines were differentiated by intense orange and blue fluorescences, respectively. Distinct Rf values with new phase composition and optimal HCl concentrations in the condensation reaction were established for each of the nine indoleamines tested. Minimal detectable doses ranged from 0.2 ng for 5-methoxytryptophol to 4 ng for 5-hydroxyindole acetic acid. The above methodology was applied in order to determine platelet serotonin. Preliminary results suggest that this method could be adapted to other selected biological samples.

Amines↗
PubMed1990

Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues.

The enzymatic methods previously described for 2-deoxyglucose (DG) and 2-deoxyglucose 6-phosphate have been refined and adapted to measurements of brain samples ranging from 50 mg wet weight to less than a microgram dry weight. Procedures for preparing such samples for assay are described. Analytical properties of the enzymes employed are given together with means for overcoming their possible short comings. Emphasis is placed on information useful for employing DG to assess rapid changes in glucose metabolism.

Animals↗
PubMed1993

An improved Bradford protein assay for collagen proteins.

A modification of the protein determination method of Bradford adapted for collagen-rich samples is described. The use of Coomassie-based protein determination methods is limited by the great variation in colour yield obtained for different proteins. This is especially important in samples containing significant amounts of collagen where direct application of the methods of Lowry and Bradford results in underestimated values. Addition of small amounts of sodium dodecyl sulphate (SDS) (0.0035%) to the diluted solutions of Coomassie Brilliant Blue G used as dye reagent in the Bradford colorimetric assay caused a 4-fold increase in the colour response of three collagen proteins (Col I, III and IV) and a decrease in absorbance for various non-collagen proteins. The presence of SDS in the reagent did not result in a significant metachromatic shift of the collagen-dye complexes. This simple modification in the preparation of the reagent for the Bradford assay allows similar response curves to be obtained for collagen and non-collagen proteins, making the modified assay of potential use for protein determination in collagen-rich samples such as pancreatic extracts.

Collagen↗
PubMed1984

On the determination of 99Tc in environmental waters.

In this paper we propose a search for 99Tc in flocs from water treatment plants. We show that preconcentration of 99Tc from environmental waters is a very advantageous method because of the high volumes involved. A radiochemical method is adapted in these kinds of samples, and 99Tc is identified in them by a previously published method. A typical value found is 0.56 +/- 0.26 mBq/g, where 1 g dry floc should be related to many liters of water.

Methods↗
PubMed1983

Regional concentrations of melatonin in the rat brain in the light and dark period.

The levels of melatonin in five brain regions, whole brain, pineal and serum samples were studied in rats adapted under a photoperiod of 12 h light and 12 h dark. It was found that the melatonin levels for all the tissues obtained in the dark period were significantly higher than those obtained in the light period. Regional study of melatonin levels in the brain in the light and dark period demonstrated a high level in the hypothalamus, intermediate levels in the mid-brain, cerebellum and pons-medulla and low level in the telencephalon. Our findings indicate that melatonin in the brain is unevenly distributed and that there are diurnal rhythms of melatonin in all the five brain regions studied.

Animals↗
PubMed1996

Fiber capillarization in flight muscle of pigeons native and flying at altitude.

We examined structural characteristics for fiber O2 supply in the highly aerobic flight muscle of 5 pigeons (Columbia livia; body mass 223--317 g) native and actively flying at altitude (La Paz, Bolivia; 3750 m). Whereas deep sites were significantly more aerobic and highly vascularized than superficial in altitude (A) but not sea-level (SL) group, both sites showed a number of similarities between the two groups. The cross-sectional area of aerobic fibers (> or = 90% of fiber number) linearly increased with body mass (r 0.84; P < 0.0005) but was not smaller in A for their body mass, and there was no reduction in the size of glycolytic fibers compared to SL. The relationships between fiber capillarization and the sectional area of aerobic fibers in transverse sections or mitochondrial volume density were not altered in A compared to SL. The results indicate that the factors which determine the relationships between fiber capillarization and ultrastructure in the muscles were not altered by the adaptation to altitude. Differences between samples were related to the relative sectional area of aerobic fibers and mitochondrial volume density, not the chronic exposure to altitude.

Altitude↗
PubMed1992

Labeled proteinase inhibitors: versatile tools for the characterization of serine proteinases in solid-phase assays.

Peptidyl chloromethyl ketones were used for the specific labeling of proteinases by attaching a biotin group to the N-terminal end of the peptide. Such labeled peptide inhibitors allowed the detection and quantitation of proteolytic enzymes immobilized on the plastic surface of a microtiter plate, as well as on nitrocellulose. The validity of these solid-phase assays was demonstrated using subtilisin Carlsberg as a model enzyme and biotinyl-epsilon-aminocaproyl-L-alanyl-L-alanyl-L-propyl-L-phenylal++ + anyl- chloromethyl ketone as a specific reagent. In addition to being usable for the screening of a particular proteinase in a large number of samples, these assays can be adapted for the analysis of specific proteolytic enzyme present in complex mixtures.

Amino Acid Sequence↗
PubMed1996

Hinge estimators of location: robust to asymmetry.

Robust estimators have been developed and tested for symmetric distributions via simulation studies. The primary objective of these robust estimators was to show that these estimators had a higher efficiency than the sample mean over these symmetric distributions. Little attention has been given to how these estimators perform on data that are from asymmetric distributions or from distributions that have inherent anomalies-so called 'messy data'. This study is intended to supplement previous studies by examining the behavior of several robust estimators over asymmetric distributions. The objective is to demonstrate several adaptive 'asymmetric' robust estimators which utilize sample selector statistics to identify the underlying distribution and to demonstrate the efficiency of these adaptive estimators. From a methodology point rather than a theoretical basis, reasonable alternatives should be available. In the asymmetric data distributions faced on a daily basis, estimators that adapt themselves to the data may be formulated and used. We recommend the use of the following algorithm in examining data sets: (a) compute the ancillary statistics-skewness and tail-length to classify the data distribution; (b) analyze each data set using at least one alternative estimator to the usual XM; (c) if the results are similar, report the XM analysis; (d) if the results are dissimilar, report the alternative analysis and the reasons for using the alternative analysis (i.e. t-tests based on a T alpha, HQ1, HQ2, or SK5).

Algorithms↗
PubMed1994

Changes in the number of active sweat glands (palmar sweat index, PSI) during a distressing film.

Changes of the number of active palmar sweat glands (palmar sweat index, PSI) as assessed by the plastic finger-print method were studied in two groups of female students (n = 21 each). In both samples experiments involved an initial adaptation period, several relaxation phases and an activation period (presentation of a movie). The film was shown 10 min earlier in Group 1, for which in turn follow-up was twice as long. Prints for determination of PSI were taken every 2.5 min from the forefinger and ring finger of the left hand, and recordings of SCL, SF (number of spontaneous fluctuations) and HR were made during the corresponding intervals. Both within- and between-groups comparisons showed an increase of PSI during the activation period and a decrease afterwards. Similar effects were observed for SCL, SF and affective and somatic arousal assessed by a state questionnaire. A decrease of PSI and parameters of electrodermal activity during the first measurements indicated an initial reaction to the assessment procedure itself. Both within-subject and between-subjects correlations between PSI from both fingers showed high parallel test reliabilities, while correlations with electrodermal variables indicated a common physiological basis.

Adult↗
PubMed1992

High-performance liquid chromatographic determination of selenocysteine with the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid.

The method described is based on derivatization of selenocysteine with N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid and responds linearly to selenocysteine spiked into plasma. Recovery is insensitive to inter-individual variation or use of serum versus plasma, but is decreased by hemolysis. The derivative is stable for at least three days. The total imprecision of determinations in plasma was 0.8-2.1% (coefficient of variation) over the range of 6-30 microM selenocysteine, with a detection limit of 0.4 microM (3 x S.D.). There was no significant interference from plasma thiols. This appears to be the first report of the selective reaction of free selenocysteine with a fluorescent reagent. This simple method works well in plasma and serum and may be adaptable to other types of samples.

Animals↗
PubMed1992

[Determination of an ELISA test on tissues of grapevine affected by yellow disease].

We report here results concerning the best part of the grapevine to section, the best sampling period and an important adaptation of the extraction media for the diagnosis of a grapevine yellow disease by ELISA. The addition of Triton-X100, or better of Chaps enabled us to get clear results for the sensitive Vitis vinifera scion varieties. Until now, results have not been obtained for the symptomless root stock varieties. However, antigen concentration by molecular filtration may be a way to solve the problem of assaying root stocks.

Detergents↗
PubMed2006

Rapid analysis of organic acids in plant extracts by capillary electrophoresis with indirect UV detection: directed metabolic analyses during metal stress.

A fast, reliable capillary zone electrophoresis (CZE) method with indirect UV detection was optimized and validated to determine the main organic acids contained in plants. Citric, malic, succinic, oxalic, formic, fumaric, acetic acids, and phosphate were quantified. A rapid separation while keeping a good resolution was obtained by optimizing capillary length, separation voltage, electrolyte composition, and pH. Analyses were performed in a 30 cm uncoated fused-silica capillary (length to the detector window) in the co-electroosmotic mode with reversed electroosmotic flow and anodic detection using a -30 kV separation voltage. The pH 9.0 electrolyte contained 3 x 10(-4)mol/L tetradecyltrimethylammonium and 10(-2)mol/L trimellitate. Separation with baseline return was achieved in 100 s. Linearity, detection limits, repeatability, reproducibility, and recoveries were evaluated. Mean precision values of 0.2 and 3.4% for migration times and time-corrected peak areas, respectively, enabled accurate identification and quantification whether in standard solutions or in samples. Such performances were perfectly adapted to high-throughput routine determinations of organic acids in research or industry. Organic acids were assayed in different plant tissues and cells, including sycamore, arabidopsis, buttercup, and pea. Citrate and malate were the most abundant in all plants tested with concentrations reaching 18.9 and 22.3 micromol/g fresh matter, respectively. Cadmium effect on pea leaves metabolism was also assessed.

Carboxylic Acids↗
PubMed2005

Abdominal surgical interventions: local and systemic consequences for the immune system--a prospective study on elective gastrointestinal surgery.

BACKGROUND: Little is known about the local accumulation and function of immune cells in peritoneal fluid after elective surgery of the upper and lower gastrointestinal tract. Our study was designed to investigate whether systemic immune cell response mirrors the local response. We focused on the cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha and on monocytes, natural killer (NK) cells, and T cells that play an important role in eliciting the innate and adaptive immune response. METHODS: Blood samples were taken prospectively from 25 patients 24 h before surgery, as well as 24 h and 48 h afterward. Abdominal drainage fluids were collected intraoperatively 1 h after the abdomen was opened and 24 h and 48 h postoperatively. Apart from the white blood cells, intracellular T-helper-cell (TH1/TH2) cytokine production (interferon-gamma, IL-2, IL-4, IL-13) and HLA-DR on monocytes were measured by four-color flow cytometry, IL-6, and TNF-alpha with the fast immunoluminescence method. RESULTS: Cells of the innate immune system (NK cells, monocytes, NK-T cells, CD5(+) B cells) rapidly decreased in abdominal fluids (P < 0.05: +24 h; +48 h) after surgery, which was paralleled by a concomitant decline in peripheral blood. The percentage of abdominal interferon-gamma, IL-2, IL-4, and IL-13-producing TH cells increased in a way that distinctly counteracted the decrease of the natural immune cells. HLA-DR expression on monocytes in peripheral blood declined significantly (P < 0.05: +24 h; +48 h). In contrast, monocytes in abdominal fluids had high HLA-DR expression. Furthermore, abdominal fluids contained significantly higher concentrations of TNF-alpha (P < 0.05: +24 h; +48 h) and IL-6 (P < 0.05: +24 h) compared with peripheral blood. CONCLUSIONS: Specific immune cell recruitment and cytokine production play an important role in post-trauma events. Measuring distinct local immune cell repertoires and cytokines provides answers as to how the different phases of postoperative immune events proceed. The evaluation of the local response may provide additional criteria for the evaluation of operative trauma. This knowledge may be helpful in detecting postoperative pathological aberrancies.

Aged↗
PubMed2004

Photometric assay for measuring the intracellular concentration of branched-chain amino acids in bacteria.

The changes in intracellular pool of branched-chain amino acids (BCAA) regulate different physiological processes in bacteria. Up to date, the only available photometric test for measuring BCAA concentration was adapted for blood and plasma samples in diagnostic purposes. We have modified this method for use on bacterial cells, and tested its applicability on several model organisms: Lactococcus lactis, Bacillus subtilis and Escherichia coli.

Amino Acids, Branched-Chain↗