RESUMEN
Glutamate receptors sensitive to N-methyl-D-aspartate (NMDA) are involved in embryonic brain development but their activity may be modulated by the kynurenine pathway of tryptophan metabolism which includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at these receptors. Our previous work has shown that prenatal inhibition of the pathway produces abnormalities of brain development. In the present study kynurenine and probenecid (both 100mg/kg, doses known to increase kynurenic acid levels in the brain) were administered to female Wistar rats on embryonic days E14, E16 and E18 of gestation and the litter was allowed to develop to post-natal day P60. Western blotting revealed no changes in hippocampal expression of several proteins previously found to be altered by inhibition of the kynurenine pathway including the NMDA receptor subunits GluN1, GluN2A and GluN2B, as well as doublecortin, Proliferating Cell Nuclear Antigen (PCNA), sonic hedgehog and unco-ordinated (unc)-5H1 and 5H3. Mice lacking the enzyme kynurenine-3-monoxygenase (KMO) also showed no changes in hippocampal expression of several of these proteins or the 70-kDa and 100-kDa variants of Disrupted in Schizophrenia-1 (DISC1). Electrical excitability of pyramidal neurons in the CA1 region of hippocampal slices was unchanged, as was paired-pulse facilitation and inhibition. Long-term potentiation was decreased in the kynurenine-treated rats and in the KMO(-/-) mice, but galantamine reversed this effect in the presence of nicotinic receptor antagonists, consistent with evidence that it can potentiate glutamate at NMDA receptors. It is concluded that interference with the kynurenine pathway in utero can have lasting effects on brain function of the offspring, implying that the kynurenine pathway is involved in the regulation of early brain development.
Asunto(s)
Galantamina/farmacología , Hipocampo/efectos de los fármacos , Quinurenina 3-Monooxigenasa/deficiencia , Quinurenina/farmacología , Plasticidad Neuronal/efectos de los fármacos , Nootrópicos/farmacología , Efectos Tardíos de la Exposición Prenatal/patología , Adyuvantes Farmacéuticos/farmacología , Animales , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Edad Gestacional , Hipocampo/citología , Técnicas In Vitro , Quinurenina 3-Monooxigenasa/genética , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/genética , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Plasticidad Neuronal/genética , Neuropéptidos/metabolismo , Embarazo , Probenecid/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/fisiología , Ratas , Ratas Wistar , Receptores de Glutamato/metabolismoRESUMEN
A chemiluminescent, microparticle-membrane capture immunoassay (CLIA/MMC) for the detection of antibody to hepatitis B core antigen (anti-HBc) is described. The assay utilizes recombinant hepatitis B core antigen coupled to carboxylated latex microparticles. Human polyclonal IgG anti-HBc labelled with acridinium competes with antibody in the sample for a limited number of binding sites on the solid phase. After a 40 min incubation at 40 degrees C, the reaction mixture is transferred to a glass fiber capture membrane and washed. A chemiluminescent signal is produced by addition of alkaline peroxide and is quantitated on a semi-automated reader as described. The CLIA/MMC assay was compared with standard EIA and RIA procedures (Corzyme and Corab, respectively, Abbott Laboratories, North Chicago, IL). Assay sensitivities were RIA greater than CLIA/MMC greater than EIA. A population of 200 normal blood donors showed nearly identical distributions with the CLIA/MMC and RIA (mean = 11% inhibition, SD = 13% for both), compared with the EIA (mean = 13% inhibition, SD = 15%). With a selected plasma population (n = 307), the CLIA/MMC immunoassay showed an excellent correlation (r = 0.94) with both the EIA and RIA procedures. Association of anti-HBc reactivity near assay cutoffs with antibody to hepatitis B surface antigen suggested relative specificity in the order RIA greater than CLIA/MMC greater than EIA. The CLIA/MMC procedure, which can be readily automated, provides a non-istopic alternative to current EIA testing with performance more nearly equivalent to RIA.
Asunto(s)
Anticuerpos contra la Hepatitis B/análisis , Antígenos del Núcleo de la Hepatitis B/inmunología , Inmunoensayo/métodos , Acridinas , Unión Competitiva/inmunología , Humanos , Inmunoensayo/instrumentación , Técnicas para Inmunoenzimas , Mediciones Luminiscentes , Microesferas , Radioinmunoensayo , Proteínas Recombinantes , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog.
Asunto(s)
Encéfalo/metabolismo , Regulación hacia Abajo/fisiología , Quinurenina/antagonistas & inhibidores , Neurogénesis/fisiología , Receptores de N-Metil-D-Aspartato/biosíntesis , Transducción de Señal/fisiología , Transmisión Sináptica/fisiología , Factores de Edad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Proteína Doblecortina , Regulación hacia Abajo/efectos de los fármacos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Femenino , Quinurenina/biosíntesis , Masculino , Neurogénesis/efectos de los fármacos , Técnicas de Cultivo de Órganos , Embarazo , Ratas , Ratas Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacología , Tiazoles/farmacologíaRESUMEN
Frequent determination of glucose concentrations in diabetic patients is an important tool for diabetes management. This requires repetitive lancing and finger bleeding. Use of noninvasive (NI) detection techniques offers several advantages, such as the absence of pain and exposure to sharp objects and biohazard materials, the potential for increased frequency of testing, and hence, tighter control of the glucose concentrations, and the potential for a closed-loop system including a monitor and an insulin pump. These potential advantages have led to considerable interest in the commercialization of NI glucose monitoring devices. Review of the scientific, patent, and commercial literature indicates that the spectroscopic basis for NI determination of glucose is not yet well established, and attempts at commercialization may be several steps ahead of our understanding the origin and characteristics of an in vivo glucose-specific or glucose-related signal. Several technologies have potential for leading to viable measuring devices, but most of the data are based on in vitro experimentation. Because of the technical complexity of in vivo glucose measurements, this review aims at discussing the gap between the established need and current technology limitations.
Asunto(s)
Automonitorización de la Glucosa Sanguínea/métodos , Análisis Espectral/métodos , Humanos , Sensibilidad y EspecificidadRESUMEN
We performed visible/near-infrared optical measurements on the forearm of human subjects. We conducted four studies: one study using a commercial diffuse reflectance spectrometer, and three studies using a breadboard temperature-controlled localized reflectance tissue photometer. Calibration relationships were established between skin reflectance signal and either the reference blood hemoglobin (Hb) concentration or the hematocrit values (Hct). Prediction results were expressed as the prediction correlation coefficient (r(p)) and the standard error for cross-validation prediction (CV-SEP). Using diffuse reflectance measurement, r(p) = 0. 8, CV-SEP = 0.9 g/dL for Hb and r(p) = 0.7, CV-SEP = 3.3% for Hct (n = 40). In a localized reflectance study involving calculating the absorption and scattering coefficients and including effect of change of skin temperature in the calibration model, the best prediction results were r(p) = 0.9, CV-SEP = 0.8 g/dL for Hb and r(p) = 0.8, CV-SEP = 3% for Hct (n = 26). In a second localized reflectance study on a population having diverse skin colors at 34 degrees C, the optimal model led to r(p) = 0.8, CV-SEP = 0.9 g/dL for Hb and r(p) = 0.9, CV-SEP = 2.1% for Hct (n = 28) using the localized reflectance values without deducing the absorption and scattering coefficients. Improvement in the correlation was more noticeable for Hct than for the case of Hb. The photometer was used to screen prospective blood donors with low Hb concentration. It was possible to predict anemic subjects in the limited prospective blood donor population.
Asunto(s)
Hematócrito , Hemoglobinas/análisis , Fotometría/métodos , Calibración , Humanos , Reproducibilidad de los Resultados , Piel , Pigmentación de la PielRESUMEN
We have developed a novel optical arrangement for the Abbott VP bichromatic analyzer, converting it into a fluorometer upon insertion of a specially designed filter carriage. Fluorescence intensity is measured by using straight-through excitation geometry and is corrected for fluctuations in light-source intensity by using the attenuated transmitted beam through the solution as a reference. The modification allows switching from the absorbance to fluorescence mode without an auxiliary light source or a major hardware change. The detection limit for an experimental prototype was estimated as 192 pmol of fluorescein per liter. In an application to a fluorescence immunoassay for theophylline in patients' sera, the data (y) correlated well with results by an enzyme immunoassay procedure (EMIT), giving a correlation equation of y = 1.849 + 0.988x (r = 0.969). A fluorometric standard curve for glucose and some performance data are also presented.
Asunto(s)
Fluorometría/instrumentación , Filtración/instrumentación , Fluoresceínas , Glucosa/análisis , Humanos , Inmunoensayo/métodos , Modelos Teóricos , Teofilina/sangreRESUMEN
We describe a reaction tray for a heterogeneous chemiluminescence (CL) immunoassay having the following features: separate sample incubation and signal detection wells; a design that allows for noncontact transfer of the reaction mixture from incubation wells to detection wells; surface features to mate with a detector and create a light-tight seal for CL detection; and self-contained means for liquid removal. The reaction mixture is transferred by injecting a wash solution from a group of nozzles into the incubation well. Quantitative transfer of microparticles (transfer efficiencies greater than 95% and CV less than 5%) is achieved by injecting two 300-microL pulses of transfer solution at a rate of 2.1 m/s. The performance of the tray and method of transfer is tested by determining the precision of CL signal for a sample containing a concentration of anti-hepatitis B core antigen (anti-HBc) or hepatitis B surface antigen (HBsAg) close to the cutoff value for the assay.
Asunto(s)
Inmunoensayo/métodos , Mediciones Luminiscentes , Unión Competitiva , Antígenos del Núcleo de la Hepatitis B/análisis , Antígenos de Superficie de la Hepatitis B/análisis , Humanos , Inmunoensayo/instrumentaciónRESUMEN
Urine samples from 358 asymptomatic males were screened for urethral inflammation by the leukocyte esterase (LE) test and for Chlamydia trachomatis by the ligase chain reaction (LCR). LE and LCR positivity rates were 7.5% (27 of 358 samples) and 2.8% (10 of 358 samples), respectively. Eight of the 10 LCR-positive samples were detected by the LE screening test. The urine LE prescreening test in combination with the LCR assay may be a reasonable approach for genitourinary chlamydial disease control.
Asunto(s)
Hidrolasas de Éster Carboxílico/orina , Infecciones por Chlamydia/diagnóstico , Chlamydia/aislamiento & purificación , Técnicas Bacteriológicas , Infecciones por Chlamydia/enzimología , Infecciones por Chlamydia/orina , Humanos , Ligasas , MasculinoRESUMEN
We describe an apparatus for measuring signals emanated from two heterogeneous chemiluminescence immunoassay (CLIA) configurations: antibody-coated polystyrene beads, in reaction tray wells, and microparticles captured by a porous matrix. An optics and fluidics design which allows the use of a common detection head for these two different assay configurations is described. The detection head moves along three Cartesian coordinates to create a localized light-tight compartment around each individual disposable reaction vessel. Reproducibility of the light seal, trigger solution delivery, and mixing is achieved for acridinium-labeled CLIA. The coated polystyrene beads configuration is tested using beta HCG, CEA, and TSH assays. The microparticle-capture configuration is tested using beta HCG and HBsAg assays. The microparticle capture CLIA has shorter incubation times and the potential for ease of automation.
Asunto(s)
Inmunoensayo/instrumentación , Mediciones Luminiscentes , Interpretación Estadística de Datos , Diseño de Equipo , Antígenos de la Hepatitis B/análisis , Inmunoensayo/métodos , Poliestirenos , Análisis de RegresiónRESUMEN
We describe a multichannel heterogeneous immunoassay analyzer in which a sample is split between disposable reaction trays in a group of linear tracks. The system's pipettor uses noninvasive sensing of the sample volume and disposable pipet tips. Each assay track has (a) a conveyor belt for moving reaction trays to predetermined functional stations, (b) temperature-controlled tunnels, (c) noncontact transfer of the reaction mixture between incubation and detection wells, and (d) single-photon counting to detect a chemiluminescence (CL) signal from the captured immunochemical product. A novel disposable reaction tray, with separate reaction and detection wells and self-contained fluid removal, is used in conjunction with the transfer device on the track to produce a carryover-free system. The linear immunoassay track has nine predetermined positions for performing individual assay steps. Assay step sequence and timing is selected by changing the location of the assay modules between these predetermined positions. The assay methodology, a combination of microparticle capture and direct detection of a CL signal on a porous matrix, offers excellent sensitivity, specificity, and ease of automation. Immunoassay configurations have been tested for hepatitis B surface antigen and for antibodies to hepatitis B core antigen, hepatitis C virus, human immunodeficiency virus I and II, and human T-cell leukemia virus I and II.