Human, rat and chicken small intestinal Na+ - Cl- -creatine transporter: functional, molecular characterization and localization.

In spite of all the fascinating properties of oral creatine supplementation, the mechanism(s) mediating its intestinal absorption has(have) not been investigated. The purpose of this study was to characterize intestinal creatine transport. [(14)C] creatine uptake was measured in chicken enterocytes and rat ileum, and expression of the creatine transporter CRT was examined in human, rat and chicken small intestine by reverse transcription-polymerase chain reaction, Northern blot, in situ hybridization, immunoblotting and immunohistochemistry. Results show that enterocytes accumulate creatine against its concentration gradient. This accumulation was electrogenic, Na(+)- and Cl(-)-dependent, with a probable stoichiometry of 2 Na(+): 1 Cl(-): 1 creatine, and inhibited by ouabain and iodoacetic acid. The kinetic study revealed a K(m) for creatine of 29 microM. [(14)C] creatine uptake was efficiently antagonized by non-labelled creatine, guanidinopropionic acid and cyclocreatine. More distant structural analogues of creatine, such as GABA, choline, glycine, beta-alanine, taurine and betaine, had no effect on intestinal creatine uptake, indicating a high substrate specificity of the creatine transporter. Consistent with these functional data, messenger RNA for CRT was detected only in the cells lining the intestinal villus. The sequences of partial clones, and of the full-length cDNA clone, isolated from human and rat small intestine were identical to previously cloned CRT cDNAs. Immunological analysis revealed that CRT protein was mainly associated with the apical membrane of the enterocytes. This study reports for the first time that mammalian and avian enterocytes express CRT along the villus, where it mediates high-affinity, Na(+)- and Cl(-)-dependent, apical creatine uptake.

Determinación de residuos de siete insecticidas organofosforados en frutas mediante cromatografía de gases con detector de nitrógenofósforo y confirmación por espectrometría de masas / Residue analysis of seven organophosphorous insecticides in fruit by GC/NPD and confirmation by mass spectrometry

Se ha estudiado la presencia de residuos de siete insecticidas organofosforados en peras y manzanas adquiridas en la provincia de León. Se ha utilizado un sistema de extracción para matrices acuosas y determinación analítica mediante cromatografía de gases (GC) y detección con un detector de nitrógeno-fósforo (NPD). Las muestras positivas se confirmaron mediante cromatografía de gases y espectrometría de masas (GC/MS). Los insecticidas analizados mediante una técnica de extracción de multiresiduos fueron: diclorvós, diazinón, metilparatión, metil-pirimifós, paratión, malatión y fentión. Se han analizado 54 muestras obtenidas en la cesta de la compra de la ciudad de León, 28 manzanas (13 de la variedad Reineta y 15 de la variedad Golden) y 26 peras (15 de la variedad Conferencia y 11 de la variedad Blanquilla), de las que aparecieron 6 (11 por ciento) unidades (1 manzana de la variedad Reineta, 3 peras de la variedad Conferencia y 2 peras de la variedad Blanquilla) contaminadas con diazinón. Ninguna de las muestras sobrepasó el límite máximo de residuos (LMR) establecido para este compuesto (0.5 ppm) por el RD 280/94 en productos vegetales. Los datos de consumo medio por habitante de Castilla y León de peras y manzanas nos han permitido conocer la ingesta diaria estimada (IDE) del diazinón (rango 0,004 0,045 µg/kg/día), que al compararlo con la ingesta diaria admisible (IDA) (2 µg/kg/día), permite estimar un margen de seguridad comprendido entre 44 y 500 (AU)

[In vitro toxicity of non-preserved artificial-tear formulations]. / Lágrimas artificiales sin conservantes: estudio comparativo in vitro.

PURPOSE: To analyse the putative toxic effect of three commercially available non-preserved artificial tear formulations on in vitro human conjunctival cells. MATERIAL AND METHOD: A conjunctival human epithelial cell line was exposed to Cellufresh, Oculotect and Acuolens formulations during 1, 3 and 24 hours. Cytotoxicity was measured by calculating the percentage of cell viability examination and scanning electron microscopy (SEM). Controls underwent exposure to supplement free DMEM-F12 (negative control) and exposure to 0.005% benzalkonium chloride solution (positive control). RESULTS: Cell viability after 1 or 3 hours incubation with Cellufresh and Oculotect was similar to that obtained for negative controls. With Acuolens incubation however, cell viability showed significant reduction after 3 and 24 hours compared to control. SEM showed that Cellufresh and Oculotect exposed cells presented similar behavior to control cells. All three cell lines presented evidence of cellular surface alteration after incubation for 1 or 3 hours compared to controls, Acuolens showing the highest rate of alterations in exposed cells and an additional increment in cell loss was observed. CONCLUSION: In the present study, non preserved artificial tears formulations showed a different degree in their in vitro toxicity, Acuolens being more toxic than Cellufresh or Oculotect.

Lágrimas artificiales sin conservantes: estudio comparativo in vitro / In vitro toxicity of non-preserved artificial-tear formulations

Objetivo: Analizar los posibles efectos tóxicos de tres preparados comerciales de lágrimas artificiales sin conservantes sobre células conjuntivales humanas in vitro. Métodos: Cultivos de células epiteliales de conjuntiva humana fueron expuestos a la acción de los preparados comerciales Cellufresh, Oculotect y Acuolens durante 1, 3 y 24 horas. Transcurrido dicho tiempo se estudió su posible efecto tóxico, expresado como porcentaje de Viabilidad Celular, y se analizó la presencia de alteraciones en la superficie de las células mediante microscopia electrónica de barrido (MEB). El estudio incluyó como control negativo de toxicidad células expuestas a medio de cultivo DMEM-F12 sin suplementos y como control positivo, células expuestas a una solución de cloruro de benzalconio al 0,005 por ciento en dicho medio de cultivo. Resultados: La Viabilidad Celular obtenida tras incubar 1 ó 3 horas con Cellufresh y Oculotect fue similar a la del control negativo, si bien disminuyó algo cuando el tiempo de incubación fue de 24 horas. Sin embargo, la Viabilidad Celular tras incubar con Acuolens fue significativamente menor para todos los tiempos. La MEB mostró que con Cellufresh y Oculotect el aspecto general de las células era muy similar al observado en las células control. Sin embargo, con Acuolens se observaron alteraciones acusadas, tanto tras 1 hora como tras 3 horas de incubación, y una notable pérdida celular. Conclusión: De las lágrimas artificiales sin conservantes en estudio, Acuolens resultó ser la más tóxica in vitro, tanto a tiempos cortos como largos (AU)

Influence of topical anesthesia on tests diagnostic of blepharitis-associated dry eye syndrome.

PURPOSE: Despite the high frequency and clinical relevance of blepharitis-associated dry-eye syndrome, no agreement exists about whether diagnostic tests should be performed with or without topical anesthesia. The aim of this study was to compare the influence of topical anesthesia on the mean values of Schirmer's test, tear lysozyme, tear lactoferrin, and tear osmolarity in patients suffering from blepharitis-associated dry eye syndrome. METHODS: The authors compared the mean values of Schirmer's test, tear osmolarity, tear lysozyme (turbidimetric assay), and tear lactoferrin (radial immunodiffusion) before and after topical anesthesia in the following groups: 56 normal subjects (group 1), 62 blepharitis patients (group 2), and 15 patients with blepharitis-associated dry eye syndrome (group 3). All clinical and laboratory tests were performed by masked observers. RESULTS: In group I, mean values of Schirmer's test decreased 24.8% (p < 0.01) when performed after application of topical anesthesia. The other tests were not significantly modified. In groups 2 and 3, significant differences were seen in Schirmer's test (25.33% and 24.19% respectively, p < 0.001) and the lysozyme determination (14.00% and 13.22% respectively, p < 0.01). Differences between the normal subjects (group I) and the patient groups increased when the tests were performed after application of topical anesthesia reaching statistical significance in group 3 for all the tests. CONCLUSIONS: Performing diagnostic tests after topical anesthesia instillation could be useful in detecting dry eye associated with blepharitis.

The rBAT gene is responsible for L-cystine uptake via the b0,(+)-like amino acid transport system in a "renal proximal tubular" cell line (OK cells).

Several studies have shown that the cRNA of human, rabbit, or rat rBAT induces in Xenopus oocytes sodium-independent, high affinity uptake of L-cystine via a system b0,(+)-like amino acid exchanger. We have shown that mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-425; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from oocytes, no other expression system has been used for transfection of functional rBAT activity. Furthermore, the b0,(+)-like transport activity has not been clearly described in the kidney or intestine. Here, we report that a "proximal tubular-like" cell line derived from opossum kidney (OK cells) expresses an rBAT transcript. Poly(A)+ RNA from OK cells induced by system b0,(+)-like transport activity in oocytes. This was hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase chain reaction-amplified cDNA fragment (approximately 700 base pairs) from OK cell RNA corresponds to an rBAT protein fragment 65-69% identical to those from human, rabbit and rat kidneys. We have also examined transport of l-cystine in OK cells and found characteristics very similar to the amino acid exchanger activity induced by rBAT cRNA in oocytes. Uptake of L-cystine was of high affinity, sodium-independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the same specificity as rBAT-induced transport activity in oocytes. Furthermore, it was localized to the apical pole of confluent OK cells. To demonstrate that the rBAT protein is functionally related to this transport activity, we have transfected OK cells with human rBAT antisense and sense sequences. Transfection with rBAT antisense, but not with rBAT sense, resulted in the specific reduction of rBAT mRNA expression and b0,(+)-like transport activity. These results demonstrate that rBAT is functionally related to the L-cystine uptake via system b0,(+)-like in the apical pole of the renal OK cell line.

Experimental model of allergic conjunctivitis to ragweed in guinea pig.

An experimental model of ocular allergy was developed in the guinea pig by exposing these animals to ragweed through topical contact on nasal and conjunctival mucosae, followed by subsequent challenge with ragweed contact on the conjunctiva. Animals developed clinical and histological signs of allergy with and without the use of interleukin 4 as adjuvant. This model mimics human hay fever conjunctivitis more naturally than do animal models previously reported, and it may be subsequently more valuable in the study of the response of allergic conjunctivitis to different therapeutic approaches.

Early and late phases of ocular anaphylaxis in actively immunized guinea pigs.

A model of ocular anaphylaxis with distinct early- and late-phase components was studied in actively immunized guinea pigs. Twenty guinea pigs were injected with dinitrophenylated (DNP) bovine gamma globulin emulsified in Freund's complete adjuvant and challenged topically with di-DNP-lysine. Clinical signs were monitored over a 48 h period. An early-phase reaction (EPR) characterized by conjunctival edema, conjunctival erythema, lid swelling, and lid redness was observed. This reaction peaked at 0.5 h after challenge and subsided to a low point at 3-4 h. Subsequently, a second episode of lid swelling and lid redness was observed at 4-8 h. All animals in both groups exhibited an EPR. In addition, 75% of the animals underwent an EPR and an LPR. No animals exhibited an isolated LPR. Of the animals that underwent a dual response, 47% were biphasic, 6% were prolonged and 47% were multiphasic. The development of an active model of ocular anaphylaxis exhibiting both EPR and LPR components will enable studies of mechanisms which regulate the frequency and magnitude of these ocular allergic responses.