bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Two Distinct Mechanisms for Induction of Apoptosis in Human Vascular Endothelial Cells
Authors: Gräfe Michael , Steinheider Gerhard , Desaga Ulrike , Warnecke Christina , Lehmkuhl Hans B. , Regitz-Zagrosek Vera , Hildebrandt Alfred G. , Fleck Eckart
Published: October 2004

Tissue homeostasis is fundamentally influenced by the functional integrity and state of endothelial cells. Survival and death of endothelial cells are encountered in cardiovascular disease and may, moreover, affect and determine the development of atherosclerosis and restenosis following intracoronary therapeutical interventions.

Apoptosis was studied in cultured human umbilical vein endothelial cells (HUVEC) to investigate the regulation of endothelial cell death following serum/growth factor depletion as well as incubation with actinomycin-D.

Apoptosis was verified by DNA fragmentation and quantified by fluorescence activated cell sorting (FACS) analysis after TdT-mediated deoxyuridine-triphosphate nick end-labeling (TUNEL). An ELISA was used for detecting intracytoplasmatic nucleosomes. Untreated HUVEC showed 16 ± 6 % TUNEL positive cells after 24 hours as analyzed by FACS. Serum/growth factor depletion increased apoptosis by 79 ± 7%, while 50 ng/ml of the pro-apoptotic drug actinomycin-D induced comparable effects (72 ± 11% ). Apoptosis by serum/growth factor depletion could be blocked completely by the anti-apoptotic agent cycloheximide (2 μg/ml), but was ineffective in blocking actinomycin-D-induced apoptosis. Pyrrolidine dithiocarbamate (PDTC) also acted as an anti-apoptotic agent by blocking apoptosis induced by actinomycin-D, but had no effect on apoptosis induced by factor depletion.

Thus, two independent mechanisms for regulation of apoptosis are suggested to be present in human vascular endothelial cells.
Lipid Peroxidation and Antioxidant Defenses in Cystic Fibrosis Patients
Authors: Benabdeslam Hassiba , Abidi Hassane , Garcia Isabelle , Bellon Gabriel , Gilly Robert , Revol André
Published: October 2004

Lipid peroxidation biomarkers and antioxidant status were measured in 76 cystic fibrosis (CF) patients and compared to 40 control subjects. Univariate and multivariate statistics were performed in this study. Results showed that indicators of lipid peroxidation were higher in CF patients than in controls; thiobarbituric acid reactants and autoantibodies against oxidized low-density lipoproteins were significantly increased in CF patients. Red blood cells and whole blood glutathione peroxidase activities were lower in CF patients than in controls. No difference in red blood cell superoxide dismutase activity was observed. Measured concentration of glutathione peroxidase in plasma showed a higher mean value of this protein in CF patients than in controls. Retinol, α-tocopherol and β-carotene concentrations were all reduced in CF patients as compared to controls; this was particularly pronounced for β-carotene. The decreased α-tocopherol concentration was associated with higher percent hemolysis in CF patients. The results of this study indicate that both lipid peroxidation biomarkers and antioxidant status were disturbed in CF patients, despite medical assistance. Measures of oxidative stress parameters, such as thiobarbituric acid reactants, glutathione peroxidase, and β-carotene concentrations can be considered as significant indicators to discriminate CF patients and control subjects.
Advances in Reverse Transcription Polymerase Chain Reaction Analysis of Cellular mRNA Levels of Transforming Growth Factor-β1 by Capillary Electrophoresis with Laser-Induced Fluorescence Detection
Authors: Beck Alexander , Lehmann Rainer , Gambaro Giovanni , Häring Hans-Ulrich , Schleicher Erwin D. , Voelter Wolfgang , Ceol Monica
Published: October 2004

The prosclerotic transforming growth factor β1 (TGF-β1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-β1 mRNA levels, the comparative kinetic reverse transcription-polymerase chain reaction strategy was used. In this method, cellular mRNA levels of the target and a house-keeping gene are reverse transcribed, amplified by the polymerase chain reaction (PCR) and the kinetics of PCR amplification are compared. Since the current determination of the PCR products, using electrophoretic separation in polyacrylamide gel, staining and scanning of the gel, is timeconsuming (≥ 5 hours) and inaccurate, we have developed a method using capillary gel electrophoresis (CGE) in combination with laser-induced fluorescence (LIF) detection for quantification of PCR-products. Using the CGE-LIF method, a minute aliquot of the PCR reaction mixture is separated and quantified within 10 min. Comparison of the values with those obtained by polyacrylamide gel electrophoresis demonstrates the improved sensitivity (> 1000 fold) and accuracy of the proposed method. The CGE-LIF procedure offers a convenient way of automated, comparative analysis of low levels of mRNA via reverse transcription PCR in low cell numbers or small amounts of tissue samples.
Physiological HEPES Buffer Proposed as a Calibrator for pH Measurement in Human Blood
Authors: Lang Werner , Zander Rolf
Published: October 2004

N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, known as HEPES buffer, with p K in the physiological range was studied for use as an alternative to conventional phosphate buffer for the calibration of pH in modern clinical analyzers. In different series of aqueous equimolar HEPES buffer, pH was measured at 37 °C with a capillary glass electrode standardized previously using phosphate, and variations due to changes in total HEPES buffer concentration (0.025 to 0.320 mol/l), and NaCl (0 to 0.250 mol/l) were monitored. For 0.05 equimolar HEPES buffer without NaCl, the pH of 7.362 ± 0.003 (n = 15) obtained coincided well with the reference pH (7.364) from the National Institute of Standards and Technology (NIST). In particular, in the preferred 0.05 equimolar HEPES buffer/0.110 mol/l NaCl, which is isotonic to human plasma (0.160 mol/l), and termed physiological HEPES buffer (PHB), the pH of 7.346 ± 0.003 (n = 84) can be related to the calculated corresponding reference pH from NIST without liquid junction (7.374), and is also compatible with the pH measured in normal arterial blood, pH = 7.403 ± 0.003 (n = 20). Hence, in the two-point calibration of clinical analyzers, PHB, which is defined operationally with respect to the glass electrode and to phosphate buffer, may be useful as a calibrator in the range of buffer adjustment control to meet the correct values for pH when measuring in blood.

Whereas Na-HEPES salt is hygroscopic and does not meet the declared purity grade (> 99 %), pure HEPES acid is non-hygroscopic and conforms to the manufacturer's purity grade (≥ 99 %). Therefore, for easy preparation of PHB, HEPES acid is the preferred starting material.
Towards Common Reference Intervals in Clinical Chemistry
Authors: Bäck Sten-Erik , Nilsson Jan-Erik , Fex Göran , Jeppson Jan-Olof , Rosén Ulf , Tryding Nils , von Schenck Henning , Norlund Lena
Published: October 2004

The population sample of the Kristianstad survey, a reference intervals survey in the county of Kristanstad, was used to establish new reference intervals in clinical chemistry at the laboratories of the Central Hospital in Kristianstad, the University Hospital in Lund and the University Hospital in Malmö.

Three-hundred and fifty nine subjects, male and female, aged 20–80+ years, were invited to participate in the study, with a participation rate of 70 %. Up to 70 analyses were performed on each subject, general clinical chemistry parameters in all three laboratories, specialized analyses where available. Separate a priori exclusion criteria were defined for each test. In addition, the test pattern of each individual was evaluated for signs of preclinical disease. Twelve cases of preclinical disease were discovered and clinically confirmed.

Details on all test methods are presented along with information concerning instruments used, calibration procedures, methods of calculation and obtained reference intervals. Although the methods were in general calibrated against acknowledged reference materials, in some instances differences were found that made common reference intervals across all laboratories impossible.

Problems relating to the practical use of international recommendations and the establishment of reliable reference intervals are discussed.
Evaluation of an Automated Method for Cytokine Measurement Using the Immulite® Immunoassay System
Authors: Berthier Franck , Lambert Claude , Genin Chrisitian , Bienvenu Jacques
Published: October 2004

Cytokines are key mediators in cell regulation and communication. The concentration of these proteins can rapidly and importantly increase during severe clinical situations. However, current techniques are not adapted to stat measurement, thus making their clinical use limited.

In this context, the commercialization of five new kits for cytokine measurement interleukin ((IL)-1, IL-6, IL-8, tumor necrosis factor (TNF)-α and IL-2R) on an automated immunoanalyzer, the Immulite®, seems to be a new approach for the determination of these markers. We report here the evaluation of the performance of these tests. The technique is based on a solid phase (bead) two site chemiluminescent enzyme immunometric assay. The analysis is performed within 60 to 90 minutes and the calibration is stable for 15 days.

The values of the between-run imprecision study were similar to those from the within-run study with coefficients of variation (CV) ranging from 2% (low values of IL-8) to 11.5 % for intermediate concentrations of IL-6 (500 pg/ml). CVs were usually around 5%.

The accuracy was determined by a linearity study using standards (except for IL-2R) provided by the National Institute for Biological Standards and Control (NIBSC). Slopes obtained during this study were close to 1 (r2 = 0.99), except for IL-6, for which the slope was 1.55. TNF-α values were close to those expected. IL-1 results were about 20 % higher. IL-6 values were over estimated above 100 pg/ml and under estimated below this value. IL-8 study seemed to be impaired by the poor stability of this molecule in the NIBSC preparation. Correlation study with standard laboratory techniques gave variable results : for IL-1 (n = 43) the slope was 0.77 (study carried out using cell culture media), for IL-6 (n = 54) the slope was 0.78, for IL-8 (n = 37) the slope was 1.64, for TNF-α (n = 40) the slope was 0.33 and the slope for IL-2R (n = 51) was 5.1. For the last cytokine, the unit in Immulite assay was different from the one used in our comparison technique. Cross-calibration results were consistent with these data and show that the bias is probably linked to a calibration problem.

The study demonstrated excellent practicality of the system, and good stability of the calibration curve (15 days). However, the sample volume required (350 μl for the IL-6 and the TNF-α) could constitute a limitation for pediatric measurements.
Characterization of Monoclonal Antibodies to Human Protein 1/Clara Cell 10 Kilodalton Protein
Authors: Yamaguchi Tetsuji , Yamada Toshiyuki , Okutani Ryuta , Shijubo Noriharu , Singh Gurmukh , Itoh Yoshihisa
Published: October 2004

Human protein 1/Clara cellMr10 000 protein consists of two identical subunits of seventy amino acid residues each. In the present study, eight clones of monoclonal antibodies against native protein 1 were prepared and their respective epitopes were immunochemically and immunohistochemically characterized using native protein 1, truncated recombinant protein 1 and synthesized peptides. Among the clones, three designated as TY-5, TY-7 and TY-8 recognized amino acid residues 7–16, residues 19–28, and residues 39–46, respectively, all of which comprise the hydrophobic cavity of protein 1, possibly associated with chemical binding function. With the exception of TY-4, the remaining clones recognized residues 61–68 which are exposed to solvent. The epitope of TY-4 remains undetermined. Proper selection and combination of clones and recombinant protein 1 may be useful for fundamental and clinical studies of protein 1.
Assessment of Package Inserts for Diagnostic Kits
Authors: Secchiero Sandra , Sciacovelli Laura , Bonvicini Piero , Zaninotto Martina , Plebani Mario
Published: October 2004

To assure the quality of service in laboratory medicine, it is necessary to implement a quality system which comprises the entire testing process. The use of quality reagents is an important aspect of the process. Despite the fact that it is the responsibility of the laboratory to ensure the quality of the analytical system (including reagents) and since it is impossible to evaluate all commercial diagnostic kits, the laboratory often depends on statements issued by the manufacturer to select the most appropriate diagnostics for a particular laboratory.

In this study we report the results of the analysis of information provided in 887 package inserts enclosed in the more widely used commercial diagnostic kits, following the Standard for the labelling of clinical laboratory materials of the European Committee for Clinical Laboratory Standards (ECCLS). Only a third of these were in agreement with the guidelines of ECCLS Standard, reporting complete and correct information. We believe that it is necessary to implement a constructive cooperation between manufacturers of diagnostic materials and clinical laboratories to produce a more uniform approach to improvements in laboratory quality assurance.
External Quality Assessment in Clinical Chemistry: Review of the Situation in Croatia with Particular Reference to Equipment
Authors: Juretić Dubravka , Čepelak Ivana , Flegar-Meštrić Zlata
Published: October 2004

The effect of the use of new flame photometers (Ciba Corning 480, Chiron, Salzburg, Austria) and multichannel analyzers (BM Hitachi 904 or BM Hitachi 911, both Boehringer GmbH, Mannheim, Germany) on analytical quality was assessed in a number of medical biochemical laboratories (n=58) at health centers and general hospitals that provide primary health care services for the respective catchment areas.

The laboratories were supplied during 1996 and 1997 with new equipment, as part of the First Croatian Health Project, Primary Health Care Subproject, carried out by the Croatian Ministry of Health and Croatian Institute of Health Insurance.

We evaluated analytical performance of these laboratories for 19 analytes, according to the results reported in the Croatian External Quality Assessment Program for medical biochemical laboratories, performed four times per year.

Changes of percentages of particular methods used by the participating laboratories were observed for inorganic phosphate, total bilirubin, urea and creatinine. Such changes were even more pronounced for enzymes, where changes in the measurement procedure such as incubation temperature and buffers were observed in the methods used.

Evaluation of method performance revealed that after the introduction of new equipment interlaboratory variation decreased, so that an increasing proportion of laboratories included in the Project produced results within the target limits.
Point-of-Care Testing: the Views of the Working Group of the Dutch Association of Clinical Chemistry
Authors: Janssen Hans W. , Bookelman Hans , Dols Jan L. S. , Elion Gerritzen Wiveka E. , de Keyzer Ries H.
Published: October 2004

There is clearly a demand for near-patient laboratory testing. Modern technological developments support its implementation, but these are not the only preconditions that need to be satisfied in order to successfully introduce decentralized testing. In part 2 of this paper we give a definition of decentralized testing. Part 3 presents the advantages and disadvantages. This list is a summary taken from the suggested literature. Part 4 examines the formal position of the clinical chemist. Of major importance to the situation in the Netherlands are the Care Institutions Quality Act and the Individual Health Care Professions Act. Part 5 discusses professional standards. Part 6 outlines practical considerations which might support the implementation of decentralized testing. The clinical chemist should play an active role in drawing up a discussion paper for his own hospital.
Antioxidant Enzyme Activities and Total Nitrite/ Nitrate Levels in the Collar Model. Effect of Nicardipine
Authors: Sözmen Eser Yildirim , Kerry Zeliha , Uysal Fevziye , Yetik Günay , Yasa Mukadder , Üstünes Levent , Onat Taner
Published: October 2004

There is a large body of literature describing the causative role of oxidative stress mediated by increased levels of reactive oxygen species in the pathogenesis of cardiovascular disease such as atherosclerosis, hypertension, and restenosis after angioplasty. The positioning of a soft silicone collar around the rabbit carotid artery elicits intimal thickening. The findings from recent studies demonstrated that both intimal thickening and atherosclerosis lead to synthesis of inducible nitric oxide synthase, resulting in abundant amounts of nitric oxide. We investigated the effects of collaring and nicardipine treatment on the activities of antioxidant enzymes, superoxide dismutase and catalase, and total nitrite/nitrate levels, stable products of nitric oxide. Placing the collar increased the total nitrite/nitrate levels and decreased superoxide dismutase activity in collared arteries. Treatment with nicardipine (20 mg/kg/day, s.c.) prevented enhanced nitric oxide degradation without affecting superoxide dismutase and catalase activities. Our results suggest that enhanced nitric oxide production and superoxide anion are generated in response to the collaring, resulting in oxidative stress within the segment in this model.
Comparison of Procalcitonin, sCD14 and Interleukin-6 Values in Septic Patients
Authors: Herrmann Wolfgang , Ecker Daniel , Quast Sabine , Klieden Marion , Rose Stefan , Marzi Ingo
Published: October 2004

The aim of the study was to investigate whether procalcitonin, soluble CD14 and interleukin-6 show advantages in predicting the outcome and specificity for bacterial infection in patients with sepsis in comparison to common C-reactive protein measurement. Laboratory parameters were measured in plasma of patients during 14 days following the diagnosis of sepsis. Patients fulfilling the ACCP/SCCM criteria for sepsis were admitted to an intensive care unit (n=35). Procalcitonin was measured with an immunoluminometric assay, and soluble CD14 and interleukin-6 were analysed by ELISA. C-reactive protein was determined nephelometrically. Measurements were performed on days 0, 1, 2, 3, 4, 7 and 14. Separating the patients into survivors (n=22) and non-survivors (n=13), it was demonstrated that non-survivors mostly exhibited, after the day of admission, increasing procalcitonin concentrations which peaked around days three and four. In contrast, the procalcitonin concentrations of survivors fell continuously to the value of 2.1 ng/ml which was reported to be important for patients prognosis. The difference between procalcitonin median values of survivors (n=22) and non-survivors (n=13) attained the level of statistical significance on day 7 and on day 14 (p=0.05). When comparing the median values of C-reactive protein, soluble CD14 and interleukin-6 between survivors and non-survivors, no significant differences were detectable. In this study, plasma concentrations of soluble CD14 and interleukin-6 showed no predictive value for patients' outcome as compared with established laboratory parameters such as C-reactive protein or leukocyte count. Monitoring of procalcitonin seemed to detect severe episodes of sepsis and may improve the laboratory monitoring of septic patients.
Analytical Performance of the Fully Automated AxSYM(r) Estradiol Assay
Authors: Reinsberg Jochen , Jost Eberhard
Published: October 2004

The test performance of the fully automated AxSYM® Estradiol available on the immunoassay analyser Abbott AxSYM® was evaluated. Imprecision, sensitivity and linearity of dilution were examined. For assessment of accuracy, the assay results measured in 12 control pools and 9 patient samples were compared with the values obtained with isotope dilution—mass spectrometry (reference method). The correlation with the manual radioimmunoassay Estradiol MAIA® was evaluated using 140 serum samples with estradiol concentrations ranging from 0 to 10 nmol/l. Imprecision studies revealed for the AxSYM® Estradiol within-run coefficients of variation of 2.8–8.4% and day-to-day coefficients of variation of 3.4–9.5% (concentration range 0.3–2.8 nmol/l). The lower limit of quantification (lowest estradiol concentration with a day-to-day coefficient of variation <20%) was <0.11 nmol/l; the lower limit of detection was <0.05 nmol/l. The estradiol concentration recovered in patient samples after dilution did not differ by more than 10% from the expected values. The estradiol concentration measured with the AxSYM® Estradiol in 12 commercial control pools in some cases grossly deviated from the reference method values; however, for 9 individual patient samples the AxSYM results deviated by not more than 22% (−11%−+22%). There was a good overall correlation (coefficient of correlation = 0.989) between the results measured with the AxSYM® Estradiol and the Estradiol MAIA® in patient samples.

The AxSYM® Estradiol assay exhibits a good precision and a sufficient degree of sensitivity for measurement of estradiol in serum of menstruating women. Although the AxSYM results measured in the control pools, in some cases did not meet the target values, the good correlation with the Estradiol MAIA® indicates that the reliability of the AxSYM® Estradiol for clinical practice is comparable with well established radioimmunoassays. Thus, the AxSYM® Estradiol offers an alternative which is comparable with respect to clinical reliability but has great advantages in view of rapidity, flexibility and convenience of analysis.
Impacts of Laboratory Methodology on Medical Thinking in the 19th Century
Authors: Büttner Johannes
Published: October 2004

During the 19th century the first laboratories at hospitals and clinics in Central Europe were established. These ‘clinical laboratories’ were devoted to chemical analyses in practical medicine and clinical research. A characteristic feature of these laboratories was the use of measuring instruments, e.g. volumetric apparatus, polarimeters, spectroscopes, colorimeters, photometers. Using these techniques new phenomena were introduced into clinical medicine which could serve as signs in the diagnosis of diseases. Many of the new diagnostic signs were quantitative data as results of measurements. Their main advantage was the greater differentiation in the description of phenomena compared with the qualitative data used before. Another important characteristic of the new diagnostic signs was the discovery of causal relations to physiological and pathological processes in the organism. The physicians and chemists in the clinical laboratory were eager not only to collect empirical data but also to find causal relationships by research work similar to that carried out in physics and chemistry. Once causal chains were been identified more general relationships became clear. One example is the concept of metabolism which comprised a dynamic view on chemical processes in the body and the variation of "Stoff" (material) in time, including a quantitative input-output analysis of the body in health and disease. In the second half of the 19th century, scientifically based diagnostic signs began to replace the traditional symptoms and signs used since antiquity.
Near-Infrared Reflectance Spectroscopy for Noninvasive Monitoring of Metabolites
Authors: Heise H. Michael , Bittner Andreas , Marbach Ralf
Published: October 2004

An important class of substances in clinical chemistry are metabolites in body fluids, which are accessible by near-infrared spectroscopy without sample treatment using reagentless, fast and readily automated in vitro assays. Furthermore, noninvasive sensing systems are under development for the determination of blood glucose, especially for diabetic patients or for monitoring in intensive care and surgery. Near-infrared diffuse reflectance spectrometry of skin was employed allowing a certain tissue volume to be integrally probed. For calibration, the partial least-squares (PLS) algorithm was used either based on wide spectral intervals or using special spectral variable selection. Capillary blood glucose reference concentrations were obtained by finger pricking and an automated laboratory method (hexokinase/G6P-DH). Clear evidence is provided for the physical effect, as manifested by the spectral glucose absorptivities, underlying the individual single-person calibration models, which still require improvements in the methodology in the normo- and hypoglycemic concentration range. In extending the potential of noninvasive blood assays by infrared spectroscopy, a novel technique is presented for probing the intravascular fluid space by using fast spectral near-infrared measurements of skin tissue. The pulsatile blood spectrum can be derived from reflectance spectra of oral mucosa by Fourier analysis (near-infrared plethysmography). Future applications and prospects for noninvasive blood assays are discussed.
Evaluation of RNA Isolation Methods and Reference Genes for RT-PCR Analyses of Rare Target RNA
Authors: Mannhalter Christine , Koizar Daniela , Mitterbauer Gerlinde
Published: October 2004

Reverse transcription polymerase chain reaction (RTPCR) analysis is increasingly becoming part of the diagnostic and prognostic evaluation for hematologic and oncologic disorders. Currently, different RNA isolation methods are used in the diagnostic laboratories. No data are available on their suitability for sensitive detection of breakpoint cluster region-abelson (BCR-ABL) gene transcripts. We have extracted RNA from mononuclear cell (MNC) fractions and from lysed blood samples of 4 patients (1 with leukocytosis, 1 with chronic myelogeneous leukemia (CML) under interferon treatment, and 2 CML patients after bone marrow transplantation) with 3 RNA isolation reagents (TRIzol™, RNAzol™, FastTube™ reagent). RNA yield was slightly higher with RNAzol™ than with TRIzol™ as indicated by agarose gel electrophoresis and spectrophotometric measurement at 260 nm. The FastTube™ reagent was unsuitable for RNA isolation from MNC, and was not evaluated for lysed blood. Quantitative competitive RT-PCR amplification of the ABL gene showed comparable results for RNA isolated with RNAzol™ and TRIzol™. In RNA samples extracted from lysed whole blood, the presence of amplifiable RNA/cDNA was confirmed by amplification of 4 selected reference genes (porphobilinogen deaminase (PBGD), ABL, the gene spanning the BCR on chromosome 22 and retinoic acid receptor alpha (RARA)) in a multiplex PCR. High quality, DNA-free RNA was obtained with RNAzol™, and 1 BCR-ABL-positive (specific for translocation t [9; 22]) cell among 2×104 normal cells was successfully detectable by single step RT-PCR. In RNA isolated with TRIzol™, major contaminations with genomic DNA were observed which significantly impaired the interpretation of the results of RT-PCR analysis.
Interferon β1a Therapy Changes Lipoprotein Metabolism in Patients with Multiple Sclerosis
Authors: Sena Armando , Pedrosa Rui , Ferret-Sena Véronique , Almeida Rita , Andrade Maria Lurdes , Morais Maria Graça , Couderc Rémy
Published: October 2004

To assess whether interferon β1a (IFNβ1a) therapy affects plasma lipoprotein metabolism, twelve patients with relapsing-remitting multiple sclerosis (MS) were studied during a two-year follow-up period. High density lipoprotein (Hdl2) cholesterol and the Hdl2/Hdl3 ratio were increased at year 2 and lipoprotein (a) was transitorily increased at year 1, in comparison to baseline levels. Apolipoprotein A-I was lower and apolipoprotein E higher at year 1, only in a subgroup of patients who experienced relapses and/or progressed during therapy. These findings suggest that IFNβ1a treatment is associated with changes in the lipoprotein metabolism. Alterations in this metabolism could be related to the immunomodulatory actions of the drug and the disease activity in multiple sclerosis patients.
On-Line Analysis of Electrolytes in Extracorporeally Circulating Blood: Application of a Rat Model to Examine the Effect of a Single Pharma-cological Dose of Melatonin on Electrolyte Levels in Blood
Authors: Heidrich Jens P. , Niemeier Anja , Seyfarth Michael , Dibbelt Leif
Published: October 2004

An experimental model was developed to study the kinetics of electrolytes under different physiological and/or pathological conditions. The model was applied to investigate in vivothe effect of a pharmacological dose of melatonin on the concentrations of Ca2+, K+, Na+, and pH in the anticoagulated blood of anaesthetized male Wistar rats (250–350 g). After the application of 0.25 mg melatonin/kg body weight, injected intraperitoneally into each of 8 rats, the electrolytes were measured by a flow-through system with highly sensitive ion-selective electrodes. The results were compared to a control group (n=8) which was treated with diluent (saline). The electrolytes were monitored continuously via an extracorporeal circulation, on-going for at least 60 min. Melatonin induced a significant increase of blood Ca2+ (p<0.02) by an average of 9.9% after 60 min. However, total calcium concentration did not increase significantly. The extracorporeal circulation provoked an elevation of K+ by hemolysis. This K+ increase was slightly diminished by melatonin (p<0.06). No melatonin effects were seen on Na+, pH and magnesium in blood and plasma, respectively. Also, the urine concentrations of the electrolytes were not altered by melatonin. The mechanism by which melatonin influences the blood concentrations of ionized calcium and potassium is not yet understood.
Human Heart-Type Cytoplasmic Fatty Acid-Binding Protein (H-FABP) for the Diagnosis of Acute Myocardial Infarction. Clinical Evaluation of H-FABP in Comparison with Myoglobin and Creatine Kinase Isoenzyme MB
Authors: Okamoto Fumio , Sohmiya Koichi , Ohkaru Yasuhiko , Kawamura Keishiro , Asayama Kumiko , Kimura Hiroshi , Nishimura Shinzo , Ishii Hiroo , Sunahara Noriyuki , Tanaka Takao
Published: October 2004

Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight cytoplasmic protein and present abundantly in the myocardium. When the myocardium is injured, as in the case of myocardial infarction, low molecular weight cytoplasmic proteins including H-FABP are released into the circulation and H-FABP is detectable in a blood sample. We have already developed a direct sandwich-ELISA for quantification of human H-FABP using two distinct types of monoclonal antibodies specific for human H-FABP. In this study we investigated the clinical validity of H-FABP as a biochemical diagnostic marker in the early phase of acute myocardial infarction (AMI). To evaluate the diagnostic usefulness of H-FABP in the early phase of AMI, blood samples were obtained from the following patients within 12 hours after the appearance of symptoms, and serum levels of H-FABP were compared with those of conventional diagnostic markers, such as myoglobin and creatine kinase isoenzyme MB (CK-MB). Blood samples were collected from patients with confirmed AMI (n=140), patients with chest pain who were afterwards not classified as AMI by normal CK-MB levels (non-AMI) (n=49) and normal healthy volunteers (n=75). The serum concentration of H-FABP was quantified with our direct sandwich-ELISA. The concentration of myoglobin mass was measured with a commercial RIA kit. The serum CK-MB activity was determined with an immuno-inhibition assay kit. The overall sensitivity of H-FA B P, within 12 hours after the appearance of symptoms, was 92.9%, while it was 88.6% with myoglobin and 18.6% with CK-MB. The overall specificity of H-FABP was 67.3%, while it was 57.1% with myoglobin and 98.0% with CK-MB. The diagnostic efficacy rates with these markers were 86.2% (H-FABP), 80.4% (myoglobin) and 39.2% (CK-MB), respectively. The diagnostic validity of H-FABP was further assessed by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) of H-FABP was 0.921, which was significantly greater than with myoglobin (AUC: 0.843) and CK-MB (AUC: 0.654). These parameters, such as sensitivity, specificity, diagnostic efficacy and diagnostic accuracy, obtained for patients with chest pain within 3 hours and/or 6 hours after the onset of symptoms were almost the same as those for patients within 12 hours after symptoms. H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis. Thus, H-FABP has great potential as an excellent biochemical cardiac marker for the diagnosis of AMI in the early phase.
Quality Control of Coated Antibodies:
New, Rapid Determination of Binding Affinity
Authors: Ricoux Rémy , Chazaud Bénédicte , Tresca Jean-Pierre , Pontet Michel
Published: October 2004

A procedure is described for the determination of the affinity constant between a fluid-phase biotinylated antigen and a solid-phase monoclonal antibody. This procedure allows evaluation of the efficiency of an antibody as a coated tool for an immunoassay. For this purpose, the biotinylation of the antigen and its further quantitative measurement by streptavidin-peroxidase led to a single reversible interaction, the binding affinity of which greatly determines the quality of the assay. The free and bound fractions of the biotinylated antigen were obtained in wells coated with a low level of immobilized antibodies. At the equilibrium state, the free antigen present in the supernatant of these wells was further transferred to high level antibody coated wells which captured all the free antigen molecules. These molecules were quantified using a standard curve established with known concentrations of biotinylated antigen, also incubated in wells coated with the high level of antibody.
Determination of Reticulocytes: Three Methods Compared
Authors: Siekmeier Rüdiger , Bierlich Alexa , Jaroß Werner
Published: October 2004

Determination of reticulocytes in peripheral blood is a valuable tool for getting information about erythropoiesis of an individual. For many years, reticulocyte numbers were quantified manually by means of a microscope after staining with supravital dyes. However, this method is tedious and shows low reproducibility. Therefore, several methods for the automated determination of reticulocytes have been established in laboratory routine within the last years. The aim of this study was to compare three of these automated methods for reticulocyte analysis. Reticulocytes were determined in 130 subsequent routine samples by means of an ABX Pentra 120 Retic haematological analyser, a Coulter EPICS XL MCL flow cytometer and a Coulter STKS haematology system, using the fluorescent dye thiazole orange or the supravital dye new methylene blue for reticulocyte staining, respectively. The reticulocyte concentrations were slightly lower for the Coulter STKS haematology system (mean ± SD 1.89 ± 1.32%) when compared with the Coulter EPICS XL MCL flow cytometer or the ABX Pentra 120 Retic haematological analyser (2.11 ± 1.25% and 2.12 ± 1.15%, respectively). The correlations between all methods were significant (rs ≥ 0.843, p < 0.001). Small intercepts were, however, observed in the correlation plots between the values obtained by means of the Coulter STKS haematology system and those obtained by the other two methods. Within-batch coefficients of variation were 6.0%, 6.9% and 7.8% for the ABX Pentra 120 Retic haematological analyser, the Coulter STKS haematology system and the Coulter EPICS XL MCL flow cytometer, respectively. The corresponding between-batch coefficient of variation values were 6.8%, 4.9% and 5.3% as well as 14.1%, 7.6% and 6.1% for the low, medium and high control levels determined by means of the ABX Pentra 120 Retic haematological analyser and the Coulter STKS haematology system, respectively. These data suggest that all three methods allow the efficient and reliable determination of reticulocyte counts under clinical routine conditions. However, although the obtained data are very similar, differences exist which should be taken into account for the normal values of the different methods.
A New Modular Chemiluminescence Immunoassay Analyser Evaluated
Authors: Ognibene Agostino , Drake Christopher J. , Jeng Kwan-Yee S. , Pascucci Terri E. , Hsu Stephen , Luceri Francesca , Messeri Gianni
Published: October 2004

Thyrotropin (TSH), free thyroxine (fT4) and testosterone assays have been used as a probe to evaluate the performances of a new modular chemiluminescence (CL) immunoassay analyser, the Abbott Architect™ i2000. The evaluation was run in parallel on other systems that use CL as the detection reaction: DPC Immulite®, Chiron Diagnostics ACS-180 and ACS Centaur (TSH functional sensitivity only). TSH functional sensitivity was 0.0012, 0.009, 0.033 and 0.039 mU/l for the Architect, Immulite, ACS Centaur and ACS-180, respectively. Testosterone functional sensitivity was 0.38, 3.7 and 2.0 nmol/l for Architect, Immulite and ACS-180, respectively. Good correlation was obtained between the ACS-180 and Architect for all assays. The Immulite correlation did not agree well with the Architect or ACS-180 for fT4 and testosterone but was in good agreement for TSH. Regarding fT4 and testosterone, equilibrium dialysis and isotopic dilution gas-chromatography mass-spectrometry (GC-MS) respectively were used as reference methods. For both within- and between-run precision, the Architect showed the best reproducibility for all three analytes (CV < 6%).
A Population Decoding Framework for Motion Aftereffects on Smooth Pursuit Eye Movements
Authors: Gardner Justin L. , Tokiyama Stefanie N. , Lisberger Stephen G.
Published: October 2004

Both perceptual and motor systems must decode visual information from the distributed activity of large populations of cortical neurons. We have sought a common framework for understanding decoding strategies for visually guided movement and perception by asking whether the strong motion aftereffects seen in the perceptual domain lead to similar expressions in motor output. We found that motion adaptation indeed has strong sequelae in the direction and speed of smooth pursuit eye movements. After adaptation with a stimulus that moves in a given direction for 7 sec, the direction of pursuit is repelled from the direction of pursuit targets that move within 90° of the adapting direction. The speed of pursuit decreases for targets that move at the direction and speed of the adapting stimulus and is repelled from the adapting speed in the sense that the decrease either becomes greater or smaller (eventually turning to an increase) when tracking targets move slower or faster than the adapting speed. The effects of adaptation are spatially specific and fixed to the retinal location of the adapting stimulus. The magnitude of adaptation of pursuit speed and direction is uncorrelated, suggesting that the two parameters are decoded independently. Computer simulation of motion adaptation in the middle temporal visual area (MT) shows that vector-averaging decoding of the population response in MT can account for the effects of adaptation on the direction of pursuit. Our results suggest a unified framework for thinking, in terms of population decoding, about motion adaptation for both perception and action.
Evidence That ΔNp73 Promotes Neuronal Survival by p53-Dependent and p53-Independent Mechanisms
Authors: Lee Anna F. , Ho Daniel K. , Zanassi Patrizia , Walsh Gregory S. , Kaplan David R. , Miller Freda D.
Published: October 2004

The p53 family member, p73, is essential for the survival of sympathetic neurons during the developmental period of naturally occurring neuronal death. Here, we have asked whether ΔNp73, which is the only p73 isoform expressed in sympathetic neurons, mediates this survival by p53-dependent and/or p53-independent mechanisms. Initially, we used a genetic approach and crossed p53+/-and p73+/-mice. Quantitation of neurons in the sympathetic superior cervical ganglion during the period of naturally occurring cell death revealed that the loss of p53 partially rescued the death of neurons seen in p73-/-animals. Moreover, exogenous expression of ΔNp73 in cultured p53-/-sympathetic neurons rescued these neurons from apoptosis after NGF withdrawal. Biochemical studies asking how ΔNp73 inhibited NGF withdrawal-induced apoptosis in wild-type neurons demonstrated that it prevented the upregulation of the direct p53 targets p21 and Apaf-1 as well as cleavage of caspase-3. It also inhibited events at the mitochondrial apoptotic checkpoint, suppressing the induction of BimEL and the release of mitochondrial cytochromec. Interestingly, ΔNp73 expression also inhibited one very early event in the apoptotic cascade, the activation of c-Jun N-terminal protein kinase (JNK), likely by binding directly to JNK. Finally, we show that neuronal cell size is decreased in p73-/-mice, and that this decrease is not rescued by the lack of p53, suggesting a role for p73 in regulating cell size that does not involve interactions with p53. Thus, ΔNp73 promotes neuronal survival via p53-dependent and -independent mechanisms, and it does so at multiple points, including some of the most proximal events that occur after NGF withdrawal.
Two Interdependent TRPV Channel Subunits, Inactive and Nanchung, Mediate Hearing inDrosophila
Authors: Gong Zhefeng , Son Wonseok , Doo Chung Yun , Kim Janghwan , Shin Dong Wook , McClung Colleen A. , Lee Yong , Lee Hye Won , Chang Deok-Jin , Kaang Bong-Kiun , Cho Hawon , Oh Uhtaek , Hirsh Jay , Kernan Maurice J. , Kim Changsoo
Published: October 2004

Hearing inDrosophiladepends on the transduction of antennal vibration into receptor potentials by ciliated sensory neurons in Johnston's organ, the antennal chordotonal organ. We previously found that aDrosophilaprotein in the vanilloid receptor subfamily (TRPV) channel subunit, Nanchung (NAN), is localized to the chordotonal cilia and required to generate sound-evoked potentials (Kim et al., 2003). Here, we show that the only otherDrosophilaTRPV protein is mutated in the behavioral mutantinactive(iav). The IAV protein forms a hypotonically activated channel when expressed in cultured cells; in flies, it is specifically expressed in the chordotonal neurons, localized to their cilia and required for hearing. IAV and NAN are each undetectable in cilia of mutants lacking the other protein, indicating that they both contribute to a heteromultimeric transduction channelin vivo. A functional green fluorescence protein-IAV fusion protein shows that the channel is restricted to the proximal cilium, constraining models for channel activation.
Optical Postsynaptic Measurement of Vesicle Release Rates for Hippocampal Synapses Undergoing Asynchronous Release during Train Stimulation
Authors: Otsu Yo , Murphy Timothy H.
Published: October 2004

Developing hippocampal neurons in microisland culture were found to undergo rapid depression of excitatory synaptic activity caused by consumption of their readily releasable pool (RRP) of vesicles in response to 20 Hz trains of stimulation. Associated with depression was a switch to an asynchronous release mode that maintained transmission at a high steady-state rate equivalent to ∼2.1 RRPs per second. We have applied postsynaptic Ca2+imaging to directly monitor these asynchronous release events to estimate both the steady rate of transmitter release and the number of quanta within the RRP at individual hippocampal synapses. Based on the frequency of asynchronous release measured at individual synapses postsynaptically using Ca2+imaging (5-17 sec after train stimulation) and with knowledge of the time course by which asynchronous release rates decay, we estimate that individual hippocampal synapses exhibit (in response to train stimulation) peak release rates of up to 21 quanta per second from an RRP that contains, on average, 10 quanta. Use-dependent block of evoked synaptic activity by MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d]cyclohepten-5,10-imine maleate] confirmed that synapses undergoing asynchronous release are not significantly different from the general population with regard to their composition of NMDA receptor and/or release probability. Given that high-frequency trains deplete the synapse of readily releasable quanta (and that these release rates can only be maintained for a few seconds), these high rates of asynchronous release likely reflect refilling of vesicles from a reserve pool and not necessarily the continuous action of a relatively slow clathrin- and endosome-dependent process.
Excessive Expression of Acetylcholinesterase Impairs Glutamatergic Synaptogenesis in Hippocampal Neurons
Authors: Dong Haiheng , Xiang Yun-Yan , Farchi Noa , Ju William , Wu Yaojiong , Chen Liwen , Wang Yutian , Hochner Binyamin , Yang Burton , Soreq Hermona , Lu Wei-Yang
Published: October 2004

Acetylcholinesterase (AChE) exerts noncatalytic activities on neural cell differentiation, adhesion, and neuritogenesis independently of its catalytic function. The noncatalytic functions of AChE have been attributed to its peripheral anionic site (PAS)-mediated protein-protein interactions. Structurally, AChE is highly homologous to the extracellular domain of neuroligin, a postsynaptic transmembrane molecule that interacts with presynaptic β-neurexins, thus facilitating synaptic formation and maturation. Potential effects of AChE expression on synaptic transmission, however, remain unknown. Using electrophysiology, immunocytochemistry, and molecular biological approaches, this study investigated the role of AChE in the regulation of synaptic formation and functions. We found that AChE was highly expressed in cultured embryonic hippocampal neurons at early culture days, particularly in dendritic compartments including the growth cone. Subsequently, the expression level of AChE declined, whereas synaptic activity and synaptic proteins progressively increased. Chronic blockade of the PAS of AChE with specific inhibitors selectively impaired glutamatergic functions and excitatory synaptic structures independently of cholinergic activation, while inducing AChE overexpression. Moreover, the PAS blockade-induced glutamatergic impairments were associated with a depressed expression of β-neurexins and an accumulation of other synaptic proteins, including neuroligins, and were mostly preventable by antisense suppression of AChE expression. Our findings demonstrate that interference with the nonenzymatic features of AChE alters AChE expression, which impairs excitatory synaptic structure and functions.