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Results for “Repeatability and Reproducibility”

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PubMed1999

Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis.

Hepatocellular carcinoma (HCC) mainly arises from the liver with chronic inflammation. Because telomere reduction reflects replicative history in somatic cells, we analyzed the possibility that liver tissues surrounding HCC consist of the cells carrying substantial reduction of telomere. We studied 20 HCC and surrounding noncancerous liver tissues (SL) obtained by surgical resection, and 10 laparoscopically obtained needle biopsy specimens of the liver with chronic inflammation including no overt HCC (CI). Five liver tissues without chronic liver diseases (ND) were also examined. Extracted genomic DNAs were blotted on a nylon membrane, and probed at first with radio-labeled d(TTAGGG)(3) and reprobed with radio-labeled d(CCT)(7). The intensity caused by d(TTAGGG)(3) was divided by that of d(CCT)(7). The ratio was defined as telomeric repeats content (TC). Dilution experiments reproducibly revealed almost the same TC. The reduction rate of telomere length through aging estimated by regression analysis of TC was 0.62% per year. Concomitant analyses of TC and average telomere length revealed that both values were significantly correlated (r =.45; P =.009). To compare TC in the liver with respect to chronic inflammation, the value was divided by TC in peripheral blood leukocytes (PBL) from the same donor. The ratio was defined as relative TC (RTC). There was a statistically significant decrease of RTC in CI compared with that in ND (P =.03). Furthermore, RTC in SL was significantly lower than that in CI (P =.0001). These observations suggest that RTC value in liver tissues may digitally indicate a replicative history of hepatocytes under chronic inflammation, and a risk of HCC development.

Adult↗

PubMed1978

Demonstration of specific cell-mediated anti-tumor immunity in lung cancer to autologous tissue extracts.

Cell-mediated immunity (CMI) of lung cancer patients to autologous tumor antigens was assessed by mixed lymphocyte tumor interactions (MLTI) as measured in a microculture (200 microliter) lymphocyte proliferation (LP) assay. Positive lymphoproleferative responses were observed with cryopreserved intact mitomycin-C-treated autologous tumor cells (8/12 or 67% patients reactive) and with hypotonic membrane extracts (HMP) of tumor cells (28/40 or 70%). Good correlation was found between reactivity to tumor cells and extracts in parallel testing. In contrast, HMP of autologous normal lung tissue elicited very little LP reactivity, with only one patient giving a weak response by the SI criterion and low level of n cpm. Upon repeat testing, many patients gave reproducibly positive LP responses to tumor HMP. Patients at all clinical stages of disease and with different histologic tumor types had a similar proportion of HMP reactivity. Most reactive patients responded to a broad range of protein concentrations of tumor HMP, and LP responses were frequently elicited with 1 microgram or less of HMP. Thus, HMP appear to afford a convenient source of reactive tumor antigen for assessing anti-tumor immunity.

Adenocarcinoma↗

PubMed2006

3D MR sialography protocol for postradiotherapy follow-up of the salivary duct system.

PURPOSE: To develop and evaluate an MR sialography protocol that reproducibly images the parotid and submandibular ducts over time, in 3D. Such a protocol is needed in order to investigate the possible radiation-induced changes to the salivary ducts in patients receiving radiotherapy to the head-and-neck. MATERIALS AND METHODS: MR sialography was performed on a 1.5-T MR scanner. Sequence parameters were optimized on 11 healthy volunteers. A 3D water-selective turbo spin echo (TSE) pulse sequence (TR/TE = 6000 msec/190 msec), using a two-element circular surface coil was applied twice in one MR session. In order to assess the reproducibility, the same procedure was repeated four to six months later. The quality of the MR sialograms was measured subjectively by developing a visibility scoring system and objectively by the means of contrast-to-noise ratio (CNR) of the ducts vs. fat (CNR(duct-fat)). RESULTS: High-quality, 3D MR sialographic images were obtained. The quality of the MR sialograms and the subjective visibility score of the salivary ducts were constant over time. The CNR(duct-fat) varied between volunteers (standard deviation, SD 26%) but it was relatively constant per volunteer (SD 5%). CONCLUSION: The MR sialography protocol presented in this study provides good quality 3D imaging of the major salivary ducts, submandibular duct, and the parotid duct and it can be used for the comparison of the salivary duct system of an individual over time.

Head and Neck Neoplasms↗

PubMed2006

Quantitative analysis of polybrominated diphenyl ethers in adipose tissue, human serum and foodstuff samples by gas chromatography with ion trap tandem mass spectrometry and isotope dilution.

A method based on gas chromatography (GC) separation followed by ion trap tandem mass spectrometry detection in EI mode (ITD-MS/MS), using isotope dilution, was developed for the determination of ten native polybrominated diphenyl ethers (PBDEs) and four (13)C(12)-labeled congeners in biological (fat tissue and human serum) and food samples. The highest-mass fragment ions were used as precursor ions for those congeners with molecular ions with m/z values higher than the maximum of the instrument. In these cases (hepta-BDEs and (13)C(12)-hexa-BDEs) no fragmentation was achieved under the experimental conditions employed. Repeatability (lower than 9%) and reproducibility (lower than 13%), expressed as relative standard deviation (RSD, n = 3 and 4, respectively), were satisfactory. Similarly, the coefficient of variation (n = 4) of the isotopic ratio between the two most abundant product ions was lower than 10 and 6% for native and labeled congeners, respectively. To evaluate the feasibility of the method, the optimized isotope dilution GC/ITD-MS/MS method was used for the quantitation of selected PBDE congeners in different samples including adipose tissue, human serum and foodstuff samples, from three inter-laboratory comparative exercises, covering a wide range of concentrations. A solid-phase extraction procedure, previously developed for PCB determination in small-size bird serum samples, was successfully applied to quantification of PBDEs in 1 mL samples of human serum.

Adipose Tissue↗

PubMed2006

Determination of fourteen non-steroidal anti-inflammatory drugs in animal serum and plasma by liquid chromatography/mass spectrometry.

The European Union has regulated the use of non-steroidal anti-inflammatory drugs (NSAIDs) in animal production and requires its member states to detect their residues in different matrices. In this work, a detailed MS and MS/MS study by ion-trap mass spectrometry of fourteen NSAIDs is described. Two multi-residue reversed-phase LC/ESI-MS/MS methods were developed, one for the determination of salicylic acid, naproxen, carprofen, flurbiprofen, ibuprofen, niflumic acid and meclofenamic acid in the negative ion mode, and the other for the determination of ketoprofen, suxibutazone, diclofenac, mefenamic acid, tolfenamic acid, phenylbutazone and its metabolite oxyphenbutazone in the positive ion mode. It was thus possible to confirm up to 14 different NSAID residues in serum and plasma samples of farmed animals, after chromatographic separation by a linear gradient. These substances were chosen as representative of different chemical subclasses of NSAIDs. The two methods were also validated in-house at three contamination levels, evaluating specificity and calculating mean recoveries, repeatability and within-laboratory reproducibility. The MS/MS product ion spectra were successfully used for the qualitative identification of all the drugs tested. All the NSAIDs, apart from salicylic acid, were recovered in high amounts, ranging between 71.6% and 100.9%.

Animals↗

PubMed1993

Use of the hydrogen clearance technique for measurements of pancreatic blood flow.

The hydrogen clearance technique (HCT) was employed for measurements of local pancreatic blood flow (PBF) in dogs, opossums, and rats under normal conditions and in opossums during biliary pancreatitis. Local PBF (ml/min/100 g) in dogs was 57.6 +/- 10.4, in opossums 60.6 +/- 9.5, and in rats 144.7 +/- 23.4, under resting conditions. Regional distribution of local PBF in dogs showed no statistical differences. Technical aspects of the HCT were described in detail. Under physiological conditions parenchymal trauma following electrode implantation was negligible in nearly all cases. More than 95% of registered clearance curves were monoexponential and showed excellent reproducibility of PBF values in repeated measurements (percentage deviation < or = 6.7). Five days after initiating biliary pancreatitis by common channel ligation in opossums local PBF was 72.1 +/- 14.7 after implantation of new electrodes. Chronically implanted electrodes, however, recorded only 39.7 +/- 15.8, due to massive fibro-proliferative alterations around the implanted electrode tips. We therefore conclude that the HCT as described is a useful tool for measuring local PBF in acute experimental settings only. Biliary pancreatitis in our model causes no significant alterations of PBF after 5 days.

Animals↗

PubMed1983

Methodological aspects of microelectrode measurements in cellular spheroids.

Different types of oxygen microelectrodes have been tested in measurements on cellular spheroids. The shape of the oxygen gradients varied strongly depending on size and type of the spheroids. No significant differences in the results were obtained when different types of electrodes were applied. All measurements were made in a perfusion chamber. The shape of the gradients did not vary with time in the perfusion chamber. The reproducibility was found good in repeated measurements using the same spheroid. No mechanical or chemical disturbances were seen during the penetration of the spheroids. Changes in the medium flow rate through the chamber did not drastically change the shape of the oxygen gradients. Almost no convection could be seen at the bottom of the chamber close to the spheroids. The composition of the medium was found to be of importance. Lock's solution containing glucose was found to be satisfactory. The potential signals in the double barrel electrodes allowed an accurate determination of the position when the electrode hit the spheroid surface. The information gained from microelectrode measurements in spheroids might be valuable for the understanding of effects of new tumor treatment modalities in which hypoxic cell sensitizers or high LET radiation are utilized.

Animals↗

PubMed1988

Determination of ethylene oxide residues in processed food products by gas-liquid chromatography after derivatization.

A simple, sensitive and fast method (taking only 4-5 h) for the determination of residues of ethylene oxide (EO) and its reaction product, ethylene chlorohydrin (ECH), is described. For the analysis sodium hydroxide is added to the sample where ECH is transformed to EO. This is followed by the distillation of EO into dilute sulphuric acid containing sodium iodide, whereby EO is converted into ethylene iodohydrin (EIH). The EIH content is determined by gas chromatography using electron capture detection. The method has proved to be applicable to the analysis of low residue levels (less than 0.05 mg/kg, calculated as EO) in various foods, including processed foods. The collaborative studies carried out with four food products in six laboratories were remarkably successful with regard to repeatability of the method and reproducibility of the results. The recoveries of ECH were 50%-60%. In the 204 food products examined EO residues were found in 96 samples at concentration levels between 0.05 mg/kg and 1800 mg/kg.

Chlorohydrins↗

PubMed1979

Reversible electrical breakdown of lipid bilayer membranes: a charge-pulse relaxation study.

Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50-200 musec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10(8) to 0.1 omega cm2), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential difference Vc. For 1 M alkali ion chlorides Vc was about 1 V. The temperature dependence of the electrical breakdown voltage Vc is comparable to that being observed with cell membranes. Vc decreases between 2 and 48 degrees C from 1.5 to 0.6 V in the presence of 1 M KCl. Breakdown experiments were also performed with lipid bilayer membrane composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability. The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).

Cell Membrane↗

PubMed1997

Inhibition of nitric oxide synthesis accelerates the recovery of polysynaptic reflex potentials after transient spinal cord ischemia in cats.

Nitric Oxide (NO) has been implicated as a mediator of neuronal injury in vascular stroke. On the other hand, NO is suggested to play a neuroprotective role by increasing blood flow during cerebral ischemia. In order to evaluate the role of NO in the spinal cord ischemia, effects of nitric oxide synthase (NOS) inhibition on the recovery of reflex potentials after a transient spinal cord ischemia were examined in urethane-chloralose anesthetized spinal cats. Spinal cord ischemia was produced by occlusion of the thoracic aorta and the both internal mammary arteries for 10 min. Regional blood flow (RBF) in the spinal cord was continuously measured with a laser-Doppler flow meter. The monosynaptic (MSR) and polysynaptic reflex (PSR) potentials elicited by electrical stimulation of the tibial nerve, were recorded from the L7 or S1 ventral root. The recovery process of spinal reflex potentials was reproducible when the oclusion was repeated twice at an interval of 120 min. Pretreatment with N(G)-monomethyl-L-arginine (L-NMMA, 10 mg/kg), a NOS inhibitor significantly accelerated the recovery of PSR potentials after spinal cord ischemia. The accelerating effect of L-NMMA on the recovery of PSR potentials was abolished by co-administration of L-arginine (1 mg/kg/min) but not by that of D-arginine (1 mg/kg/min). L-NMMA failed to improve RBF in the spinal cord during ischemia and reperfusion. Nitroprusside (10 microg/kg/min), a NO donor, retarded the recovery of PSR potentials after spinal cord ischemia. These results suggest that NO production has a significant influence on the functional recovery after transient spinal cord ischemia.

Animals↗

PubMed2006

Multi-analyte SPR immunoassays for environmental biosensing of pesticides.

Multi-analyte detection of environmentally relevant pesticides is performed by using a two-channelled surface plasmon resonance (SPR) biosensor. The special design of the SPR instrument allows the determination of several analytes (DDT, chlorpyrifos and carbaryl) via different immobilization formats. First, simultaneous pesticide monitoring is possible by flowing chlorpyrifos, carbaryl or DDT samples separately over each channel of the SPR system, wherein their corresponding recognition element was previously immobilized. The second approach is based on the multiple and combined immobilization of several analyte recognition elements on the sensing surface of one individual flow cell. In this format, the analysis time for all three pesticides varied from 40 to 60 min depending on the number of regeneration cycles. In most cases, similar detection limits were attained for the target analyte irrespective of the assay format, with sensitivity values at the nanogram per litre level (18-50 ng L(-1)). The assay reproducibility was proved through the repeated use of the same sensor surface for over more than 200 assay cycles, whereas the absence of biosensor response to non-related analytes showed the specificity and reliability of the analysis. The SPR instrument, including optics, electronics and microfluidics, is already commercialised by the company SENSIA, SL.

Antibodies↗

PubMed1998

Assay of centromere function using a human artificial chromosome.

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1-5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes.

Autoantigens↗

PubMed1990

Assay for 2-oxoglutarate decarboxylating enzymes based on the determination of [1-14C]succinate: application to prolyl 4-hydroxylase.

A method for the assay of 2-oxoglutarate decarboxylating enzymes based on determination of the reaction product [1-14C]succinate after precipitation of remaining 2-oxo[5-14C]glutarate with 2,4-dinitrophenyl hydrazine is reported. It is particularly useful for the study of the 2-oxoglutarate-coupled dioxygenase prolyl 4-hydroxylase (EC 1.14.11.2); it is superior to previously described assay methods of this enzyme with respect to simplicity of the procedure, speed, cost, and radiochemical safety. The results are highly reproducible, the standard deviation of repeated measurements being about 2% of the mean. The commercially available 2-oxo[5-14C]glutarate used in this study contained approximately 3% of radioactivity coeluting with succinate in HPLC and 1.5% of an unidentified radioactive compound as impurities, which contributed to the background.

Animals↗

PubMed1992

Chemically synthesized 182-235 segment of tau protein and analogue peptides are efficient in vitro microtubule assembly inducers of low apparent sequence specificity.

A 54-amino acid peptide reproducing the first and second repeats and intervening spacer sequence of the tubulin binding motif (residues 182-235) of murine tau protein, and several congeners representing different degrees of sequence scrambling have been prepared by solid phase methods and fully characterized chemically. These double-repeat peptides have been shown to induce microtubule formation at concentrations about one order of magnitude lower than single-repeat controls, under conditions close to the critical concentration needed for tubulin self-assembly. On the other hand, partial loss of microtubule-inducing capacity was observed for peptides with primary structures increasingly disorganized with respect to the canonical peptide. These results call into question the assumption that a high degree of primary structure specificity is involved in the tau-tubulin interaction leading to in vitro microtubule formation.

Amino Acid Sequence↗

PubMed1995

Reflex reversal of apnoeic episodes by electrical stimulation of upper airway in cats.

Respiratory effects of electrical stimulation of the upper airways (UAW) before and during apnoeic episodes induced by nitrogen inhalation were studied in 9 anaesthetized cats. In eupnoeic animals these electrically-evoked reflexes comprise rapid and powerful inspiratory efforts characterized by strong maximal airway occlusion pressures (Pmax = 635 +/- 39 mm H2O) and rapid peak inspiratory flow rates (PIF = 536 +/- 36 ml.sec-1) similar to the sniff-like aspiration reflex elicited mechanically. Electrical stimulation of the UAW mucosa can elicit reflex inspirations and sniff-like aspiration reflexes even during reversible hypoxic apnoea but their intensity and reproducibility are transiently reduced. When repeated adequately, the electrically-induced reflexes can increase the reactivity of respiratory centre and interrupt or terminate apnoeic episodes as do other types of UAW stimulation. Reflex mechanisms and respiratory centre activations seem to be involved in these effects. The results suggest that electrical stimulation of UAW could be useful for testing the respiratory centre reactivity as well as for reflex reversal of apnoeic episodes and restoration of normal breathing in animal experiments and clinico-physiological studies. Such investigation of the role of UAW reflexes in the pathogenesis and therapy of apnoeic syndromes might also be possible by using a cardiostimulator adapted as respiratory pacemaker.

Animals↗

PubMed1985

Programmed aging or error catastrophe? An examination by two-dimensional polyacrylamide gel electrophoresis.

We have examined newly synthesized proteins in the young adult and in older populations of the nematode Caenorhabditis elegans using two-dimensional polyacrylamide gel electrophoresis (2D PAGE). A temperature-sensitive mutant strain, DH26, with a mean life span of about 15 days, under our conditions, was used to block progeny development. Nematodes of several different ages were pulse-labeled for 5 h, in vivo, with 35S-labeled E. coli, A subsequent 30-min chase with unlabeled E. coli served to rid the worms of endogenous labeled E. coli proteins. We resolve 700 or more proteins by 2D PAGE polyacrylamide gel electrophoresis of extracts of young nematodes. The patterns of these proteins are highly reproducible in comparisons of independent repeats of identical experiments. No new major proteins are synthesized at any time during the adult phase (4-22 days) nor are any of the most abundant proteins not made during this period. At our level of detectability (estimated as a satellite spot containing 4% of the amount of label in a major spot) we see no misincorporation of radioactive amino acids into newly synthesized proteins. These data are inconsistent with predictions by any one of several, so called, "error catastrophe" models of senescence and also show that modulation of the highest abundancy classes of proteins are also not involved in senescence.

Aging↗

PubMed1984

The up-and-down method for the determination of nociceptive thresholds in rats.

The up-and-down method applied to the jump-flinch technique provided estimates of shock thresholds for jump and flinch with high statistical efficiency and a much reduced amount of experimentation compared with traditional methods. Reproducible results were obtained after repeated testing and flinch and jump thresholds were significantly increased by treatment with morphine or oxotremorine.

Animals↗

PubMed1993

Predicting the leakage performance of bodyworn disposable incontinence pads using laboratory tests.

An international multi-centre project (The ISO Pad Leakage Project) was conducted to study the leakage performance of large bodyworn incontinence pads for heavily incontinent users and to create international standards for measuring their absorption capacities in the laboratory. This was achieved by recruiting 13 user test centres through which over 100 incontinent subjects tested each of six different products (each in three different sizes) for a period of about a week to a common protocol. Over 10,000 used pads were collected and weighed and the severity of leakage from each of them recorded. Correlations were sought between these data and the results from some 50 technical tests performed in a total of 16 technical test centres in order to discover the impact of different technical parameters on clinical pad performance. It was found that at low urine weights (less than 50 g, say) pad shaping was the most important predictor of pad leakage performance: shaped pads leaked less. With increasing urine weight, absorption capacity and absorption time increased in importance until at 350 g of urine these two parameters and shaping were of about equal significance: shaped pads with high absorption capacity and fast absorption time leaked least. A second series of analyses identified two absorption capacity tests which produced data correlating well with the overall leakage performance of pads, considering all urine weights together. Both tests were checked for repeatability (precision within laboratories) and reproducibility (precision between laboratories) and have been written up as working draft standards.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗