Hepatocitos Humanos Upcytes como modelo experimental in vitro para el estudio del daño hepático inducido por fármacos a largo plazo / Human Hepatocytes Upcytes (R) as in vitro experimental model to study long-term drug-induced liver injury

La determinación de la hepatotoxicidad a largo plazo de nuevos fármacos es esencial para el desarrollo farmacéutico pero está limitada por la falta de modelos celulares adecuados que mantengan una expresión prolongada de la funcionalidad hepática. En el presente trabajo se ha explorado la idoneidad de un nuevo modelo celular llamado Hepatocitos Humanos Upcytes (HHU) cuyas células preservan funciones hepáticas y capacidad replicativa. La caracterización exhaustiva de los principales enzimas de Fase I y II implicados en el metabolismo hepático de fármacos en HHU de tres donantes independientes a diferentes tiempos de cultivo (hasta 21 días) reveló que estas células muestran perfiles de expresión (mRNA) y niveles de actividades enzimáticas más cercanos a los hepatocitos humanos que las células HepG2. Dada la estabilidad fenotípica de los HHU, tanto a nivel transcripcional como a nivel funcional, se exploró su utilidad potencial para evaluar la hepatotoxicidad a largo plazo. Para ello las células se trataron durante diferentes periodos (hasta 21 días) con varias concentraciones de tres fármacos modelo y se evaluaron los efectos sobre la viabilidad celular, la acumulación de lípidos y fosfolípidos, el calcio intracelular y los niveles de GSH. Nuestro estudio permitió detectar efectos tóxicos crónicos, como la esteatosis o la fosfolipidosis, a concentraciones en las que la viabilidad celular no estaba comprometida, confirmando la idoneidad de este nuevo modelo celular para el estudio de la hepatotoxicidad tras tratamientos prolongados con los fármacos (AU)

Determining long-term hepatotoxicity of new drugs is essential for the pharmaceutical industry development; however, it is limited by the lack of suitable cell-based models able to maintain hepatic functions over time. In the present work we explored the suitability of a new cellular model called Human Hepatocytes Upcytes (HHU) which preserves liver functions and replicative capacity. The exhaustive characterization of the major Phase I and II enzymes involved in hepatic drug metabolism in HHU from three independent donors at different culture times (up to 21 days) revealed that these cells show expression profiles (mRNA) and activity levels enzymes that are closer to human hepatocytes than those of HepG2 cells. Given the phenotypic stability of HHU, both at the transcriptional and functional level, their potential utility to assess long-term hepatotoxicity was explored. Cells were treated for various periods (up to 21 days) with several concentrations of three model drugs and the effects on cell viability, lipid and phospholipid accumulation, intracellular calcium and GSH levels were evaluated. Our study allowed us to detect chronic toxic effects, such as steatosis or phospholipidosis, at concentrations that did not compromise celular viability, confirming the suitability of this new cellular model for the study of long-term drug-induced hepatotoxicity (AU)

A rate-based transcutaneous CO2 sensor for noninvasive respiration monitoring.

The pain and risk of infection associated with invasive blood sampling for blood gas measurements necessitate the search for reliable noninvasive techniques. In this work we developed a novel rate-based noninvasive method for a safe and fast assessment of respiratory status. A small sampler was built to collect the gases diffusing out of the skin. It was connected to a CO2 sensor through gas-impermeable tubing. During a measurement, the CO2 initially present in the sampler was first removed by purging it with nitrogen. The gases in the system were then recirculated between the sampler and the CO2 sensor, and the CO2 diffusion rate into the sampler was measured. Because the measurement is based on the initial transcutaneous diffusion rate, reaching mass transfer equilibrium and heating the skin is no longer required, thus, making it much faster and safer than traditional method. A series of designed experiments were performed to analyze the effect of the measurement parameters such as sampler size, measurement location, subject positions, and movement. After the factor analysis tests, the prototype was sent to a level IV NICU for clinical trial. The results show that the measured initial rate of increase in CO2 partial pressure is linearly correlated with the corresponding arterial blood gas measurements. The new approach can be used as a trending tool, making frequent blood sampling unnecessary for respiratory status monitoring.

Cell-based models to predict human hepatotoxicity of drugs / Modelos celulares para predecir la hepatotoxicidad humana de fármacos

Drug-induced liver injury is a significant leading cause of liver disease and post-market attrition of approved drugs. Several hepatic cell-based models have been used for early safety risk assessment during drug development. Their capacity to predict hepatotoxicity depends on cells' functional performance. Cultured hepatocytes have contributed to increase knowledge of the metabolic patterns and mechanisms involved in drug toxicity. A major limitation of monolayer hepatocytes is that they undergo rapid loss of hepatic functionality over time, particularly drug metabolising capability. The sandwich culture model promotes polarised cell surface and stabilises hepatocyte functionality, particularly transport systems, better than monolayer cultures. As 3D spatial organisation and complex heterotypic cell interactions are essential for the functional homeostasis of the liver, hepatocyte models (3D cultures, co-cultures with NPCs and microfluidic systems) that mimic cell-cell, cell- matrix interactions and nutrient flow characteristic of the liver microenvironment have been shown to improve the metabolic competency of hepatocytes and have been proposed for better in vitro predictions of drug hepatotoxicity. In addition to hepatocytes, other cell-based models have been proposed for hepatotoxicity studies. Hepatoma cell lines are metabolically poor compared to hepatocytes, but offer key advantages, such as unlimited life span, reproducibility, high availability and easy handling, which make them useful for screening purposes. Alternatively, hepatic cell lines engineered for stable or transient expression of key drug-metabolising enzymes have also been used. Finally, stem cell-derived hepatocytes are emerging in vitro systems that would provide a stable source of hepatocytes from individuals with highly valuable particular polymorphic characteristics for preclinical drug metabolism and toxicity prediction of new drugs (AU)

La lesión del hígado por fármacos es una de las causas principales de enfermedad hepática y de retirada del mercado de fármacos autorizados. Son varios los modelos de células hepáticas utilizados durante el desarrollo de fármacos para la valoración temprana de su seguridad. Los estudios basados en hepatocitos cultivados han contribuido al conocimiento de los mecanismos implicados en la toxicidad por fármacos. Una limitación fundamental de los hepatocitos cultivados en monocapa es la pérdida temprana de funciones hepáticas, en particular la capacidad para metabolizar fármacos. El cultivo tipo sándwich mantiene la polaridad de los hepatocitos y los sistemas de transporte y estabiliza su funcionalidad mejor que el cultivo en monocapa. Puesto que la organización espacial 3D y las interacciones celulares heterotípicas son esenciales para la homeostasis funcional del hígado, los hepatocitos cultivados en sistemas que reproducen las interacciones entre células, célula-biomatriz y el flujo de nutrientes característicos del microambiente hepático (cultivos 3D, co-cultivos con células no parenquimales, sistemas microfluidicos) presentan mayor capacidad metabólica y han sido propuestos para mejorar la predicción in vitro de la hepatotoxicidad. Otras células hepáticas han sido propuestas como alternativa a los hepatocitos para evaluar la hepatotoxicidad. Si bien las líneas celulares de hepatoma tienen menor capacidad metabólica que los hepatocitos, presentan ventajas clave para el cribado de fármacos (vida ilimitada, reproducibilidad, gran disponibilidad, fácil manejo). También se utilizan células manipuladas para la expresión estable o transitoria de enzimas de biotransformación. Por último, los hepatocitos procedentes de células madre son sistemas in vitro emergentes que proporcionarían una fuente estable de hepatocitos, a partir de individuos con características polimórficas especiales, sumamente valiosa para la predicción preclínica de la toxicidad de nuevos fármacos (AU)

Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay: a quantitative method for oxidative stress assessment of nanoparticle-treated cells.

No consensus exists on how to address possible toxicity of nanomaterials as they interfere with most in vitro screening tests based on colorimetric and fluorimetric probes such as the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay for detection of oxidative species. In the present research, nanomaterial interaction with DCFH-DA was studied in relation to its nature and/or assay conditions (cell-based and time exposure) by incubating Rhodamine (Rhd)-labeled 25nm and 50nm silica (SiO2), naked and oleic acid coated magnetite, (Fe3O4) and maghemite (Fe2O3) iron oxide, titanium dioxide (TiO2) and poly(ethylene oxide)-poly(lactide/glycolide) acid (PLGA-PEO) nanoparticles (NPs) with metabolically active rat hepatocytes for 4 and 24-h periods. Data indicated that nanoparticle uptake correlated with quenching of dye fluorescence emission. In spite of their masking effect, the oxidative potential of NPs could be detected at a limited threshold concentration when exposed for periods of time longer than those frequently used for this test. However, changes in the experimental conditions did not systematically result in free radical formation for all nanomaterials tested. Overall data indicate that despite the quenching effect of nanoparticles on DCFH-DA assay, it can be considered as a useful tool for quantitative measurement of NPs-induced oxidative stress by minor modifications of standardized protocols.

High Resolution Non-contact Fluorescence Based Temperature Sensor for Neonatal Care.

To date, thermistors are used to continuously monitor the body temperature of newborn babies in the neonatal intensive care unit. The thermistor probe is attached to the body with a strong adhesive tape to ensure that the probe stays in place. However, these strong adhesives are shown to increase microbial growth and cause serious skin injuries via epidermal stripping. The latter compromises the skin's ability to serve as a protective barrier leading to increase in water loss and further microbial infections. In this article a new approach is introduced that eliminates the need for an adhesive. Instead, two kinds of fluorophores are entrapped in a skin friendly chitosan gel that can be easily wiped on and off of the skin, and has antimicrobial properties as well. A CCD camera is used to detect the temperature dependent fluorescence of the fluorophore, tris(1,10-phenthroline)ruthenium(II) while 8-aminopyrene-1,3,6-trisulfonic acid serves as the reference. This temperature sensor was found to have a resolution of at least 0.13°C.

[Validity and reliability of bladder ultrasound imaging for noninvasive estimation of urine volume in a major outpatient surgery department]. / Validez y fiabilidad de la ecografía vesical en la estimación no invasiva del volumen de orina en una Unidad de cirugía mayor ambulatoria.

OBJECTIVE: To assess the validity and reliability of bladder ultrasound imaging for noninvasive estimation of urine volume, residual volume after voiding, volume before anesthetic induction and after surgery, and volume on sensing an urge to void. PATIENTS AND METHODS: Study of a prospective series of 47 ASA 1-3 patients aged 18 to 79 years undergoing major outpatient surgical procedures under general anesthesia (n = 24) or regional anesthesia and sedation (n = 23). Urine volume was measured at baseline and on recovery (bladder volume by ultrasound and voided volume in a flask) and at the end of surgery (ultrasound only). The reliability and validity of the ultrasound estimation was calculated. RESULTS: Agreement (intraclass correlation coefficient [ICC]) between 4 ultrasound-image estimations at baseline and the measured amount collected in a flask ranged from 0.70 to 0.86. The inter- and intra-measurement reliability was high, with ICC values greater than 0.80. The median error of estimation by ultrasound, with respect to measurement in the flask, was 23% at baseline and 29% after recovery. The amount in the flask was greater. CONCLUSIONS: Ultrasound monitoring of urine in the bladder is reliable and valid, particularly for small volumes. The procedure is tolerated by patients.

Validez y fiabilidad de la ecografía vesical en la estimación no invasiva del volumen de orina en una Unidad de cirugía mayor ambulatoria / Validity and reliability of bladder ultrasound imaging for noninvasive estimation of urine volume in a major outpatient surgery department

OBJETIVOS: Evaluar la validez y fiabilidad de la ecografíavesical en la estimación no invasiva del volumende orina y del volumen residual postmiccional, antes dela inducción anestésica, tras la intervención y al sentirnecesidad de orinar.PACIENTES Y MÉTODOS: Serie prospectiva de 47 pacientesASA I-III, de 18 a 79 años de edad, intervenidos enrégimen de cirugía mayor ambulatoria y sometidos aanestesia general (24) o a anestesia regional y sedación(23). Se midió el volumen de orina mediante ecografía yen matraz en los tiempos basal y postquirúgico, medianteecografía en el postquirúrgico inmediato. Se estimaronla fiabilidad y la validez de las mediciones.RESULTADOS: La concordancia entre las cuatro tomasbasales realizadas con el ecógrafo en el primer tiempo yentre cada una de ellas, con las determinaciones delmatraz oscila entre 0,70 y 0,86 (coeficiente de correlaciónintraclase). La fiabilidad inter e intramedida es elevada,con coeficientes de correlación intraclase superiores a0,80. Las medianas de error entre el volumen medido conel ecógrafo y con el matraz, en el momento basal y en lareadaptación, fueron del 23 y el 29% a favor del volumenmedido con el matraz.CONCLUSIONES: La monitorización por ultrasonidos delcontenido vesical es un método fiable y válido, especialmentepara volúmenes pequeños, y aceptado por lospacientes(AU)

OBJECTIVE: To assess the validity and reliability ofbladder ultrasound imaging for noninvasive estimationof urine volume, residual volume after voiding, volumebefore anesthetic induction and after surgery, and volumeon sensing an urge to void.PATIENTS AND METHODS: Study of a prospective series of47 ASA 1-3 patients aged 18 to 79 years undergoing majoroutpatient surgical procedures under general anesthesia (n= 24) or regional anesthesia and sedation (n = 23). Urinevolume was measured at baseline and on recovery (bladdervolume by ultrasound and voided volume in a flask)and at the end of surgery (ultrasound only). The reliabilityand validity of the ultrasound estimation was calculated.RESULTS: Agreement (intraclass correlation coefficient[ICC]) between 4 ultrasound-image estimations at baselineand the measured amount collected in a flask rangedfrom 0.70 to 0.86. The inter- and intra-measurement reliabilitywas high, with ICC values greater than 0.80. Themedian error of estimation by ultrasound, with respect tomeasurement in the flask, was 23% at baseline and 29%after recovery. The amount in the flask was greater.CONCLUSIONS: Ultrasound monitoring of urine in thebladder is reliable and valid, particularly for small volumes.The procedure is tolerated by patients(AU)

Tumor necrosis factor alpha and interferon gamma cooperatively induce oxidative stress and motoneuron death in rat spinal cord embryonic explants.

The accumulation of reactive microglia in the degenerating areas of amyotrophic lateral sclerosis (ALS) tissue is a key cellular event creating a chronic inflammatory environment that results in motoneuron death. We have developed a new culture system that consists in rat spinal cord embryonic explants in which motoneurons migrate outside the explant, growing as a monolayer in the presence of glial cells. The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) have been proposed to be involved in ALS-linked microglial activation. In our explants, the combined exposure to these cytokines resulted in an increased expression of the pro-oxidative enzymes inducible nitric oxide synthase (iNOS), the catalytic subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91(phox) and cyclooxygenase-2 (COX-2), as compared to each cytokine alone. This effect was related to their cooperation in the activation of the transcription factor nuclear factor kappa B (NF-kappaB). TNF-alpha and IFN-gamma also cooperated to promote protein oxidation and nitration, thus increasing the percentage of motoneurons immunoreactive for nitrotyrosine. Apoptotic motoneuron death, measured through annexin V-Cy3 and active caspase-3 immunoreactivities, was also found cooperatively induced by TNF-alpha and IFN-gamma. Interestingly, these cytokines did not affect the viability of purified spinal cord motoneurons in the absence of glial cells. It is proposed that the proinflammatory cytokines TNF-alpha and IFN-gamma have cooperative/complementary roles in inflammation-induced motoneuron death.

Rheumatoid arthritis in UK primary care: incidence and prior morbidity.

OBJECTIVES: To estimate the incidence of rheumatoid arthritis (RA) in primary care and to investigate associations with consultation behaviour, risk factors, and comorbidities, using the UK General Practice Research Database (GPRD). METHODS: Subjects with a first-ever diagnosis of RA between 1 January 1996 and 31 December 1997 (n = 579) were identified from a cohort of 1 206 918 subjects aged 20-79 years without cancer. Controls from the same cohort were frequency-matched to the RA group by age, sex, and calendar year (n = 4234). Odds ratios (ORs) and 95% confidence intervals (CIs) of being diagnosed with RA in association with a range of factors were estimated using logistic regression analysis. RESULTS: RA incidence was 0.15 per 1000 person-years, was higher in women than in men, and increased with age in both sexes. Consultations and use of non-steroidal anti-inflammatory drugs (NSAIDs) prior to diagnosis were increased in subjects with RA. An increased risk of RA was observed in association with anaemia in the previous year (OR 2.63, 95% CI 1.54-4.48) and with smoking (1.33, 1.07-1.67). A decreased risk of RA was observed in association with infectious diseases (0.68, 0.50-0.94) and pregnancy in the previous year (0.22, 0.06-0.77), diabetes (0.45, 0.26-0.78), and hypertension (0.74, 0.57-0.94). We found no association with alcohol intake, obesity, or use of low-dose aspirin, oral contraceptives, or hormone replacement therapy (HRT). CONCLUSIONS: Smoking was identified as the only significant lifestyle-related risk factor for RA. Infection in the previous year was associated with a reduced likelihood of RA.

Vascular endothelial growth factor protects motoneurons from serum deprivation-induced cell death through phosphatidylinositol 3-kinase-mediated p38 mitogen-activated protein kinase inhibition.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective degeneration and death of motoneurons in the spinal cord, brainstem and motor cortex which causes progressive muscle weakness and paralysis. Although the molecular mechanisms causing the disease remain unknown, excitotoxicity and loss of trophic support have been proposed as causes of degeneration. The present study was designed to elucidate the mechanisms of motoneuron death induced by serum deprivation and the potential neuroprotective effects of vascular endothelial growth factor (VEGF) in dissociated and organotypic rat spinal cord cultures. Serum withdrawal induced apoptotic cell death in dissociated spinal cord cultures, which was prevented in the presence of VEGF. In organotypic spinal cord cultures, low serum-induced motoneuron death was mediated by the stress-related kinase p38 mitogen-activated protein kinase (p38MAPK), as it was reversed by the p38MAPK inhibitor SB203580. In these cultures, exposure to VEGF blocked p38MAPK phosphorylation and prevented the demise of motoneurons. These effects of VEGF were mediated through the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway, as they were blocked in the presence of the PI3-K inhibitor LY294002. In addition, serum deprivation induced down-regulation of the anti-apoptotic protein Bcl-2 and this effect was prevented both by SB203580 and by VEGF via the PI3-K/Akt pathway. Therefore, Bcl-2 could also play an important role in the neuroprotection induced by VEGF in spinal cord cultures. Together, these findings indicate that VEGF prevents motoneuron death induced by serum deprivation blocking the activity of p38MAPK via the PI3-K/Akt signaling pathway.

[The use of psychotropic drugs in prison (CP Madrid III)]. / Uso de Psicofármacos en prisión (CP Madrid III).

The use of psychotropic drugs in primary care has exponentially increased and prisons are no exception. These drugs are often prescribed in order to find therapeutic uses in the fields of personality disorders, addictions, and dysfunctional behaviours that have not been accepted as indications (compassive use). This study enabled us to make a detailed description of the use of psychiatric drugs at the Madrid III prison, a centre with one of the lowest levels of pharmaceutical expenditure in the region. For a two-week period, all prescriptions of psychotropic drugs were collected and registered along with data of several possible conditioning factors. 20.5% of the population was receiving some kind of psychiatric drug; 76% of those inmates undergoing treatment were receiving one or two psychotropic drugs; 65% were taking sedatives, 38% antidepressants and 27% antipsychotic medication. The total amount of psychotropics consumed was 9,840 DDDs, 46% of which were sedatives, 17% of those being antidepressants and the other 14% antipsychotics. The total cost of the fortnight's treatment was 5,379 euros, 72% of which was spent on antipsychotic medication. There are signs that compassive use of the latest generation of antipsychotics and antiepileptics, and the newer antidepressants are a main cause of the dramatic increase in cost, and cost efficiency has not always been clearly demonstrated. One of the key influencing factors on amount, type and cost of treatment was the prescriptor. An unexpected result was that of finding no relationship between age, nationality, grade or other individual variables and prescription of different kind of medication, with the exception of benzodiazepines.

Uso de Psicofármacos en prisión (CP Madrid III) / Theuse of psychotropic drugs in prison ()CP Madrid III)

El uso de psicofármacos en el entorno de atención primaria está aumentando exponencialmente y el medio penitenciario no constituye una excepción a esta tendencia. Con alguna frecuencia estos tratamientos se prescriben buscando utilidades terapéuticas en relación con trastornos de personalidad, dependencias y conductas disfuncionales que no han sido aceptadas como indicaciones (uso compasivo). El presente estudio ha permitido una descripción pormenorizada del uso de psicofármacos en el Centro Penitenciario (CP) Madrid III, uno de los centros con menor gasto farmacéutico de la Comunidad Autónoma. Durante dos semanas se registraron todas las prescripciones de psicofármacos junto con diversos parámetros que pudiesen resultar condicionantes. Un 23,46% de la población recibía algún tipo de psicofármaco; el 76% de los internos a tratamiento recibía uno o dos psicofármacos, un 65% tenía prescritos ansiolíticos, un 38% antidepresivos y un 27% antipsicóticos. El consumo total de psicofármacos ascendió a 9.840 Dosis Diarias de Mantenimiento, un 46% de las cuales correspondían con ansiolíticos, un 17% con antidepresivos y un 14% con antipsicóticos. En dos semanas, el gasto total sumó 5.379 euros y los antipsicóticos acumularon un 72% del coste (3.857 euros). Hay indicios de que el uso compasivo de nuevos antipsicóticos y antiepilépticos supone un porcentaje sustancial del incremento del gasto con dudosa utilidad. Los resultados del estudio apuntan al médico prescriptor como agente primordial en relación con qué, cuánto y con qué coste se prescribe. Al contrario de lo que cabía esperar, no se encontró asociación entre variables como la edad, nacionalidad, grado o tipo de condena de los internos y la prescripción de los distintos subgrupos de psicofármacos con la excepción de las benzodiacepinas

The use of psychotropic drugs in primary care has exponentially increased and prisons are no exception. These drugs are often prescribed in order to find therapeutic uses in the fields of personality disorders, addictions, and dysfunctional behaviours that have not been accepted as indications (compassive use). This study enabled us to make a detailed description of the use of psychiatric drugs at the Madrid III prison, a centre with one of the lowest levels of pharmaceutical expenditure in the region. For a two-week period, all prescriptions of psychotropic drugs were collected and registered along with data of several possible conditioning factors. 20.5% of the population was receiving some kind of psychiatric drug; 76% of those inmates undergoing treatment were receiving one or two psychotropic drugs; 65% were taking sedatives, 38% antidepressants and 27% antipsychotic medication. The total amount of psychotropics consumed was 9,840 DDDs, 46% of which were sedatives, 17% of those being antidepressants and the other 14% antipsychotics. The total cost of the fortnight’s treatment was 5,379 euros, 72% of which was spent on antipsychotic medication. There are signs that compassive use of the latest generation of antipsychotics and antiepileptics, and the newer antidepressants are a main cause of the dramatic increase in cost, and cost efficiency has not always been clearly demonstrated. One of the key influencing factors on amount, type and cost of treatment was the prescriptor. An unexpected result was that of finding no relationship between age, nationality, grade or other individual variables and prescription of different kind of medication, with the exception of benzodiazepines

Diseño y análisis de un estudio de validez de pruebas diagnósticas en investigación clínica / Design and analysis of a validity study for clinical research diagnostic test

El diagnóstico es u n proceso por la incertidumbre, al que el investigador puede aproximarse a través de conocimientos basados en la teoría de la probabilidad y con tal, forma parte del proceso general de la toma de decisiones. La utilización de una prueba es un proceso que ayuda a confirmar o refutar la probabilidad inicial de que el paciente esté enfermo. Se abordan los principios generales que rigen el diseño y ejecución de un estudio de evaluación de pruebas diagnósticas, la estructura que siguen estos estudios y fundamentalmente, el análisis, cuyo propósito es evaluar en qué medida el test diagnóstico discrimina entre sujetos con y sin la enfermedad de estudio

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Obtención, caracterización y evaluación de la actividad antimicrobiana de extractos de propóleos de Campeche / The collection, characterization and evaluation of antimicrobial activity of propolis extracts from Campeche, Mexico

Entre los productos que se pueden obtener de la colmena se encuentran la cera, la miel, la jalea real y el propóleo. Este último es una mezcla de composición química compleja que contiene bálsamos, aceites etéreos, polen, vitaminas, algunos minerales y proteínas, sustancias que le confieren una variedad de propiedades biológicas de gran interés para fines terapéuticos. Por esta razón en el presente estudio se obtuvieron y caracterizaron extractos etanólicos y acuosos de propóleos de diferentes localidades del estado de Campeche, México, probando posteriormente su efectividad antimicrobiana sobre las bacterias Staphylococcus aureus, Salmonella typhi, Pseudomonas aeruginosa y Streptococcus pyogenes. Los extractos presentaron colores que variaron del ámbar claro al café oscuro, encontrándose un rendimiento en sólidos solubles totales superior en los etanólicos que en los acuosos. Se identificaron como metabolitos: lactonas, saponinas, fenoles, triterpenos, taninos, alcaloides, flavonoides, sustancias aminadas y leucoantocianidinas, estas últimas sólo en los extractos acuosos. La efectividad antimicrobiana de los extractos depende del solvente empleado, la procedencia del propóleo y de la especie bacteriana evaluada, siendo los extractos etanólicos los más efectivos, en particular los obtenidos de propóleos procedentes de Hampolol. La especie bacteriana más sensible resultó ser la P. aeruginosa y la S. typhi la más resistente (AU)

Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein.

We describe a glucose sensor based on a mutant glucose/galactose binding protein (GGBP) and phase-modulation fluorometry. The GGBP from Escherichia coli was mutated to contain a single cysteine residue at position 26. When labeled with a sulfhydryl-reactive probe 2-(4'-iodoacetamidoanilino)naphthalene-6-sulfonic acid, the labeled protein displayed a twofold decrease in intensity in response to glucose, with a dissociation constant near 1 microM glucose. The ANS-labeled protein displayed only a modest change in lifetime, precluding lifetime-based sensing of glucose. A modulation sensor was created by combining ANS26-GGBP with a long-lifetime ruthenium (Ru) metal-ligand complex on the surface of the cuvette. Binding of glucose changed the relative intensity of ANS26-GGBP and the Ru complex, resulting in a dramatic change in modulation at a low frequency of 2.1 MHz. Modulation measurements at 2.1 MHz were shown to accurately determine the glucose concentration. These results suggest an approach to glucose sensing with simple devices.

Polarization-based sensing of glucose using an oriented reference film.

We describe a new approach to glucose sensing using polarization measurements in the presence of a stretch-oriented reference film. The method relies on measurement of the polarized emission from the reference film and of a fluorophore which changes intensity in response to glucose. A glucose-sensitive fluorescent signal was provided by the glucose/galactose binding protein from E. coli. This protein was labeled with an environmentally sensitive fluorophore at a single genetically inserted cysteine residue, and displayed decreased fluorescence upon glucose binding. Using the protein and the reference film we observed glucose-sensitive polarization values for micromolar glucose concentrations. This method of polarization-based sensing is generic and can be used for any sensing fluorophore which displays a change in intensity. In principle, one can construct simple and economical devices for this type of glucose measurement. © 1999 Society of Photo-Optical Instrumentation Engineers.

ADVANCES IN FLUORESCENCE SPECTROSCOPY: MULTI-PHOTON EXCITATION, ENGINEERED PROTEINS, MODULATION SENSING AND MICROSECOND RHENIUM METAL-LIGAND COMPLEXES.

The technology and applications of fluorescence spectroscopy are rapidly advancing. In this overview presentation we summarize some recent developments from this laboratory. Two and three-photon excitation have been observed for a wide variety of intrinsic and extrinsic fluorophores, including tryptophan, tyrosine, DNA stains, membrane probes, and even alkanes. It has been possible to observe multi-photon excitation of biopolymers without obvious photochemical or photo-thermal effects. Although not de-scribed in our lecture, another area of increasing interest is the use of engineered proteins for chemical and clinical sensing. We show results for the glucose-galactose binding protein from E. coli. The labeled protein shows spectral changes in response to micromolar concentrations of glucose. This protein was used with a novel sensing method based on the modulated emission of the labeled proteins and a long lifetime reference fluorophore. And finally, we describe a recently developed rhenium complex which displays a lifetime near 3 µs in oxygenated aqueous solution. Such long life-time probes allow detection of microsecond dynamic processes, bypassing the usual nanosecond timescale limit of fluorescence. The result of these developments in protein engineering, sensing methods, and metal-ligand probe chemistry will be the increased use of fluorescence in clinical chemistry and point-of-care analyses.

Low-frequency modulation sensors using nanosecond fluorophores.

We describe a new approach to fluorescence sensing based on a mixture of fluorophores, one of which is sensitive to the desired analyte. If a long-lifetime analyte-insensitive fluorophore is mixed with a short-lifetime analyte-sensitive fluorophore, the modulation of the emission at conveniently low frequencies becomes equal to the fractional fluorescence intensity of the sensing fluorophore. Under these conditions, the modulation can be used to determine the analyte concentration. This can be used with any fluorophore that changes intensity in response to analyte and does not require the sensing fluorophore to display a change in lifetime. The feasibility of modulation-based sensing was demonstrated using mixtures of 6-carboxyfluorescein and [Ru 2,2'-(bipyridyl)3]2+ as a pH sensor and of the calcium probe Fluo-3 and [Ru 2,2'-(bipyridyl)3]2+ as a calcium sensor.

Lifetime-based sensing of glucose using energy transfer with a long lifetime donor.

We describe an optical assay for glucose based on the luminescence decay time of a long lifetime metal-ligand complex. Concanavalin A was covalently labeled with Ruthenium metal-ligand complex (RuCon A) which served as the donor. The acceptor was malachite green which was covalently linked to insulin. The malachite green insulin was also covalently labeled with maltose (MIMG) to provide binding affinity to RuCon A. Binding of RuCon A to MIMG resulted in a decreased intensity and decay time of RuCon A. Glucose was detected by competitive displacement of MIMG from RuCon A, resulting in increased intensity and decay time. This glucose assay has several favorable features. The long lifetime of RuCon A allows phase-modulation decay time measurements using an amplitude-modulated bluelight-emitting diode as the light source. Reversibility of the assay can be controlled by the extent of sugar labeling of the insulin. Finally, the glucose-sensitive range can be adjusted by selection of the sugar structure and extent of labeling of the insulin.