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PubMed2004

Quantification of the contribution of surface outgrowth to biocatalysis in sol-gels: oxytetracycline production by Streptomyces rimosus.

A technique was developed for differentiating the activity of microbes solely within sol gels by using the contribution of biomass outgrowth. Streptomyces rimosus was immobilised in colloidal silica gels and biomass growth, oxytetracycline synthesis, pH and carbohydrate consumption were compared for UV surface-sterilised gels, untreated gels, and liquid cultures. Absolute and biomass specific oxytetracycline yields were higher for non-sterile gels than for liquid culture. Biomass solely within colloidal silica gels (1.7 mg ml(-1)), and gels obtained from colloidal silica modified by addition of larger silica particles (1.2 mg ml(-1)) yielded 27 and 21 microg ml(-1) oxytetracycline compared with 97 and 104 microg ml(-1) for unsterilised gels (3.6 and 5.2 mg ml(-1) biomass) displaying outgrowth. It was therefore apparent that biomass and antibiotic production within the gels was limited and that optimisation requires gel modification.

Bioreactors↗

PubMed2003

Cloned plasmid DNA fragments as calibrators for controlling GMOs: different real-time duplex quantitative PCR methods.

Analytical real-time PCR technology is a powerful tool for implementation of the GMO labeling regulations enforced in the EU. The quality of analytical measurement data obtained by quantitative real-time PCR depends on the correct use of calibrator and reference materials (RMs). For GMO methods of analysis, the choice of appropriate RMs is currently under debate. So far, genomic DNA solutions from certified reference materials (CRMs) are most often used as calibrators for GMO quantification by means of real-time PCR. However, due to some intrinsic features of these CRMs, errors may be expected in the estimations of DNA sequence quantities. In this paper, two new real-time PCR methods are presented for Roundup Ready soybean, in which two types of plasmid DNA fragments are used as calibrators. Single-target plasmids (STPs) diluted in a background of genomic DNA were used in the first method. Multiple-target plasmids (MTPs) containing both sequences in one molecule were used as calibrators for the second method. Both methods simultaneously detect a promoter 35S sequence as GMO-specific target and a lectin gene sequence as endogenous reference target in a duplex PCR. For the estimation of relative GMO percentages both "delta C(T)" and "standard curve" approaches are tested. Delta C(T) methods are based on direct comparison of measured C(T) values of both the GMO-specific target and the endogenous target. Standard curve methods measure absolute amounts of target copies or haploid genome equivalents. A duplex delta C(T) method with STP calibrators performed at least as well as a similar method with genomic DNA calibrators from commercial CRMs. Besides this, high quality results were obtained with a standard curve method using MTP calibrators. This paper demonstrates that plasmid DNA molecules containing either one or multiple target sequences form perfect alternative calibrators for GMO quantification and are especially suitable for duplex PCR reactions.

Calibration↗

PubMedDate not supplied

Quantification of leukocyte migration: improvement of a method.

Eighteen different permeable membrane supports with and without confluent endothelial cell monolayers were incubated with normal donor derived neutrophils in the upper chambers of a 24 multiwell double chamber system. In order to study transmembrane or transendothelial leukocyte migration leukocytes were stimulated by chemoattractants, or endothelial cells were activated by IL-1. After coincubation the membrane supports building the upper chambers were discarded. Using this technique, leukocytes that had migrated into the lower chamber were exposed to the fluorescent dye calcein AM without additional washing or transfer steps. Absolute cell counts were determined computer assisted using dilution series of calcein AM labeled leukocytes as standards. Serial dilutions of neutrophils exposed to calcein AM showed reproducible linear fluorescence intensity, and relative fluorescence intensity correlated significant with cell counts (r2 = 0.974, p < 0.0001). Out of 18 membrane supports only one was suitable for our assay set up. Best technical and optical performance was achieved with a membrane made of polyethylene terephtalate with a pore size of 3 mm at a pore density of 0.8 x 10(6)/cm2. Stimulation of leukocytes or endothelium by FMLP or IL-1 revealed an increase of transendothelial migration to 7.2 +/- 1.8 x 10(5) PMN and 5.1 +/- 0.7 x 10(5) PMN respectively if compared with medium (0.6 +/- 0.2 x 10(5) PMN). IL-1 induced migration of neutrophils was inhibited by anti IL-1 autoantibodies derived from chronic renal failure patients (IL-1: 100% of PMN migrated, anti IL-1 antibody: 39% of PMN migrated, control antibody: 84% of PMN migrated). In summary, a simple fluorimetric assay was established for the quantification of transmembrane and transendothelial leukocyte migration.

Autoantibodies↗

PubMed2005

Measurement of cerebrospinal fluid oxygen partial pressure in humans using MRI.

Fluid-attenuated inversion recovery (FLAIR) images obtained during the administration of supplemental oxygen demonstrate a hyperintense signal within the cerebrospinal fluid (CSF) that is likely caused by T1 changes induced by paramagnetic molecular oxygen. Previous studies demonstrated a linear relationship between the longitudinal relaxation rate (R1 = 1/T1) and oxygen content, which permits quantification of the CSF oxygen partial pressure (P(csf)O2). In the current study, CSF T1 was measured at 1.5 T in the lateral ventricles, third ventricle, cortical sulci, and basilar cisterns of eight normal subjects breathing room air or 100% oxygen. Phantom studies performed with artificial CSF enabled absolute P(csf)O2 quantitation. Regional P(csf)O2 differences on room air were observed, from 65 +/- 27 mmHg in the basilar cisterns to 130 +/- 49 mmHg in the third ventricle. During 100% oxygen, P(csf)O2 increases of 155 +/- 45 and 124 +/- 34 mmHg were measured in the basilar cisterns and cortical sulci, respectively, with no change observed in the lateral or third ventricles. P(csf)O2 measurements in humans breathing room air or 100% oxygen using a T1 method are comparable to results from invasive human and animal studies. Similar approaches could be applied to noninvasively monitor oxygenation in many acellular, low-protein body fluids.

Adult↗

PubMed2006

Determination of gabapentin in human plasma using hydrophilic interaction liquid chromatography with tandem mass spectrometry.

A rapid, sensitive and selective method for the determination of gabapentin in human plasma was developed using hydrophilic interaction liquid chromatography/tandem mass spectrometry (HILIC/MS/MS). The devised method involved protein precipitation with acetonitrile followed by separation on an Atlantis HILIC silica column using an acetonitrile/ammonium formate mobile phase (100 mM, pH 3.0) (85:15, v/v). Analytes were detected using an electrospray ionization mass spectrometer in the multiple-reaction monitoring mode. The standard curve was linear (r = 1.000) over the concentration range of 50.0-10000 ng/mL. The lower limit of quantification for gabapentin was 50.0 ng/mL (ca. 20 pg gabapentin) using a 10-microL plasma sample. The coefficients of variation and relative errors for intra- and inter-assay at four QC levels (i.e., 50.0, 125, 750, and 7500 ng/mL) were 4.7 to 9.4% and -4.1 to 1.6%, respectively. Absolute and relative matrix effects for gabapentin and metformin were practically absent. Gabapentin and metformin recoveries were 98.5% and 99.0%, respectively. This method was successfully applied to a bioequivalence study of gabapentin in humans.

Amines↗

PubMed1992

On the influence of spatial resolution and of the size and form of regions of interest on the measurement of regional cerebral metabolic rates by positron emission tomography.

Factors that affect the accuracy of the positron emission tomographic (PET) quantification of cerebral metabolic rates include the spatial resolution of the employed imaging device and the method used for extraction of regional metabolic values from the PET data set. The present article reviews (i) how and to what extent these two factors are presumed to influence the measurement of absolute values of cerebral metabolic rates and their ratios, and (ii) whether and how these factors may affect comparisons of regional metabolic rates between groups of subjects.

Brain↗

PubMed1997

[Hibernating myocardium and the 'no reflow' phenomenon: a study of absolute regional myocardial perfusion and glucose metabolism using positron emission tomography in chronic and acute heart disorders].

Positron emission tomography (PET) is a powerful tool for in vivo measurements of physiologic processes such as regional myocardial blood flow and metabolism. Myocardial blood flow is often studied using radioactive labeled ammonia (13NH3) while myocardial metabolism can be investigated using 18F-fluorodeoxyglucose (FDG). Moreover, the use of appropriate kinetic models allows quantification of these processes. In this study, myocardial viability in both chronic and acute heart disease was investigated by the use of positron emission tomography. In this context, viable refers to dysfunctioning areas of the myocardium in which functional recovery is observed after revascularization. In patients suffering chronic coronary artery disease, PET findings of flow and metabolism were correlated with myocardial ultrastructure. In dysfunctional myocardial segments, normal 13NH3 uptake or decreased 13NH3 uptake with relatively increased FDG uptake (PET mismatch) indicates the possibility for functional recovery after bypass surgery. Since absence of scar tissue in these segments is likely to be required for functional recovery, it was not surprising that little fibrosis was found in myocardial biopsies taken in PET mismatch areas. The biopsies also revealed the presence of viable myocardial cells showing a variable loss of contractile material. The contractile material was replaced by glycogen. One could wonder about the time course needed for functional recovery after restoration of blood flow in the presence of a considerable amount of cells lacking a normal contractile apparatus. It would therefore be interesting to study functional recovery at different time points in patients with variable amounts of these myolytic cells. Probably, recovery of contractility would be slower in myocardial areas with a larger amount of abnormal cells. Another question that arises is the meaning of the increased FDG signal in dysfunctional, though viable myocardium. At first sight, glycogen storage in myolytic cells seems an excellent candidate to explain the increased intake of FDG in PET mismatch areas. However, in this study, in areas considered nonviable by PET, similar amounts of myolytic cells were found. Histologically altered cells might represent a structural and protective adaptation to long term hypoperfusion or to repetitive episodes of ischemia. Another possibility for the increased FDG uptake is an enhancement of glucose utilization in the mismatch areas not only in the myolytic cells, but also in the morphologically normal cell fractions. In patients with a PET mismatch pattern, significant recovery of flow and function was observed after surgery with a significant decrease in glucose utilization. Although it would have been interesting to histologically study the fate of myolytic cells in these recovered areas, this was not possible for obvious ethical reasons. In areas considered non viable by PET expressing a concordant decrease of 13NH3 and FDG uptake (PET match), no recovery of function, flow or metabolism was noted at follow-up. Another study was conducted in our department in infarct patients in which regional myocardial blood was measured within 24 hours after successful thrombolysis. The aim was to investigate the presence of impaired tissue perfusion in the acute stage and to evaluate its effect on recovery of flow, metabolism and function. In about 30% of patients with a TIMI 3 patent vessel, seriously impaired tissue flow was observed in the acute stage. Whether this impairment was due to irreversible damage to capillaries or myocytes, to reperfusion injury or to the presence of multiple distal thrombi remains unknown. Most patients showing severely impaired regional myocardial blood flow in the acute stage revealed absence of viable myocardium on follow-up PET NH3/FDG scans.

Acute Disease↗

PubMed1999

A flow cytometric procedure for the quantification of cell adhesion in complex mixtures of cells.

We present a simple non-radioactive cytometry-based assay that permits the simultaneous quantitation of cell adhesion of distinct subsets of cells contained in a mixture without any previous fractionation. The procedure is simple and highly reproducible and has the advantage of confining the quantitation of cell adhesion to live cells only. This new approach is based on counting the absolute number of cells. This is done by adding known numbers of distinguishable beads to the cell suspension and counting beads and cells in a cytometer. Quantitation of adhesion is accomplished by counting each subpopulation of cells before and after the adhesive process. To illustrate this methodology we determined adhesion of Ramos cells to monolayers of endothelial cells and its inhibition by specific antibodies. Also, we determined adhesion to endothelial cells of B lymphocytes and subsets of T lymphocytes present in a preparation of unfractionated human mononuclear cells. The results presented here demonstrate that the new assay has the required properties to be used in the quantitation of cell adhesion.

Antibodies, Monoclonal↗

PubMed2006

A rapid confirmatory method for analyzing tetracycline antibiotics in bovine, swine, and poultry muscle tissues: matrix solid-phase dispersion with heated water as extractant followed by liquid chromatography-tandem mass spectrometry.

A rapid, specific, and sensitive procedure for determining four widely used tetracycline antibiotics and three related epimers in bovine, swine, and poultry muscle tissues is presented. The method is based on the matrix solid-phase dispersion technique with heated water as the extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissues with 5 mL of water heated at 70 degrees C. After acidification and filtration, 100 microL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, selecting two precursor ion to product ion transitions for each target compound. Heated water appeared to be an excellent extractant, since the absolute recovery data ranged between 70 and 78%. The accuracy of the method was determined at three spike levels, using minocycline as a surrogate analyte, in any different kind of muscle tissues considered and varied between 88 and 109% with relative standard deviations ranging between 3 and 11%. Limits of quantification were estimated to range between 1 (chlortetracycline) and 9 ng/g (4-epioxytetracycline), based on a signal-to-noise ratio of 10, and are well below the tolerance levels set by the European Union. The effects of the extraction temperature, volume of the extractant, and washing of the material supporting the biological matrix with ethylenediamine tetraacetic disodium salt on the analyte recovery were studied.

Animals↗

PubMed2002

Primary prevention protects public health.

It is widely accepted that epidemiological data provide the only reliable evidence of a carcinogenic effect in humans, but epidemiology is unable to provide early warning of a cancer risk. The experimental approach to carcinogenicity can ascertain and predict potential cancer risks to humans in time for primary prevention to be successful. Unfortunately, only in rare instances were experimental data considered sufficiently convincing per se to stimulate the adoption of preventive measures. The experimental testing of environmental agents is the second line of defense against potential human carcinogens. The first line is the testing of synthesized agents, be these pesticides, medical drugs, or industrial chemical/physical agents, at the time of their development. We do not know, however, how many substances have been prevented from entering the environment because most tests are carried out by commercial or private laboratories and results are rarely released. A better understanding of the mechanisms underlying the sequence of events of the carcinogenesis process will eventually lead to a more accurate characterization and quantification of risks. However, the ways that mechanistic data have been used lately for evaluating evidence of carcinogenicity have not necessarily meant that the evaluations were more closely oriented toward public health. A tendency has surfaced to dismiss the relevance of long-term carcinogenicity studies. In the absence of absolute certainty, rarely if ever reached in biology, it is essential to adopt an attitude of responsible caution, in line with the principles of primary prevention, the only one that may prevent unlimited experimentation on the entire human species.

Attitude to Health↗

PubMed1986

Classification of psychotropic drugs by rat EEG analysis: learning set development.

EEG recordings from 5 different brain areas of freely moving rats were performed under controlled vigilance stage conditions. Effects of antidepressant and neuroleptic drugs were assessed following their intraperitoneal injection. Quantification of the drug effects was achieved by analysis of variance following on-line fast-Fourier transformation of the EEG signal. This resulted in so-called drug profiles. From the individual antidepressant and neuroleptic drug profiles, an antidepressant and neuroleptic drug class profile was calculated. Using the absolute power for the calculation of the antidepressant and neuroleptic drug class profiles gave a better discrimination between the two drug class profiles than using the relative power. The antidepressant and neuroleptic drug class profiles derived from the n. amygdala and hippocampus did not show significant differences. The antidepressant and neuroleptic drug class profiles showed significant differences (p less than 0.05) in rather small frequency bands from the n. caudatus (16-14 Hz), n. accumbens septum (19-24 and 27-31 Hz) and cortex (10-20 Hz).

Amygdala↗

PubMedDate not supplied

Down syndrome: MR quantification of brain structures and comparison with normal control subjects.

For quantification of brain structures from MR scans, a novel, powerful stereologic tool known as Cavalieri's principle was applied. This tool enables an objective estimation of volume. The method was applied to detect differences in various brain structures between persons with Down syndrome and control subjects. On the basis of absolute values, smaller volumes for the whole brain, cerebral cortex, white matter, and cerebellum were seen in persons with Down syndrome. Similar results were observed when a normalization procedure, based on the volume of cranial cavity, was used. Stereologic determinations of the volumes of brain structures from MR images can reliably identify volume differences between persons with Down syndrome and control subjects.

Adult↗

PubMed2006

The quantification of prion gene expression in sheep using real-time RT-PCR.

Determination of the transcription level of cellular prion protein (PrP(C)) is essential for understanding its poorly explained role in organisms. Scrapie in sheep is the prototype of all prion diseases. However, the expression of prion protein (PrP) mRNA in sheep has not been quantified in great detail. Herein we report on measurement of sheep PrP mRNA using absolute quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR). Total RNA was isolated from seven different regions of the central nervous system (CNS) and six peripheral organs of 18 sheep and PrP mRNA was quantified by real-time RT-PCR using an externally calibrated standard curve constructed with the recombinant PrP plasmid. The results showed that high levels of PrP mRNA were expressed in all seven regions of the brain examined, with obex and neocortex expressing the highest PrP, followed by cerebellum, spinal cord, hippocampi, conarium and thalamus, In peripheral organs examined, lymph node showed a level of PrP expression similar to that in overall brain, whereas spleen, heart, liver and lung showed moderate level of expression and kidney showed the lowest expression. Our study provided the first quantitative, tissue-specific data of PrP mRNA expression in sheep for further studies of pathogenesis of prion diseases.

Animals↗

PubMed1979

Liquid chromatography in pharmaceutical analysis X: Determination of chlorzoxazone and hydroxy metabolite in plasma.

A method for the high-pressure liquid chromatographic determination of chlorzoxazone and its hydroxy metabolite in human plasma samples is presented. The separation of the compounds is achieved on an octadecylsilane column with a mobile phase of absolute methanol-distilled water (40:60) at a flow rate of 2.0 ml/min (3100 psig). The chromatographic separation is achieved within 10 min. The overall analysis time is about 45 min, which includes extraction of the drug and metabolite from plasma followed by high-pressure liquid chromatographic separation and quantification. The accuracy of the procedure is in the 1-5% range.

Benzoxazoles↗

PubMed1997

Contact patterns at the tarsal joints.

OBJECTIVE: To determine the contact patterns at the tarsal joints under two levels of loading (150 and 600 N) with the foot in the neutral position, plantarflexion, dorsiflexion, inversion and eversion. DESIGN: In vitro measurements quantifying the size, location and patterns of contact. BACKGROUND: Several methods have been employed to study contact areas and patterns in joints such as the hip and knee; in contrast there is limited data on the joints of the foot. METHODS: Contact areas and patterns were determined by injecting coloured dyes, one for each level of loading, into the joint spaces in 23 pairs of feet: three pairs in the neutral position, five pairs in each of plantarflexion and dorsiflexion, and ten pairs in inversion (right foot) and eversion (left foot). RESULTS: Contact area maps were constructed for the ankle, subtalar, talonavicular, calcaneocuboid, cuneonavicular and the intercuneiform joints. Quantification of the surface areas was performed using a digitizer. The extent of the changes in contact area appeared to be related to the mobility of the joints under consideration, with the greatest changes in contact pattern and area being associated with the most mobile joints. CONCLUSION: The small change in absolute contact area with increased loading reflects the role of the foot, in providing a stable support for the body during gait.

Journal Article↗

PubMed2006

Biomechanical effects of splint types on traumatized tooth: a photoelastic stress analysis.

This study was undertaken to assess the effect of splint type on stresses occurring around traumatized tooth by photoelastic stress analysis. Three semi-rigid splint types--a wire-composite splint, fiberglass splint and titanium trauma splint--were utilized for comparisons. Extracted left upper central and lateral incisors and the canine tooth of an otherwise healthy patient were embedded equidistantly in photoelastic resin. For all cases studied, a static axial and 20 degrees oblique force of 100 N was applied on the lateral incisor in separate sessions. The experiments were undertaken without any splint application (unsplinted, control) after which the splints, adhesively bonded to the labial aspects of teeth, were consecutively tested. During each loading sequence, generation of isochromatic fringes was observed in the field of a polariscope, and photographed by a digital camera. Quantification of fringes was performed on magnified images, transferred to a PC. Under vertical loading, the highest stresses in the apical regions were observed for the unsplinted and ribbond-splint groups, whereas the lowest fringes occurred with the use of orthodontic wire as a splinting medium. Titanium trauma splint had absolutely no effect on reduction of stresses, as the fringe orders were slightly higher than the unsplinted lateral tooth. The use of orthodontic wire resulted in lowest fringe orders around the traumatized tooth.

Biomechanical Phenomena↗

PubMed1994

Measurement of urinary androgen sulfates without previous hydrolysis: a tool to investigate adrenarche. Validation of a commercial radioimmunoassay for dehydroepiandrosterone sulfate.

A commercial 125I radioimmunoassay designed for the quantification of dehydroepiandrosterone sulfate (DHEAS) in blood samples was tested for its direct applicability to 24-hour urine samples from children and adults. Average recoveries in parallelism and spiking experiments were found to be near 100%. Intra- and inter-assay coefficients of variation were below 10%. Urinary DHEAS concentrations determined directly by the radioimmunoassay (x) differed only slightly from corresponding radioimmunoassay values (y) obtained after C18 reversed-phase extraction and LH-20 chromatography (y = 0.85x + 0.12; r = 0.99). Cross-reactivity data from related steroids suggested only a small contribution to the DHEAS titer by other steroids. In 8-year-old children compared to preadrenarchal children (4 years old) a clearly increased median daily urinary DHEAS output could be observed both for absolute excretion data (0.163 versus 0.05 mumol/d, P < 0.01) and for excretion values related to body surface area (0.181 versus 0.071 mumol/d/1.73 m2, P < 0.05). However, this "onset of adrenarche" was no longer statistically significant when urinary creatinine was taken as adjustment parameter for renal androgen sulfate output. After correction with the individual body surface area, rises of urinary DHEAS from childhood to adulthood were nearly in the order of the literature data on age-corresponding serum increases of DHEAS. In conclusion, the direct radioimmunological quantification of DHEAS in 24-hour urine samples with subsequent correction for individual body surface area appears to present a physiologically meaningful way to assess the adrenal gland's secretory activity for this androgen sulfate.

Adrenal Glands↗

PubMed2000

AgNOR staining and quantification.

Nucleolar organiser regions (NORs) are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver-staining, the NORs can be easily identified as black dots exclusively localised throughout the nucleolar area, and are called "AgNORs". The NORs' argyrophilia is due to a group of nucleolar proteins, which have a high affinity for silver (AgNOR proteins). A number of studies carried out in different tumour types demonstrated that malignant cells frequently present a greater AgNOR protein amount than corresponding non-malignant cells. Moreover, in cancer tissues AgNOR protein expression was found to be strictly related to the cell duplication rate. Over the past 12 years, the "AgNOR method" has been applied in tumour pathology for both diagnostic and prognostic purposes. However, the lack of a standardised silver-staining protocol has led to much misinterpretation of actual structures evaluated in individual studies. Indeed, the absolute AgNOR scores reported by different authors for the same types of tumour are scarcely comparable and the results produced by these investigations sometimes seem to be conflicting. In order to achieve definitive standardisation of the AgNOR method and produce comparable data in all laboratories, the "International Committee on AgNOR Quantitation" was founded, and during the first Workshop "AgNORs in Oncology" held in Berlin in 1993 guidelines for AgNOR protein evaluation were first defined. The present paper discusses the main technical aspects of NOR silver-staining, and critically evaluates the methods commonly employed for AgNOR protein quantification in routine cyto-histopathology.

Humans↗