RESUMEN
Phagocyte functions are markedly inhibited after infection with the intracellular protozoan parasite Leishmania. This situation strongly favors the installation and propagation of this pathogen within its mammalian host. Previous findings by us and others have established that alteration of several signaling pathways (protein kinase C-, Ca2+- and protein-tyrosine kinases-dependent signaling events) were directly responsible for Leishmania-induced macrophage (MO) dysfunctions. Here we report that modulation of phosphotyrosine-dependent events with a protein tyrosine phosphatases (PTP) inhibitor, the peroxovanadium (pV) compound bpV(phen) (potassium bisperoxo(1,10-phenanthroline)oxovanadate(Vi)), can control host-pathogen interactions by different mechanisms. We observed that the inhibition of parasite PTP resulted in an arrest of proliferation and death of the latter in coincidence with cyclin-dependent kinase (CDK1) tyrosine 15 phosphorylation. Moreover the treatment of MO with bpV(phen) resulted in an increased sensitivity to interferon-gamma stimulation, which was reflected by enhanced nitric oxide (NO) production. This enhanced IFN-gamma-induced NO generation was accompanied by a marked increase of inducible nitric oxide synthase (iNOS) mRNA gene and protein expression. Finally we have verified the in vivo potency of bpV(phen) over a 6-week period of daily administration of a sub-toxic dose. The results revealed its effectiveness in controlling the progression of visceral and cutaneous leishmaniasis. Therefore PTP inhibition of Leishmania and MO by the pV compound bpV(phen) can differentially affect these eukaryotic cells. This strongly suggests that PTP plays an important role in the progression of Leishmania infection and pathogenesis. The apparent potency of pV compounds along with their relatively simple and versatile structure render them attractive pharmacological agents for the management of parasitic infections.
Asunto(s)
Interferón gamma/farmacología , Leishmaniasis/enzimología , Activación de Macrófagos , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacología , Leishmaniasis/patología , Ratones , Ratones Endogámicos BALB C , Vanadatos/farmacologíaRESUMEN
Three organophosphorus acid anhydrases have been isolated from E. coli by gel filtration and ion exchange column procedures, and further identified by gel electrophoresis. All three have molecular weights in the 120,000-140,000 range. Two of them hydrolyze racemic 1,2,2-trimethylpropylmethylphosphonofluoridate (soman) to completion at a single rate and, in parallel with this, detoxify soman at a comparable rate. The third enzyme appears to show stereoselectivity with respect to the two pairs of isomers of soman in that it hydrolyzes the racemic mixture at a fast and a slow rate, the latter approaching the non-enzymatic rate, and detoxifies soman only at the slower rate. In the past, organophosphorus acid anhydrases from bacterial and mammalian sources have been assayed either as crude sonicates or homogenates, or as cold ethanol precipitated fractions. Major discrepancies among laboratories have probably been due either to the assay of mixtures of varying proportions of these three enzymes depending on the various organs or organisms used as the source, or to the purification of one of the enzymes at the expense of the others. For E. coli, a fourth organophosphorus acid anhydrase is also present but at a considerably lower activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Escherichia coli/enzimología , Monoéster Fosfórico Hidrolasas/metabolismo , Soman/farmacocinética , Arildialquilfosfatasa , Proteínas Bacterianas/aislamiento & purificación , Cromatografía en Gel , Electroforesis , Semivida , Hidrólisis , Inactivación Metabólica , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Soman/metabolismo , EstereoisomerismoRESUMEN
An enzyme that hydrolyzes soman (1,2,2-trimethylpropyl methylphosphonofluoridate) and two other phosphonofluoridates, but does not hydrolyze DFP (diisopropylphosphorofluoridate), has been partially purified from a rod-shaped spore-forming gram-positive OT (obligate thermophilic) bacterium. The enzyme shows a marked Mn2+ stimulation, and in this and its substrate preference does not resemble the organophosphorus acid anhydrolase (sometimes termed DFPase) found in squid. Like the squid enzyme, it is not inhibited by mipafox (N,N'-diisopropylphosphordiamidofluoridate), is not inactivated by ammonium sulfate, and does hydrolyze the acetylcholinesterase-inhibitory pair of diastereoisomers of soman as well as the relatively noninhibitory pair, thus detoxifying soman. In these three properties the OT enzyme does not resemble the ubiquitous organophosphorus acid anhydrolase often purified from mammalian and bacterial sources by cold ethanol fractionation. Thus this phosphono-specific OT enzyme may have a natural substrate and a physiological role distinct from other organophosphorus acid anhydrolases.