In vitro metabolism of an orally active O-methyl amidoxime prodrug for the treatment of CNS trypanosomiasis.

A new aza-analogue of furamidine, 6-[5-(4-amidinophenyl)-furan-2-yl]nicotinamidine (DB820), has potent in vitro antitrypanosomal activity; however, it suffers from poor oral activity because of its positively charged amidine groups. The dimethoxyamidine prodrug of DB820, N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine (DB844), has potent oral activity in mouse models of both early-stage and CNS African trypanosomiasis. Metabolism of DB844 in human liver microsomes (HLM) was investigated using liquid chromatography-mass spectrometry (LC-MS/MS). The metabolism of DB844 in HLM was NADPH-dependent and resulted in the production of eight metabolites over a 90?min incubation. O-Demethylation and N-dehydroxylation reactions resulted in the metabolic conversion of DB844 to its active DB820 metabolite. Chromatographic conditions used for LC-MS analysis allowed for the separation and identification of all metabolites including positional isomers. Demethylation of either the phenyl or pyridine side of DB844 (DB844 m/z 366.2) resulted in the production of two metabolites (M1A, M1B), each with a molecular ion of m/z of 352.3 and MS(2) fragments of 288.1, 305.2, 321.2 and 335.2. However, the intensities of the MS(2) fragments were different among the two isomeric metabolites, and comparison to an authentic standard allowed for the structural determination of each metabolite. The isomeric metabolites M2A and M2B, resulting from amidoxime reductions of M1A and M1B, were also chromatographically separated and had distinguishable MS(2) profiles that allowed for their structural assignments when compared to an authentic standard. The di-amidoxime product resulting from O-demethylation of either side of DB844 was also identified as an abundant metabolite during microsomal incubations. The active antitrypanosomal metabolite, DB820, was the last metabolite to be formed and thus provides evidence that DB844 may effectively be metabolized to its active metabolite in vivo.

Nuclear magnetic resonance analysis of [1-13C]dimethylsulfoniopropionate (DMSP) and [1-13C]acrylate metabolism by a DMSP lyase-producing marine isolate of the alpha-subclass of Proteobacteria.

The prominence of the alpha-subclass of Proteobacteria in the marine bacterioplankton community and their role in dimethylsulfide (DMS) production has prompted a detailed examination of dimethylsulfoniopropionate (DMSP) metabolism in a representative isolate of this phylotype, strain LFR. [1-(13)C]DMSP was synthesized, and its metabolism and that of its cleavage product, [1-(13)C]acrylate, were studied using nuclear magnetic resonance (NMR) spectroscopy. [1-(13)C]DMSP additions resulted in the intracellular accumulation and then disappearance of both [1-(13)C]DMSP and [1-(13)C]beta-hydroxypropionate ([1-(13)C]beta-HP), a degradation product. Acrylate, the immediate product of DMSP cleavage, apparently did not accumulate to high enough levels to be detected, suggesting that it was rapidly beta-hydroxylated upon formation. When [1-(13)C]acrylate was added to cell suspensions of strain LFR it was metabolized to [1-(13)C]beta-HP extracellularly, where it first accumulated and was then taken up in the cytosol where it subsequently disappeared, indicating that it was directly decarboxylated. These results were interpreted to mean that DMSP was taken up and metabolized by an intracellular DMSP lyase and acrylase, while added acrylate was beta-hydroxylated on (or near) the cell surface to beta-HP, which accumulated briefly and was then taken up by cells. Growth on acrylate (versus that on glucose) stimulated the rate of acrylate metabolism eightfold, indicating that it acted as an inducer of acrylase activity. DMSP, acrylate, and beta-HP all induced DMSP lyase activity. A putative model is presented that best fits the experimental data regarding the pathway of DMSP and acrylate metabolism in the alpha-proteobacterium, strain LFR.

Phylogenetic analysis of culturable dimethyl sulfide-producing bacteria from a spartina-dominated salt marsh and estuarine water.

Dimethylsulfoniopropionate (DMSP), an abundant osmoprotectant found in marine algae and salt marsh cordgrass, can be metabolized to dimethyl sulfide (DMS) and acrylate by microbes having the enzyme DMSP lyase. A suite of DMS-producing bacteria isolated from a salt marsh and adjacent estuarine water on DMSP agar plates differed markedly from the pelagic strains currently in culture. While many of the salt marsh and estuarine isolates produced DMS and methanethiol from methionine and dimethyl sulfoxide, none appeared to be capable of producing both methanethiol and DMS from DMSP. DMSP, and its degradation products acrylate and beta-hydroxypropionate but not methyl-3-mecaptopropionate or 3-mercaptopropionate, served as a carbon source for the growth of all the alpha- and beta- but only some of the gamma-proteobacterium isolates. Phylogenetic analysis of 16S rRNA gene sequences showed that all of the isolates were in the group Proteobacteria, with most of them belonging to the alpha and gamma subclasses. Only one isolate was identified as a beta-proteobacterium, and it had >98% 16S rRNA sequence homology with a terrestrial species of Alcaligenes faecalis. Although bacterial population analysis based on culturability has its limitations, bacteria from the alpha and gamma subclasses of the Proteobacteria were the dominant DMS producers isolated from salt marsh sediments and estuaries, with the gamma subclass representing 80% of the isolates. The alpha-proteobacterium isolates were all in the Roseobacter subgroup, while many of the gamma-proteobacteria were closely related to the pseudomonads; others were phylogenetically related to Marinomonas, Psychrobacter, or Vibrio species. These data suggest that DMSP cleavage to DMS and acrylate is a characteristic widely distributed among different phylotypes in the salt marsh-estuarine ecosystem.

Metabolism of acrylate to beta-hydroxypropionate and its role in dimethylsulfoniopropionate lyase induction by a salt marsh sediment bacterium, Alcaligenes faecalis M3A.

Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, we report on the mechanism of DMSP-acrylate metabolism by Alcaligenes faecalis M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, beta-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme. (1)H and (13)C nuclear magnetic resonance analyses were used to identify the products resulting from [1-(13)C]acrylate metabolism. The results indicated that A. faecalis first metabolized acrylate to beta-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to beta-hydroxypropionate in the aerobic beta-Proteobacterium A. faecalis has been described.

Evidence for Intracellular and Extracellular Dimethylsulfoniopropionate (DMSP) Lyases and DMSP Uptake Sites in Two Species of Marine Bacteria.

The kinetics of dimethylsulfoniopropionate (DMSP) uptake and dimethylsulfide (DMS) production from DMSP in two bacterial species, Alcaligenes sp. strain M3A, an isolate from estuarine surface sediments, and Pseudomonas doudoroffii, from seawater, were investigated. In Alcaligenes cells induced for DMSP lyase (DL) activity, DMS production occurred without DMSP uptake. In DL-induced suspensions of P. doudoroffii, uptake of DMSP preceded the production of DMS, indicating an intracellular location of DL; intracellular DMSP levels reached ca. 7 mM. DMSP uptake rates in noninduced cells showed saturation at three concentrations (K(inft) [transport] values, 3.4, 127, and 500 (mu)M). In DL-induced cells of P. doudoroffii, DMSP uptake rates increased ca. threefold (V(infmax), 0.022 versus 0.065 (mu)mol of DMSP taken up min(sup-1) mg of cell protein(sup-1)), suggesting that the uptake binding proteins were inducible. DMSP uptake and DL activity in P. doudoroffii were both inhibited by CN(sup-), 2,4-dinitrophenol, and membrane-impermeable thiol-binding reagents, further indicating active uptake of DMSP by cell surface components. The respiratory inhibitors had limited or no effect on DL activity by the Alcaligenes sp. Of the structural analogs of DMSP tested for their effect on DMSP metabolism, glycine betaine (GBT), but not methyl-3-mercaptopropionic acid (MMPA), inhibited DMSP uptake by P. doudoroffii, suggesting that GBT shares a binding protein with DMSP and that MMPA is taken up at a separate site. Two models of DMSP uptake, induction, and DL location found in marine bacteria are presented.

Valor de la punción biopsia dirigida en la estadificación del carcinoma colo-rectal / Value of the biopsy puncture for colorectal carcinoma stage

Objetivo: Analizar retrospectivamente los resultados que se obtuvieron con la punción guiada por Tomografía Computada en pacientes con metástasis de carcinoma colorrectal. Diseño: Análisis retrospectivo de una serie de punciones guiadas por Tomografía Computada. Lugar de realización: Servicio de Cirugía General, Hospital Español de Buenos Aires. Pacientes y métodos: de 131 punciones biopsias abdominales realizadas entre enero de 1992 y junio de 1996 se realizaron 21 punciones efectuadas en 20 pacientes con metástasis de carcinoma colorrectal. La edad promedio fue de 66 años. Se utilizó un Tomógrafo Computado Philips Tomoscán 60 y un Tomógrafo helicoidal Toshiba Xvision. El procedimiento fue realizado con anestesia local y agujas modelo Turner con calibres variables según la lesión entre 19 y 23 Gauge. En todos los casos se realizó control tomográfico post-punción. En todos los casos se realizó análisis citológico del material intraprocedimiento. Resultados: En el 100 por ciento de los casos sólo fue necesario realizar una sola muestra. No se registraron complicaciones relacionadas con el método. En el 90 por ciento de los pacientes la punción decidió terapéutica oncológica concreta. La sensibilidad general en todas las localizaciones fue del 85 por ciento. La especificidad y el valor predictivo positivo fueron del 100 por ciento siendo los resultados comparables con otras series más numerosas. Conclusión: La punción aspiración con agujas finas guiadas por Tomografía Computada resulta un método seguro y útil, participando en forma decisiva en la toma de decisiones terapéuticas en aquellos pacientes con lesiones secundarias de carcinoma colorrectal.

Evidence for Intracellular and Extracellular Dimethylsulfoniopropionate (DMSP) Lyases and DMSP Uptake Sites in Two Species of Marine Bacteria.

Volume 63, no. 8, p. 3183, column 1, line 3 from the bottom: "0.012 versus 0.730" should read "0.022 versus 0.065." [This corrects the article on p. 3182 in vol. 63.].