Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania ( Viannia ) and its evaluation as a drug target / Caracterización bioquímica de la enzima bifuncional dihidrofolato reductasa-timidilato sintasa de Leishmania ( Viannia ) y su evaluación como blanco molecular

Introduction. Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania ( Viannia ) species . Materials and methods. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. Results. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed K m and V max values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H 2 F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity ( Ki = 22.0 µM) than trimethoprim ( Ki = 33 µM) and pyrimethamine ( Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. Conclusion. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

Introducción. La dihidrofolato reductasa (DHFR) se ha utilizado como blanco molecular en tratamientos antibacterianos, anticancerígenos y antipalúdicos. También, actúa como blanco molecular en Leishmania ; sin embargo, no existen reportes de la enzima bifuncional en especies de Leishmania ( Viannia ). Materiales y métodos. Se ha aislado y expresado en Escherichia coli el gen que codifica para la enzima bifuncional DHFR y la timidilato-sintasa (TS) de Leishmania braziliensis . La enzima recombinante se purificó y caracterizó, y se evaluó el efecto inhibitorio de algunos antifolatos, así como de cuatro alcaloides aporfínicos. Resultados. El gen se compone de aproximadamente 1.560 pb y codifica un péptido de 58 kDa que contiene los dominios DHFR y TS ligados en una sola cadena polipeptídica. La enzima recombinante DHFR-TS, utilizando el dihidrofolato (H2F) como sustrato, presentó valores de K m y V max de 55,35 ± 4,02 (media ± el error estándar de la media) y de 0,02 ± 5,34 x 10 -4 , respectivamente. La enzima rDHFR-TS de L. braziliensis presentó valores de Ki para los antifolatos en el rango de micras. El metotrexato fue el inhibidor más potente de la actividad enzimática ( Ki =22,0 mM) en comparación del trimetoprim ( Ki =33 mM) y la pirimetamina ( Ki =68 mM). Estos valores de Ki son significativamente más bajos en comparación con los obtenidos para los alcaloides aporfínicos. Conclusión. Los resultados muestran el efecto inhibitorio de los antifolatos sobre la actividad enzimática, lo cual indica que la rDHFR-TS de L. braziliensis podría ser un modelo para estudiar moléculas antiprotozoarias potenciales.

Aging impairs insulin-stimulated glucose uptake in rat skeletal muscle via suppressing AMPKalpha

Insufficient intracellular fat oxidation is an important contributor to aging-related insulin resistance, while the precise mechanism underlying is unclear. AMP-activated protein kinase (AMPK) is an important regulator of intracellular fat oxidation and was evidenced to play a key role in high-glucose and high-fat induced glucose intolerance. In the present study, we investigated whether altered AMPK expression or activity was also involved in aging-related insulin resistance. Insulin sensitivity of rats' skeletal muscles was evaluated using in-vitro glucose uptake assay. Activity of alpha subunit of AMPK (AMPKalpha) was evaluated by measuring the phosphorylation of both AMPKalpha (P-AMPKalpha) and acetyl-CoA carboxylase (P-ACC), while expression of AMPKalpha was assessed by determining the mRNA levels of AMPKalpha1 and AMPKalpha2, and protein contents of AMPKalpha. Compared with 4-month old rats, 24-month old rats exhibited obviously impaired insulin sensitivity. At the same time, AMPKalpha activity significantly decreased, while AMPKalpha expression did not alter during aging. Glucose transporter 4 expression also decreased in old rats. Compared with 24-month old rats, administration of the specific activator of AMPK, 5-aminoimidazole-4-carboxamide riboside (AICAR), significantly elevated AMPKalpha activity and GluT4 expression. Also, aging-related insulin resistance was significantly ameliorated by AICAR treatment. In conclusion, aging-related insulin resistance is associated with impaired AMPKalpha activity and could be ameliorated by AICAR, thus indicating a possible role of AMPK in aging-induced insulin resistance.

Inhibition of rac1 Reduces PDGF-induced Reactive Oxygen Species and Proliferation in Vascular Smooth Muscle Cells

In vascular smooth muscle cells, reactive oxygen species (ROS) were known to mediate platelet-derived growth factor (PDGF)-induced cell proliferation and NADH/NADPH oxidase is the major source of ROS. NADH/NADPH oxidase is controlled by rac1 in non-phagocytic cells. In this study, we examined whether the inhibition of rac1 by adenoviral-mediated gene transfer of a dominant negative rac1 gene product (Ad.N17rac1) could reduce the proliferation of rat aortic vascular smooth muscle cells (RASMC) stimulated by PDGF via decreasing intracellular ROS. RASMC were stimulated by PDGF (80 ng/mL) with or without N-acetylcysteine 1 mM or infected with 100 mutiplicity of infection of Ad.N17rac1. Intracellular ROS levels were measured at 12 hr using carboxyl-2', 7'-dichlorodi-hydrofluorescein diacetate confocal microscopy. At 72 hr, cellular proliferation was evaluated by cell number counting and XTT assay. Compared with control, ROS levels were increased by 2-folds by PDGF. NAC and Ad.N17rac1 inhibited PDGF-induced increase of ROS by 77% and 65%, respectively. Cell number was increased by PDGF by 1.6-folds compared with control. NAC and Ad.N17rac1 inhibited PDGF-induced cellular growth by 45% and 87%, respectively. XTT assay also showed similar results. We concluded that inhibition of rac1 in RASMCs could reduce intracellular ROS levels and cellular proliferation induced by PDGF.