RESUMEN

Trichomonas vaginalis causes trichomoniasis, an inflammatory process related to an increased rate of HIV transmission. In order to study T. vaginalis infection response in a microorganism-free environment, an infection model was established providing a host­parasite interaction system useful to study the interplay between immune cells and the parasite. Infected mice peritoneal cells were immunophenotyped at different times after infection using flow cytometry. Neutrophils and macrophages showed the most relevant increase from third to 12th day post-infection. A high number of B lymphocytes were present on 15th day post-infection, and an increase in memory T cells was observed on sixth day post-infection. The levels of NO increased at day 10 post-infection; no significant influence was observed on T. vaginalis clearance. Increased viability of T. vaginalis was observed when the NETs inhibitors, metformin and Cl− amidine, were administrated, highlighting the importance of this mechanism to control parasite infection (43 and 86%, respectively). This report presents a comprehensive cell count of the immune cells participating against trichomoniasis in an in vivo interaction system. These data highlight the relevance of innate mechanisms such as specific population changes of innate immune cells and their impact on the T. vaginalis viability.

Asunto(s)

Tricomoniasis , Trichomonas vaginalis , Animales , Cinética , Ratones , Neutrófilos , Peritoneo

RESUMEN

Post-transcriptional control has emerged as a major regulatory event in gene expression and often occurs at the level of translation initiation. Although overexpression or constitutive activation of tyrosine kinases (TKs) through gene amplification, translocation or mutation are well-characterized oncogenic events, current knowledge about translational mechanisms of TK activation is scarce. Here, we report the presence of translational cis-regulatory upstream open reading frames (uORFs) in the majority of transcript leader sequences of human TK mRNAs. Genetic ablation of uORF initiation codons in TK transcripts resulted in enhanced translation of the associated downstream main protein-coding sequences (CDSs) in all cases studied. Similarly, experimental removal of uORF start codons in additional non-TK proto-oncogenes, and naturally occurring loss-of-uORF alleles of the c-met proto-oncogene (MET) and the kinase insert domain receptor (KDR), was associated with increased CDS translation. Based on genome-wide sequence analyses we identified polymorphisms in 15.9% of all human genes affecting uORF initiation codons, associated Kozak consensus sequences or uORF-related termination codons. Together, these data suggest a comprehensive role of uORF-mediated translational control and delineate how aberrant induction of proto-oncogenes through loss-of-function mutations at uORF initiation codons may be involved in the etiology of cancer. We provide a detailed map of uORFs across the human genome to stimulate future research on the pathogenic role of uORFs.

Asunto(s)

Sistemas de Lectura Abierta/fisiología , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas/metabolismo , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Redes Reguladoras de Genes/fisiología , Células HEK293 , Células HeLa , Humanos , Proteínas Tirosina Quinasas/genética , Proto-Oncogenes Mas

RESUMEN

INTRODUCCIÓN. Analizar la concordancia y validación a diez años de dos ecuaciones de riesgo coronario que utilizan la función de Framingham calibrada para población española (REGICOR y DORICA) en pacientes diabéticos tipo 2. PACIENTES Y MÉTODOS. Estudio descriptivo, longitudinal, de seguimiento de una cohorte durante 10 años. Un total de 131 pacientes diabéticos de un centro de salud urbano, de 35 a 64 años de edad, sin antecedentes de cardiopatía isquémica, a quienes se les pudo calcular el riesgo coronario antes del 1-01-1995. RESULTADOS. El porcentaje real de eventos coronarios fue del 9,9% (8,8% en varones y 11,1% en mujeres). El riesgo coronario global calculado en la ecuación de Framingham-REGICOR fue del 8,9%, ajustándose perfectamente al riesgo coronario en varones (8,8%) e infraestimándolo en mujeres (9,0%). En cambio, la ecuación de Framingham-DORICA sobreestimó el riesgo global de la cohorte (17,3% frente al 9,9% de eventos), tanto en varones (19,7%) como en mujeres (14,8%), siendo sus riesgos significativamente distintos (p < 0,05). La concordancia entre las dos funciones fue aceptable (índice Kappa = 0,5). La aplicación inicial de la función de Framingham-DORICA, con un umbral para riesgo coronario alto ≥ 10%, y posteriormente la de Framingham-REGICOR a los pacientes catalogados como de riesgo coronario no alto (< 10%), permitieron clasificar correctamente el riesgo coronario de los pacientes diabéticos. CONCLUSIONES. La concordancia entre las funciones de Framingham-REGICOR y DORICA es aceptable, siendo REGICOR la que más se aproximó al riesgo real de la cohorte. La aplicación secuencial de ambas ecuaciones clasifica correctamente el riesgo coronario de los pacientes diabéticos

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INTRODUCTION. Analyze the concordance and validation at ten years of two coronary risk equations that use the calibrated Framingham function for Spanish population (REGICOR and DORICA) in type 2 diabetic patients. PATIENTS AND METHODS. Descriptive, longitudinal, follow-up study of a cohort over 10 years. A total of 131 diabetic patients from an urban health care center, from 35 to 65 years of age, without a background of ischemic heart disease, in whom coronary risk could be calculated before 1-01-1995. RESULTS. The real percentage of coronary events was 99% (8.8% in males and 11.1% in women). Global coronary risk calculated in the Framingham-REGICOR equation was 8.9%, it being perfectly adjusted to coronary risk in males (8.8%) and underestimated in women (9.0%). On the contrary, the Framingham-DORICA equation overestimated the global risk of the cohort (17.3% versus 9.9% of events) in both males (19.7%) and women (14.8%), their risks being significantly different (p < 0.05). Concordance between the two functions was acceptable (Kappa index = 0.5). Initial application of the Framingham-DORICA function with a threshold for high coronary risk ≥ 10% and then the Framingham-REGICOR to patients listed as not high coronary risk (< 10%) made it possible to correctly classify the coronary risk of the diabetic patients. CONCLUSIONS. Concordance between the Framingham-REGICOR and DORICA is acceptable, the REGICOR being that which approached the real risk of the cohort most. The sequential application of both equations correctly classified the coronary risk of diabetic patients

Asunto(s)

Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Diabetes Mellitus/complicaciones , Enfermedades Cardiovasculares/epidemiología , Modelos Cardiovasculares , Ajuste de Riesgo/métodos , Factores de Riesgo , Estudios Prospectivos

RESUMEN

The exacerbation of asthma during viral infections is mainly explained by neutrophils infiltrating into the airways. However, enhanced functions of eosinophils are also observed. The aim of this study was to reveal the mechanism of how eosinophils are activated during and after viral infection of the airways, using a model of viral infection. A synthetic double-stranded RNA, poly inosinic-cytidyric acid (poly(IC)), was transfected to a human airway epithelial cell line (BEAS-2B) and the primary bronchial epithelial cells, to mimic a viral infection. The production of chemokines from the cells was investigated. The transfection of poly(IC), alone, marginally affected the eotaxin-3 production of the cells. However, the transfection of poly(IC) prior to interleukin (IL)-4 stimulation enhanced eotaxin-3 production. Poly(IC) transfection increased mRNA and protein expressions of IL-4 receptor (R)alpha and IL-2Rgamma, components of the IL-4R. In BEAS-2B cells, IL-4-mediated phosphorylation of signal transducer and activator of transcription six was enhanced in poly(IC) transfected cells. This was reversed by the addition of anti-IL-4Ralpha antibody, suggesting the role of an increased number of IL-4 receptors in enhanced IL-4-induced eotaxin-3 production. Poly(IC)-induced upregulation of IL-4Ralpha was inhibited by treatment with cycloheximide or dexamethasone. In conclusion, these results suggest that viral airway infection may enhance interleukin-4-induced eotaxin-3 production through upregulation of the interleukin-4 receptor in airway epithelial cells.

Asunto(s)

Quimiocinas CC/metabolismo , Células Epiteliales/metabolismo , Receptores de Interleucina-4/metabolismo , Mucosa Respiratoria/metabolismo , Infecciones del Sistema Respiratorio/metabolismo , Virosis/metabolismo , Células Cultivadas , Quimiocina CCL26 , Quimiocinas CC/genética , Humanos , ARN Bicatenario/genética , Infecciones del Sistema Respiratorio/complicaciones , Transfección/métodos , Regulación hacia Arriba , Virosis/complicaciones

RESUMEN

Standard random Boolean networks display an order-disorder phase transition. We add to the standard random Boolean networks a disconnection rule that couples the control and order parameters. In this way, the system is driven to the critical line transition. Under the influence of perturbations the system points out self-organized critical behavior. Several numerical simulations have been done and compared with a proposed analytical treatment.

Asunto(s)

Red Nerviosa , Animales , Fenómenos Biofísicos , Biofisica , Simulación por Computador , Humanos , Modelos Neurológicos , Modelos Estadísticos , Modelos Teóricos , Dinámicas no Lineales , Factores de Tiempo

RESUMEN

Retinal plasticity has been shown in the adult visual nervous system in mammals. Following a retinal lesion (scotoma) there is a reorganization of the cortical receptive field distribution: cortical neurons selective to visual stimuli in the area of the visual field corresponding to the retinal lesion, become selective to other parts of the visual field. In this work, we study this effect with a self-organizing neural network. In a first stage, the network reaches a pattern of connectivity that represents normal development of neuronal selectivity. The scotoma is simulated by perturbing accordingly the properties of a region of the input layer representing the retina. The system evolves to a new receptive field distribution mainly by means of the reorganization of the intra cortical connectivity. No major change of the geniculo cortical connectivity is detected. This may explain the surprisingly short time scale of the event.

Asunto(s)

Simulación por Computador , Modelos Neurológicos , Plasticidad Neuronal/fisiología , Corteza Visual/fisiología , Factores de Edad , Animales , Mamíferos , Escotoma/fisiopatología , Campos Visuales/fisiología

RESUMEN

Interleukin (IL)-4, IL-10, and IL-13, Th2 cell-derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B(4) in human polymorphonuclear leukocytes (PMNs). Production of LTB(4) was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), and LTA(4) hydrolase, which were involved in the synthesis of LTB(4), was determined by reverse transcription-polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB(4) synthesis and increased mRNA expression and protein synthesis of LTA(4) hydrolase, but not those of cPLA(2) or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB(4), a potent chemotactic factor to PMNs, at the enzyme level of LTA(4) hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation. (Blood. 2000;96:601-609)

Asunto(s)

Epóxido Hidrolasas/biosíntesis , Interleucina-13/farmacología , Interleucina-4/farmacología , Neutrófilos/enzimología , Araquidonato 5-Lipooxigenasa/genética , Northern Blotting , Western Blotting , Calcimicina/farmacología , Cromatografía Líquida de Alta Presión , Inducción Enzimática , Epóxido Hidrolasas/genética , Humanos , Interleucina-10/farmacología , Ionóforos/farmacología , Leucotrieno B4/biosíntesis , Fosfolipasas A/genética , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

The nitration of methylnaphthalenes with NO(2)BF(4) and NOBF(4) was examined in order to shed light on the controversial aromatic nitration mechanism, electrophilic vs charge-transfer process. The NO(2)(+) nitration of 1,8-dimethylnaphthalene showed a drastic regioselectivity change depending on the reaction temperature, where ortho-regioselectivity at -78 degrees C and para-regioselectivity at 0 degrees C were considered to reflect the electrophilic and the direct or alternative charge-transfer process, respectively, because the NO(+) nitration through the same reaction intermediates as in the NO(2)(+) nitration via a charge-transfer process resulted in para-regioselectivity regardless of the reaction temperature. The NO(2)(+) nitration of redox potential methylnaphthalenes higher than 1,8-dimethylnaphthalene gave a similar ortho-regioselectivity enhancement to 1,8-dimethylnaphthalene at lower temperature, thus reflecting the electrophilic process. On the other hand, the NO(2)(+) nitration of redox potential methylnaphthalenes lower than 1,8-dimethylnaphthalene showed para-regioselectivity similar to the NO(+) nitration, indicating the direct or alternative charge-transfer process. In the presence of strong acids where the direct charge-transfer process will be suppressed by protonation, the ortho-regioselectivity enhancement was observed in the NO(2)(+) nitration of 1,8-dimethylnaphthalene, suggesting that the direct charge-transfer process could be the main process to show para-regioselectivity. These experimental results imply that the NO(2)(+) nitration proceeds via not only electrophilic but also direct charge-transfer processes, which has been considered to be unlikely because of the high energy demanding process of a bond coordination change between NO(2)(+) and NO(2). Theoretical studies at the MP2/6-31G(d) level predicted ortho- and para-regioselectivity for the NO(2)(+) nitration via electrophilic and charge-transfer processes, respectively, and the preference of the direct charge-transfer process over the alternative one, which support the experimental conclusion

RESUMEN

AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had only weak inhibitory action on the release of beta-HEX. The results indicate that anti-allergic action of AS-35 is predominantly attributable to its inhibition of LT synthesis by suppressing three consecutive enzymes for LTC(4) synthesis.

Asunto(s)

Antagonistas de Leucotrieno/farmacología , Leucotrieno B4/antagonistas & inhibidores , Leucotrieno B4/biosíntesis , Leucotrieno C4/antagonistas & inhibidores , Leucotrieno C4/biosíntesis , Piridinas/farmacología , Pirimidinonas/farmacología , Tetrazoles/farmacología , Animales , Ácido Araquidónico/metabolismo , Sistema Libre de Células , Citosol/enzimología , Leucotrieno A4/metabolismo , Fosfolipasas A/metabolismo , Ratas , Especificidad por Sustrato , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Células Tumorales Cultivadas/metabolismo , beta-N-Acetilhexosaminidasas/metabolismo

RESUMEN

La indentación escleral mediante un explante circunferencial de silicona sólida, es un procedimiento habitualmente realizado en la cirugía del desprendimiento de retina. Proponemos un método sencillo para calcular la longitud que debe tener la banda de silicona que deseamos colocar. Para ello medimos el diámetro horizontal del globo ocular, a la altura de la inserción de los músculos rectos horizontales. Tras obtener dicha medida, calculamos la longitud teórica de la circunferencia correspondiente a ese diámetro. A dicha longitud restamos 8 mm., y la longitud obtenida finalmente, es la que asignamos a la banda de silicona que colocamos en el globo ocular para obtener una buena identación escleral (AU)

Asunto(s)

Humanos , Siliconas/uso terapéutico , Desprendimiento de Retina/cirugía , Pesos y Medidas

RESUMEN

Polymorphonuclear leukocytes (PMNs) produce arachidonic acid (AA) metabolites including thromboxane A2 (TXA2). These cells are the first line of defense against bacterial invasion, which often causes endotoxin shock. TXA2 which plays an important role in the pathogenesis of endotoxin shock is synthesized by three consecutive enzyme activation, cytosolic phospholipase A2 (cPLA2), prostaglandin H2 synthase (PHS type 1 and type 2) and TXA2 synthase. Among them, cPLA2- and PHS-2 activity is known to be transcriptionally and/or posttranscriptionally up-regulated by various bioactive substances including lipopolysaccharide (LPS), a bacterial endotoxin, in many cell types. We investigated the action of LPS on TXA2 synthesis in human PMNs. A23187-stimulated production of thromboxane B2 (TXB2, a stable metabolite of TXA2), assayed by specific radioimmunoassay (RIA), was significantly increased from 566.7+/-44.1 pg/10(6) cells to 966.7+/-44.1 pg/10(6) cells (p<0.05) after 6 h-exposure to LPS at the concentration of 100 ng/ml. Messenger RNA for PHS-2, PHS-1, TXA2 synthase and cPLA2, which was assessed by reverse transcription-polymerase chain reaction (RT-PCR), was expressed in PMNs without LPS stimulation. Although PHS-2 was putatively an inducible enzyme, abundance of mRNA for PHS-2 in PMNs without LPS stimulation was detectable. Messenger RNA abundance for PHS-2 and cPLA2, but not for PHS-1 and TXA2 synthase, was enhanced by LPS-treatment, indicating that the increased production of TXB2 was attributable to the up-regulation of cPLA2 and PHS-2. We conclude that (1) PHS-2 plays a more important role than PHS-1 in the production of TXA2 in human PMNs and (2) TXA2 synthesis in human PMNs is transcriptionally up-regulated by new induction of cPLA2 as well as PHS-2, when the cells encounter endotoxin producing bacteria.

Asunto(s)

Isoenzimas/biosíntesis , Lipopolisacáridos/farmacología , Neutrófilos/efectos de los fármacos , Fosfolipasas A/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Aspirina/farmacología , Calcimicina/farmacología , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Inducción Enzimática/efectos de los fármacos , Fosfolipasas A2 Grupo IV , Humanos , Isoenzimas/genética , Proteínas de la Membrana , Neutrófilos/enzimología , Nitrobencenos/farmacología , Fosfolipasas A/genética , Fosfolipasas A2 , Prostaglandina-Endoperóxido Sintasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfonamidas/farmacología , Tromboxano B2/biosíntesis , Tromboxano-A Sintasa/biosíntesis , Tromboxano-A Sintasa/genética

RESUMEN

Six native Spanish cattle breeds have been characterized by using 30 microsatellite markers. The studied populations can be divided into three groups: Brown orthoid (Asturian Mountain, Asturian Lowland and the Nord-west Brown Group), Red convex (Pyrenean and Menorquina) and the Iberian bovine (Fighting bull). Allele frequencies were calculated and used for the characterization of the breeds and the study of their genetic relationships. Different genetic distance measures were calculated and used for dendogram construction. The closest populations were those representing Asturian breeds, the most divergent being Menorquina and Fighting Bull. The latter also showed the lowest diversity values (mean number of alleles per locus and heterozygosity). Genetic distances obtained between the other populations under analysis were similar to those reported for different European cattle breeds. This work analyzes the recent origin of these populations and contributes to the knowledge and genetic characterization of European native breeds.

Asunto(s)

Bovinos/genética , Repeticiones de Microsatélite , Alelos , Animales , Frecuencia de los Genes , Variación Genética , Heterocigoto , Filogenia , Polimorfismo Genético , España , Especificidad de la Especie

RESUMEN

We have observed an inhibitory action of magnolol on the production of leukotriene (LT) C4 and LTB4, important lipid mediators in allergy and inflammation. IgE- and A23187-stimulated production of LTC4 and LTB4 was measured by radio-immunoassay (RIA) in the absence or presence of various concentrations of magnolol in intact rat basophilic leukemia (RBL)-2H3 cells. Magnolol dose-dependently inhibited synthesis of LTC4 and LTB4. Magnolol inhibited the IgE-mediated increase of intracellular calcium ion concentration, resulting in the inhibition of cytosolic phospholipase A2 (cPLA2) and possibly 5-lipoxygenase (5-LO), both calcium ion-dependent enzymes. In cell-free studies magnolol inhibited LTC4 synthase activity. LTA4 hydrolase activity was only inhibited at the higher concentration (2.5 x 10(-5)M). These results indicate that magnolol inhibits production of LTs by inhibiting PLA2, 5-LO, LTC4 synthase and LTA4 hydrolase which are essential for LT-synthesis. Magnolol may have anti-allergic effect by blocking LT-synthesis.

Asunto(s)

Compuestos de Bifenilo/farmacología , Leucemia Basofílica Aguda/metabolismo , Leucotrieno B4/biosíntesis , Leucotrieno C4/biosíntesis , Lignanos , Animales , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Glutatión Transferasa/antagonistas & inhibidores , Leucemia Basofílica Aguda/enzimología , Leucemia Basofílica Aguda/patología , Inhibidores de la Lipooxigenasa , Ratas , Células Tumorales Cultivadas

RESUMEN

There are substantial numbers of reports showing that leukotrienes (LTs) play important roles in adult asthma. No definite evidence has been demonstrated that LTs are involved in asthma attacks in children, although it is highly expected. In this report, we demonstrated that the levels of LTB4 and LTC4 but not thromboxane B2 (TXB2), a stable metabolite of TXA2, were significantly elevated in the bronchoalveolar lavage fluid, which was obtained from intubated and mechanically ventilated children with severe asthma attacks. This is direct evidence that LTB4 and LTC4 predominantly participate in asthma attacks in pediatric patients.

Asunto(s)

Asma/fisiopatología , Leucotrieno B4/fisiología , Leucotrieno C4/fisiología , Tromboxano A2/fisiología , Adolescente , Preescolar , Femenino , Humanos , Masculino

RESUMEN

To determine the inhibitory mechanisms of terfenadine on the synthesis of leukotriene C4 (LTC4), an important mediator in allergic diseases, we evaluated the action of terfenadine on the IgE-dependent production of LTC4 in rat basophilic leukaemia 2H3 cells. Rat IgE-loaded cells were stimulated with anti-IgE in the presence or absence of various concentrations of terfenadine and the level of LTC4 released into the medium was measured by performing a specific radio immunoassay. Terfenadine inhibited the synthesis of LTC4 to 67.2% at a concentration of 5 microg/ml. LT synthesis was directly suppressed by inhibition of 5-lipoxygenase (5-LO) through calcium ion-independent mechanisms, and was also possibly suppressed by inhibition of cytosolic phospholipase A2 and 5-LO by blocking the influx of intracellular calcium ion that was initiated by IgE-related stimulation.

Asunto(s)

Antialérgicos/farmacología , Antagonistas de Leucotrieno , Leucotrienos/biosíntesis , Terfenadina/farmacología , Animales , Antibacterianos/farmacología , Anticuerpos Antiidiotipos/farmacología , Ácido Araquidónico/metabolismo , Calcimicina/farmacología , Calcio/metabolismo , Inmunoglobulina E/inmunología , Inmunoglobulina E/farmacología , Leucotrieno A4/metabolismo , Leucotrieno B4/biosíntesis , Leucotrieno B4/metabolismo , Leucotrieno C4/biosíntesis , Leucotrieno C4/metabolismo , Especificidad por Sustrato , Tritio , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

Human leukemia (HL) 60 cells were differentiated by dimethylsulfoxide (DMSO) treatment to granulocyte-like cells, leukotriene (LT) synthesizing activity of which was increased in response to the differentiation of the cells. Four synthesizing enzymes, cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), LTA4 hydrolase and LTC4 synthase, and an enzyme associated protein, 5-lipoxygenase activating protein (FLAP) are involved in the generation of LTC4 and LTB4. We examined the expression of messenger RNA (mRNA) for these LT synthesizing enzymes and an associated protein in DMSO differentiated HL-60 cells by reverse transcriptase polymerase chain reaction (RT-PCR). The production of LTC4 and LTB4, measured by radioimmunoassay (RIA), was increased after the incubation with DMSO for more than 3 days. Messenger RNA abundance for 5-LO, LTC4 synthase and LTA4 hydrolase was increased, that for FLAP was stable, but that for cPLA2 was decreased. These results indicate that DMSO induced increase of LT synthesis is associated with the increase of mRNA expression of 5-LO, LTC4 synthase and LTA4 hydrolase, although the precise regulatory mechanisms of the increased mRNA expression are not determined. We also investigated an action of dexamethasone (DEX) on DMSO-induced enhancement of LT synthesis. DEX suppressed DMSO induced increase of LTC4 synthesis, but rather enhanced DMSO induced LTB4 production. The DEX attenuated the DMSO-induced increase of mRNA expression for LTC4 synthase, but showed no effect on that for LTA4 hydrolase. The inhibition of LTC4 synthesis is associated with the suppression of mRNA expression for LTC4 synthase. However, increased LTB4 synthesis by DEX is regulated by the mechanisms which are independent from mRNA level of LTA4 hydrolase.

Asunto(s)

Dexametasona/farmacología , Dimetilsulfóxido/farmacología , Leucotrienos/biosíntesis , Proteínas Activadoras de la 5-Lipooxigenasa , Araquidonato 5-Lipooxigenasa/metabolismo , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Células HL-60 , Humanos , Leucotrieno A4/metabolismo , Leucotrieno C4/metabolismo , Proteínas de la Membrana/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismo , Factores de Tiempo

Asunto(s)

Proteínas de Unión al ADN/genética , Perros/genética , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN/genética , Femenino , Factores de Transcripción de Tipo Kruppel , Masculino , Reacción en Cadena de la Polimerasa , Análisis para Determinación del Sexo , Factores de Transcripción , Cromosoma X/genética , Cromosoma Y/genética

RESUMEN

We examined the action of Shinpi-To (Formula divinita; TJ-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC4 and LTB4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C4 and LTB4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A2 (PLA2), 5-lipoxygenase (5-LO). LTC4 synthase, and LTA4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA2 activity. It also suppressed production of LTC4 and LTB4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC4 and LTB4 when LTA4-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca2+ ([Ca2+]i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA2 and 5-LO activities without affecting the mobilization of [Ca2+]i.

Asunto(s)

Medicamentos Herbarios Chinos/farmacología , Inmunoglobulina E/inmunología , Leucotrieno B4/biosíntesis , Leucotrieno C4/biosíntesis , Análisis de Varianza , Animales , Ácido Araquidónico/metabolismo , Asma/tratamiento farmacológico , Broncodilatadores/farmacología , Calcimicina/toxicidad , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Medicamentos Herbarios Chinos/uso terapéutico , Efedrina/análogos & derivados , Efedrina/farmacología , Ionóforos/toxicidad , Marcaje Isotópico , Leucemia Basofílica Aguda/patología , Leucotrieno A4/biosíntesis , Leucotrieno B4/metabolismo , Leucotrieno C4/metabolismo , Inhibidores de la Lipooxigenasa/farmacología , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Radioinmunoensayo , Ratas , Tritio , Células Tumorales Cultivadas

Asunto(s)

Endopeptidasas/fisiología , Entamoeba histolytica/patogenicidad , Animales , Biomarcadores/química , Membrana Celular/enzimología , Entamoeba histolytica/enzimología , Entamoeba histolytica/ultraestructura , Virulencia

RESUMEN

The effects of honokiol, a diphenyl compound extracted from a Chinese herbal medicine, on leukotriene (LT) synthesis were evaluated in rat basophilic leukemia (RBL) cells. The production of LTC4 and LTB4 stimulated by the Ca2+ ionophore A23187 was measured in RBL-1 cells by high-performance liquid chromatography. Honokiol inhibited the production of LTC4 and LTB4 stimulated by A23187 in RBL-1 cells. Honokiol did not inhibit either phospholipase A2 activity, measured by the release of 3H-arachidonic acid (AA), or LTC4 synthase and LTA4 hydrolase activities, measured with LTA4-free acid as substrate. The synthesis of LTC4 and LTB4 from AA in RBL-1 cell lysates in the presence of Ca2+ was inhibited by honokiol. These results indicate that honokiol blocks LT synthesis by inhibiting 5-lipoxygenase activity. Honokiol also inhibited immunoglobulin E-mediated production of these LTs in RBL-2H3 cells, which was measured by a specific radioimmunoassay (RIA). These results suggest that honokiol may exhibit antiallergic actions by inhibiting LT synthesis in immediate-type hyperreactivity.

Asunto(s)

Antialérgicos/farmacología , Compuestos de Bifenilo/farmacología , Leucemia Basofílica Aguda/metabolismo , Antagonistas de Leucotrieno , Leucotrienos/biosíntesis , Lignanos , Animales , Leucemia Basofílica Aguda/enzimología , Ratas , Células Tumorales Cultivadas