Plasmodium falciparum, the causative agent of malaria, relies extensively on glycolysis coupled with homolactic fermentation during its blood-borne stages for energy production. Selective inhibitors of the parasite lactate dehydrogenase (LDH), central to NAD ؉ regeneration, therefore potentially provide a route to new antimalarial drugs directed against a novel molecular target. A series of heterocyclic, azole-based compounds are described that preferentially inhibit P. falciparum LDH at sub-micromolar concentrations, typically at concentrations about 100-fold lower than required for human lactate dehydrogenase inhibition. Crystal structures show these competitive inhibitors form a network of interactions with amino acids within the active site of the enzyme, stacking alongside the nicotinamide ring of the NAD ؉ cofactor. These compounds display modest activity against parasitized erythrocytes, including parasite strains with known resistance to existing anti-malarials and against Plasmodium berghei in BALB/c mice. Initial toxicity data suggest the azole derivatives have generally low cytotoxicity, and preliminary pharmocokinetic data show favorable bioavailability and circulation times. These encouraging results suggest that further enhancement of these structures may yield candidates suitable for consideration as new therapeutics for the treatment of malaria. In combination these studies also provide strong support for the validity of targeting the Plasmodium glycolytic pathway and, in particular, LDH in the search for novel anti-malarials.Plasmodium parasites are believed to lack a functional Krebs (citric acid) cycle for at least part of their life cycle and hence rely extensively on ATP generation via the anaerobic fermentation of glucose (see Ref. 1 for review). The energy requirement of the parasitized erythrocyte is such that utilization of glucose is up to 100 times greater than in nonparasitized erythrocytes (2, 3), and virtually all glucose can be accounted for by production of lactate (2). Lactate dehydrogenase (LDH), 1 the last enzyme in the glycolytic pathway in Plasmodium falciparum, is a 2-hydroxy acid oxidoreductase that converts pyruvate to lactate and simultaneously the conversion of NADH to NAD ϩ . As a constant supply of NADH is a prerequisite for glycolysis, and LDH acts as the primary source in Plasmodium for the regeneration of NADH from NAD ϩ , inhibition of LDH is expected to stop production of ATP, with subsequent P. falciparum cell death. Any compound that blocks the LDH enzyme is a potentially potent antimalarial with a different mode of action to existing drugs. As such, P. falciparum lactate dehydrogenase (pfLDH) has been suggested as a drug target by several authors (4 -6). One well recognized difficulty is that the drug must potently inhibit pfLDH yet show much less activity against the three human LDH (hsLDH) isoforms.A comparison of the crystal structures of both P. falciparum and human LDH (7,8) shows the following two key differences: namely positioning of the NADH factor, re...

The p75 neurotrophin receptor (p75, also known as NGFR) is a multifaceted signalling receptor that regulates neuronal physiology, including neurite outgrowth, and survival and death decisions. A key cellular aspect regulating neurotrophin signalling is the intracellular trafficking of their receptors; however, the post-endocytic trafficking of p75 is poorly defined. We used sympathetic neurons and rat PC12 cells to study the mechanism of internalisation and postendocytic trafficking of p75. We found that p75 internalisation depended on the clathrin adaptor protein AP2 and on dynamin. More surprisingly, p75 evaded the lysosomal route at the level of the early endosome, instead accumulating in two different types of endosomes, Rab11-positive endosomes and multivesicular bodies (MVBs) positive for CD63, a marker of the exosomal pathway. Consistently, depolarisation by KCl induced the liberation of previously endocytosed full-length p75 into the extracellular medium in exosomes. Thus, p75 defines a subpopulation of MVBs that does not mature to lysosomes and is available for exosomal release by neuronal cells.

S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS) isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2), SERCA2 and the L-Type Ca2+ channel (LTCC). In the isolated rat heart, isoproterenol (10 nM, 3-min) increased S-nitrosylation of total cardiac proteins (+46±14%) and RyR2 (+146±77%), without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC) and Nω-propyl-l-arginine decreased basal contractility and relaxation (−25–30%) and basal S-nitrosylation of total proteins (−25–60%), RyR2, SERCA2 and LTCC (−60–75%). NOS-1 inhibition reduced (−25–40%) the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (−85±7%). Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethyl)ornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1) normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2) β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect.

The biomedical potential of the edible red seaweed Agarophyton chilense (formerly Gracilaria chilensis) has not been explored. Red seaweeds are enriched in polyunsaturated fatty acids and eicosanoids, which are known natural ligands of the PPARγ nuclear receptor. PPARγ is the molecular target of thiazolidinediones (TZDs), drugs used as insulin sensitizers to treat type 2 diabetes mellitus. Medical use of TZDs is limited due to undesired side effects, a problem that has triggered the search for selective PPARγ modulators (SPPARMs) without the TZD side effects. We produced Agarophyton chilense oleoresin (Gracilex®), which induces PPARγ activation without inducing adipocyte differentiation, similar to SPPARMs. In a diet-induced obesity model of male mice, we showed that treatment with Gracilex®improves insulin sensitivity by normalizing altered glucose and insulin parameters. Gracilex®is enriched in palmitic acid, arachidonic acid, oleic acid, and lipophilic antioxidants such as tocopherols and β-carotene. Accordingly, Gracilex® possesses antioxidant activity in vitro and increased antioxidant capacity in vivo in Caenorhabditis elegans. These findings support the idea that Gracilex® represents a good source of natural PPARγ ligands and antioxidants with the potential to mitigate metabolic disorders. Thus, its nutraceutical value in humans warrants further investigation.

Las hospitalizaciones por Slndrome Diarreico agudo en el lactante afectan en un porcentaje importante a lactantes desnutrtdos, y a menores de 6 meses, siendo a su vez estos grupos los que con mayor frecuencia presentan intolerancia secundaria a disacaridos, y evolucionan t6rpidamente> Por ello requieren una estada mas prolongada en el establecimiento hospitalario, con el consiguiente aumento de los riesgos de infecci6n intrahospitalaria, aparte del mayor costo que esto implica.En el Servicio de Pediatrfa del Hospital Josefina Martinez de Ferrari se ha usado en los ultimos arios en forma empirica y con buenos resultados aparentes, una f6rmula para realimentaci6n de diarrea en base a glucosa, quesillo de babeurre (Eled6n), zanahoria y aceite.Es una f6rmula pobre en lactosa y sacarosa, de costo similar a la leche entera en polvo, bien tolerada, con propiedades astringentes y que aporta nutrientes en cantidades aceptables para cubrir los requerimientos basal es del nino durante el periodo de realimentaci6n.Se considerb necesario efectuar un estudio comparativo de la evoluci6n clfnica de los laclantes realimentados con dicha formula en relaci6n a la leche entera en polvo (Purita) reconstituida al 8%, ya que, aunque se habfa efectuado anteriormente en el Servicio una invesrjgaci6n similar que mostr6 resultados bastante satisfactorios a favor de la crema de zanahoria, estos resultados no fueron publicados en su oportunidad. OBJETIVOSEl presente trabajo tuvo como objefeivo evaluar las posibles ventajas o inconvenientes de la crema de zanahorias con diarrea aguda y deshidratacion. MATERIAL Y METODOLa crema de zanahorias se prepara sometiendo a ebullici6n por un minuto Eled6n preparado al 10%, para que coagulen las proteinas, separando luego el cuajo en gasa y eliminando el sobrenadante. Se agrega zanahoria cocida lieu ad a, glucosa al 5%, aceite y agua, hasta reconstituir el volumen inicial.El aporte de proteinas en la crema de zanahoria II es de 2.1 g%, comparable al de f6rmu-las con leche de vaca diluidas al 2/3, teniendo un p% de 11.5 que es adecuado para f6rmulas del lactante. En hidratos de carbono (glucosa) el aporte es tambien apropiado, sucediendo lo mismo con los lfpidos. De modo que la f6rmula es, en general, bien proporcionada en cuanto a nutrientes basicos, otorgando 72.7 cal. por 100 ml. (Tabla N.° 1).En la Tabla N.° 2 se efectiia una comparaci6n en el aporte de aminoacidos esenciales y no esenciales de la leche humana, leche de vaca y crema de zanahorias.Puede apreciarse que en isoleucina los valores son algo inferiores a los de la leche humana, en histidina no son concluyentes, quedando, ademas, sin informaci6n respecto a triptofano. De modo que no se pueden sacar conclusiones definitivas de la CZ II en cuanto a su aporte en aminoacidos esenciales.5