RESUMEN
L-nucleosides selectively enter malaria infected erythrocytes and have the unique ability to be metabolised by the malarial adenosine deaminase. This has allowed us to design novel L-nucleosides as potential anti-malarials.
Asunto(s)
Adenosina Desaminasa/metabolismo , Antimaláricos/farmacología , Nucleósidos de Purina/farmacología , Animales , Antimaláricos/farmacocinética , Cromatografía Líquida de Alta Presión , Eritrocitos/parasitología , Plasmodium falciparum/enzimología , Nucleósidos de Purina/farmacocinéticaRESUMEN
The L-stereoisomer analogues of D-coformycin selectively inhibited P. falciparum adenosine deaminase (ADA) in the picomolar range (L-isocoformycin, Ki 7 pM; L-coformycin, Ki 250 pM). While the L-nucleoside analogues, L-adenosine, 2,6-diamino-9-(L-ribofuranosyl)purine and 4-amino-1-(L-ribofuranosyl)pyrazolo[3,4-d]-pyrimidine were selectively deaminated by P. falciparum ADA, L-thioinosine and L-thioguanosine were not. This is the first example of 'non-physiological' L-nucleosides that serve as either substrates or inhibitors of malarial ADA and are not utilised by mammalian ADA.
Asunto(s)
Inhibidores de la Adenosina Desaminasa , Antimaláricos/farmacología , Antimetabolitos/farmacología , Coformicina/análogos & derivados , Inhibidores Enzimáticos/farmacología , Nucleósidos/farmacología , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Antimaláricos/síntesis química , Antimaláricos/química , Antimetabolitos/síntesis química , Antimetabolitos/química , Bovinos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Eritrocitos/enzimología , Eritrocitos/parasitología , Humanos , Estructura Molecular , Nucleósidos/síntesis química , Nucleósidos/química , Plasmodium falciparum/efectos de los fármacos , Estereoisomerismo , Especificidad por SustratoRESUMEN
A new and simple colorimetric method has been developed for determining activity in vitro against Giardia intestinalis. The microtitre plate assay is based upon the nucleoside hydrolase activity released from G. intestinalis by lysis. Action of the nucleoside hydrolase on the substrate analogue, 4-nitrophenyl beta-D-ribofuranoside (NPR), gives rise to a coloured product which may be determined directly by the change in absorbance. A number of other such nucleoside analogues can be similarly used, but NPR is the preferred substrate, since it gives high enzymic activity at a relatively low substrate concentration. The IC50 values determined using this method for the known anti-giardials metronidazole, tinidazole and furazolidone were consistent with previously published values. The method is simple, does not involve radioisotopes or complex instrumentation, and thus provides a convenient method for screening potential anti-giardial agents.
Asunto(s)
Compuestos Cromogénicos/química , Colorimetría/métodos , Giardia lamblia/crecimiento & desarrollo , Giardiasis/tratamiento farmacológico , Nucleotidasas/química , Animales , Antitricomonas/farmacología , Antitricomonas/uso terapéutico , Furazolidona/farmacología , Furazolidona/uso terapéutico , Giardia lamblia/efectos de los fármacos , Giardia lamblia/enzimología , Concentración 50 Inhibidora , Metronidazol/farmacología , Metronidazol/uso terapéutico , Ribonucleósidos/química , Sensibilidad y Especificidad , Tinidazol/farmacología , Tinidazol/uso terapéuticoRESUMEN
The expression of purine-specific nucleoside and base transporters of Crithidia luciliae has been demonstrated in Xenopus laevis oocytes. Poly(A)+-mRNA from C. luciliae, cultured in either purine-replete or purine-starved conditions, was microinjected into X. laevis oocytes. For "purine-replete" mRNA, expression of adenosine and hypoxanthine uptake in microinjected X. laevis oocytes was increased on average 9- and 3-fold above water-injected controls, respectively. Expression of adenosine and hypoxanthine uptake in oocytes microinjected with "purine-starved" mRNA was 8 and 3-fold above water-injected controls, respectively. Substrate competition indicated an adenosine/deoxyadenosine transporter and a separate base transporter specific for hypoxanthine. In contrast to C. luciliae in vivo, where the level of activity of adenosine and hypoxanthine transport was regulated by the level of purines in the medium, the heterologous expression of these transporters (from both purine replete and deplete cultures) in X. laevis oocytes was independent of the extracellular purine concentration. These results may suggest that the presence of specific transporter message is independent of the extracellular purine content, indicating that the regulation of activation and expression of these transporters in C. luciliae may not be under transcriptional control.
Asunto(s)
Adenosina/metabolismo , Proteínas Portadoras/biosíntesis , Crithidia/metabolismo , Hipoxantina/metabolismo , Proteínas de la Membrana/biosíntesis , Animales , Proteínas Portadoras/genética , Crithidia/genética , Desoxiglucosa/metabolismo , Expresión Génica , Proteínas de la Membrana/genética , Microinyecciones , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas de Transporte de Nucleósidos , Oocitos/metabolismo , ARN Mensajero/metabolismo , Xenopus laevisRESUMEN
When the malarial parasite Plasmodium falciparum multiplies in erythrocytes it dramatically increases uptake of essential metabolic precursors (nucleosides, nucleobases and glucose) and export of lactic acid by undefined mechanisms. The first evidence is provided here, by a detailed study in Xenopus laevis oocytes, that several specific nutrient transporters are the product of P. falciparum genes. We report the expression of nucleoside, nucleobase, hexose and monocarboxylate transport systems in Xenopus oocytes when injected with mRNA isolated from asexual stages of developing P. falciparum parasites. Their properties are distinct from transport events occurring at the infected erythrocyte membrane or the electrophysiologically identified channel localised to the parasitophorous vacuolar membrane. These novel transporters are substrate-specific and stereoselective, and represent a key regulatory step in the acquisition and export of metabolites by intraerythrocytic P. falciparum.
Asunto(s)
Proteínas Portadoras/biosíntesis , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Adenosina/metabolismo , Animales , Transporte Biológico , Desoxiglucosa/metabolismo , Eritrocitos/parasitología , Expresión Génica , Hipoxantina/metabolismo , Modelos Biológicos , Oocitos , Permeabilidad , ARN Mensajero/genética , ARN Protozoario/genética , Especificidad por Sustrato , Xenopus laevisRESUMEN
How parasitic protozoa survive varying nutrient levels is a key issue in parasitology. Here, Annette Gero explains how the Trypanosomatid Crithidia luciliae responds to purine stress by increasing the rates of transport of nucleosides and bases from the environment and by increasing the activity of the ectoenzyme 3'-nucleotidase (3'NTase), which breaks down external nucleotides so that they can be salvaged as nucleosides. The increase in activity of the purine transporters, and the 3'NTase activity is simultaneous with a general increase in the purine metabolic pathway, hence ensuring that purines are readily available to the parasite during purine stress.
Asunto(s)
Adenosina/metabolismo , Eritrocitos/parasitología , Plasmodium falciparum/metabolismo , Respirovirus/fisiología , Timidina/metabolismo , Adenosina/química , Animales , Transporte Biológico , Permeabilidad de la Membrana Celular , Eritrocitos/metabolismo , Eritrocitos/virología , Estereoisomerismo , Timidina/químicaRESUMEN
Influx of the purine nucleoside, adenosine, was assessed in erythrocytes from both normal subjects and from subjects with a range of genetically determined erythrocyte disorders from Myanmar. The latter included alpha-thalassemia major (Myanmar variant), beta-thalassemia major (Myanmar variant), beta-thalassemia trait, HbEE and HbAE erythrocytes and two variants of glucose-6-phosphate dehydrogenase (G6PDH) deficiency. Significant reductions (p < 0.01) of adenosine influx were observed in erythrocytes from individuals with alpha- and beta-thalassemia major and severe G6PDH deficiency. Abnormal erythrocytes infected with the malarial parasites, Plasmodium falciparum or Plasmodium vivax, demonstrated a reduction in adenosine transport which correlated with the proportion of abnormal erythrocytes present in the samples obtained. The effect of nitrobenzylthioinosine (NBMPR) on adenosine influx was explored in normal and abnormal erythrocytes. In all these cases, NBMPR completely inhibited the transport of adenosine. However, transport of adenosine into P. falciparum and P. vivax-infected normal erythrocytes and abnormal cells was only inhibited 50-60% by NBMPR. The combination of tubercidin and NBMPR completely blocked adenosine transport into both normal and abnormal erythrocytes infected with either P. falciparum or P. vivax.
Asunto(s)
Adenosina/sangre , Eritrocitos Anormales/parasitología , Eritrocitos/parasitología , Deficiencia de Glucosafosfato Deshidrogenasa/parasitología , Hemoglobinopatías/genética , Malaria Falciparum/sangre , Malaria Vivax/sangre , Adulto , Marcadores de Afinidad/farmacología , Niño , Eritrocitos/metabolismo , Eritrocitos Anormales/metabolismo , Femenino , Deficiencia de Glucosafosfato Deshidrogenasa/metabolismo , Hemoglobinopatías/sangre , Humanos , Masculino , Mianmar , Tioinosina/análogos & derivados , Tioinosina/farmacología , Tubercidina/farmacologíaRESUMEN
Nutritional insufficiency is a common environmental extreme to which parasitic protozoa are routinely exposed. In this study of purine salvage mechanisms we illustrate some successful adaptations of the parasite Crithidia luciliae to its environment, particularly in the case of purine stress. In purine-depleted conditions, the insect trypanosome C. luciliae has the ability to increase the rates of transport of adenosine, guanosine and hypoxanthine and the activity of the exoenzyme 3'nucleotidase (3'NTase) during the growth cycle. The dramatic increase in these activities appears after a 72-h period in culture. The increased activity of the purine transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine or hypoxanthine (100 microM). Under conditions where the concentration of purines in the medium could be closely regulated, C. luciliae grown in purine-replete medium (> or = 75 microM purine) exhibited low rates of purine transport and activity of 3'NTase. In comparison, parasites transferred to medium with a low purine source (< or = 7.5 microM adenosine) had levels of adenosine, guanosine and hypoxanthine transport elevated 25-40-fold. The results link the simultaneous increase in activity of the nucleoside and base transporters, 3'NTase activity and a general increase in the purine salvage of C. luciliae to the concentration of purines available at any time to the parasite.
Asunto(s)
Adenosina/metabolismo , Crithidia/metabolismo , Guanosina/metabolismo , Hipoxantina/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animales , Transporte Biológico , Crithidia/crecimiento & desarrollo , Medio de Cultivo Libre de Suero , Cicloheximida/farmacología , Desoxiglucosa/metabolismo , Inosina Monofosfato/metabolismo , Nucleotidasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacologíaRESUMEN
The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids, lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the nonmetabolized alanine analogue alpha-aminoisobutyrate. alpha-Aminoisobutyrate efflux was activated within a few seconds of a reduction of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of efflux of alpha-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration. Hypotonically activated alpha-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional influx rates for alpha-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated alpha-aminoisobutyrate influx showed no tendency to saturate up to an extracellular concentration of 50 mM. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells.
Asunto(s)
Aminoácidos/metabolismo , Crithidia/metabolismo , Presión Osmótica , Ácidos Aminoisobutíricos/metabolismo , Animales , Aniones/metabolismo , Soluciones Hipotónicas/farmacología , Técnicas In Vitro , Transporte Iónico/efectos de los fármacosRESUMEN
During the growth cycle of the protozoan parasite Crithidia luciliae, there was a dramatic concomitant increase in the rate of adenosine and guanosine transport and 3' nucleotidase (3'NTase) activity after 72-94 hr. The simultaneous increased activities of the nucleoside transporters and 3'NTase could be suppressed by addition to the medium of a purine supplement such as adenosine (100 microM). C. luciliae grown in purine-replete medium (> or = 75 microM adenosine) exhibited low rates of adenosine and guanosine transport whilst parasites transferred to a defined serum-free medium containing < or = 7.5 microM adenosine demonstrated elevated levels of both adenosine and guanosine transport up to 25- to 40-fold. The increased activity of the nucleoside transporters was inhibited by cycloheximide (10 microM). Under conditions of purine depletion 3'AMP and 3'GMP inhibited the adenosine and guanosine transporters, respectively. However, in the presence of a purine supplement (100 microM), neither 3'AMP nor 3'GMP was an effective inhibitor of nucleoside transport. Our results link the increased activity of the nucleoside transporters to the increased activity of the 3'NTase, indicating the activation of a purine salvage system not previously reported in other organisms.
Asunto(s)
Adenosina/metabolismo , Crithidia/metabolismo , Guanosina/metabolismo , Nucleósidos de Purina/farmacología , Adenosina/farmacología , Adenosina Monofosfato/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Crithidia/enzimología , Crithidia/crecimiento & desarrollo , Cicloheximida/farmacología , Guanosina Monofosfato/farmacología , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Nucleósidos , Nucleotidasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacologíaRESUMEN
A mechanism which mediates the transport of the nonphysiological nucleoside, L-adenosine, was demonstrated in Plasmodium falciparum infected erythrocytes and naturally released merozoites. L-Adenosine was not a substrate for influx in freed intraerythrocytic parasites or in normal human erythrocytes nor was L-adenosine transported in a variety of cell types including other parasitic protozoa such as Crithidia luciliae, Trichomonas vaginalis, Giardia intestinalis, or the mammalian cells, Buffalo Green Monkey and HeLa cells. L-Adenosine transport in P. falciparum infected cells was nonsaturable, with a rate of 0.13 +/- 0.01 pmol/microliter cell water per s per microM L-adenosine, yet the transport was inhibited by furosemide, phloridzin and piperine with IC50 values between 1-13 microM, distinguishing the transport pathway from simple diffusion. The channel-like permeation was selective as disaccharides were not permeable to parasitised cells. In addition, an unusual metabolic property of parasitic adenosine deaminase was found in that L-adenosine was metabolised to L-inosine by both P. falciparum infected erythrocytes and merozoites, an activity which was inhibited by 50 nM deoxycoformycin. No other cell type examined displayed this enzymic activity. The results further substantiate that nucleoside transport in P. falciparum infected cells was significantly altered compared to uninfected erythrocytes and that L-adenosine transport and metabolism was a biochemical property of Plasmodium infected cells and merozoites and not found in normal erythrocytes nor any of the other cell types investigated.
Asunto(s)
Proteínas Portadoras/metabolismo , Eritrocitos/metabolismo , Malaria Falciparum/metabolismo , Proteínas de la Membrana/metabolismo , Nucleósidos/metabolismo , Adenosina/metabolismo , Transporte Biológico , Células Cultivadas , Medios de Cultivo , Eritrocitos/parasitología , Humanos , Cinética , Proteínas de Transporte de Nucleósidos , Especificidad por SustratoRESUMEN
The incorporation of purine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in nucleotide salvage biosynthetic pathways were investigated by radioactive tracer analysis and nuclear magnetic resonance spectroscopy. The organism took up the nucleobases adenine, guanine and hypoxanthine, and the nucleosides adenosine, guanosine and deoxyadenosine. Any incorporation of deoxyguanosine by the cells was below the detection limits of the methods employed. The activities of adenine-, guanine- and hypoxanthine-phosphoribosyl transferases were established. The bacterium showed high levels of adenosine and guanosine nucleosidase activities and lesser activity for deoxyadenosine; no hydrolysis of deoxyguanosine was detected. Phosphorylase activities were not observed with any of the nucleosides. Phosphotransferase activities with similar rates were demonstrated for adenosine, guanosine and deoxyadenosine; and a weaker activity was detected for deoxyguanosine. No nucleoside kinase activities were observed with any of the nucleosides. The presence of adenylate kinase was established, but no guanylate kinase activity was observed. The study provided evidence for the presence in H. pylori of salvage pathways for the biosynthesis of purine nucleotides.
Asunto(s)
Helicobacter pylori/metabolismo , Nucleótidos de Purina/biosíntesis , Purinas/metabolismo , Adenina/metabolismo , Adenilato Quinasa/análisis , Desoxirribonucleósidos/metabolismo , Desoxirribonucleótidos/biosíntesis , Guanina/metabolismo , Helicobacter pylori/enzimología , Espectroscopía de Resonancia Magnética , N-Glicosil Hidrolasas/análisis , Pentosiltransferasa/análisis , Isótopos de Fósforo , Ribonucleósidos/metabolismoRESUMEN
The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and 31P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.
Asunto(s)
Ligasas de Carbono-Nitrógeno , Helicobacter pylori/metabolismo , Nucleótidos de Pirimidina/biosíntesis , Aspartato Carbamoiltransferasa/análisis , Ácido Aspártico/metabolismo , Bicarbonatos/metabolismo , Desoxicitidina Quinasa/análisis , Helicobacter pylori/enzimología , Marcaje Isotópico , Ligasas/análisis , Espectroscopía de Resonancia Magnética , Orotato Fosforribosiltransferasa/análisis , Pentosiltransferasa/análisis , Isótopos de FósforoRESUMEN
Analogues of purine nucleosides and deoxynucleosides were tested for toxicity against the intraerythrocytic parasite Plasmodium falciparum in vitro culture. Sangivamycin (7-deaza-7-amido-adenosine) (IC37 of 0.3 microM), tubercidin (7-deaza-adenosine) (IC37 of 0.7 microM), 6-methylamino-deoxyadenosine (IC37 of 10 microM), 8-aza-2-amino-deoxy-adenosine (IC37 of 11 microM) and 2-chloro-adenosine (IC37 of 11 microM) were found to be the most toxic towards the parasite. Structure-activity analysis suggested that alteration of the purine ring at the 7 or 8 position significantly increased the toxicity of the compound against P. falciparum. Analysis by HPLC of parasite lysates which had been subjected to the cytotoxic compounds confirmed that alterations in the flux of the purine salvage pathways of the parasite had occurred. Comparison of the toxicity of these compounds against P. falciparum with the toxicity against a similar intraerythrocytic parasite, Babesia bovis, or human melanoma cell lines indicated a differential toxicity, in that many of the compounds toxic towards P. falciparum were relatively non-toxic towards human melanoma cell lines or B. bovis and vice versa. The mechanism of toxicity of the deoxyadenosine and adenosine analogues, whose normal metabolism involves transport, metabolism and incorporation into nucleic acids appears to vary significantly between P. falciparum, B. bovis and mammalian cells.
Asunto(s)
Adenosina/análogos & derivados , Plasmodium falciparum/efectos de los fármacos , Adenosina/química , Adenosina/farmacología , Animales , Babesia bovis/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Humanos , Melanoma , Relación Estructura-Actividad , Células Tumorales CultivadasAsunto(s)
Adenosina/análogos & derivados , Adenosina/toxicidad , Antimaláricos/toxicidad , Plasmodium falciparum/efectos de los fármacos , Animales , Antineoplásicos/toxicidad , Desoxiadenosinas/toxicidad , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Melanoma , Relación Estructura-Actividad , Células Tumorales CultivadasAsunto(s)
Adenosina/metabolismo , Alcaloides , Antimaláricos/toxicidad , Eritrocitos/metabolismo , Eritrocitos/parasitología , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Adenosina/sangre , Marcadores de Afinidad , Animales , Benzodioxoles , Benzoquinonas/farmacología , Transporte Biológico/efectos de los fármacos , Ciona intestinalis/metabolismo , Crithidia/metabolismo , Dilazep/farmacología , Eritrocitos/efectos de los fármacos , Furosemida/farmacología , Células HeLa , Humanos , Malaria Falciparum/sangre , Florizina/farmacología , Piperidinas/farmacología , Plasmodium falciparum/metabolismo , Alcamidas Poliinsaturadas , Estereoisomerismo , Tioinosina/análogos & derivados , Tioinosina/farmacología , Trichomonas vaginalis/metabolismoRESUMEN
Nucleoside transport was evaluated in the trypanosomatid Crithidia luciliae by a rapid sampling technique. C. luciliae was shown to possess two independent nucleoside transporters, one which transported adenosine, deoxyadenosine, tubercidin, sangivamycin and the pyrimidine nucleoside thymidine, while the second was specific for guanosine, inosine and deoxyguanosine. The rapid influx occurred by a process of facilitated transport. The apparent Km values for adenosine and guanosine were 9.34 +/- 1.30 and 10.6 +/- 2.60 microM, respectively. The pyrimidine nucleoside thymidine was transported at a rate approximately 50% lower than the purine nucleosides, whilst uridine, deoxyuridine and deoxycytidine were not transported. The optical isomer, L-adenosine entered the organism by simple diffusion rather than by facilitated transport. In contrast to mammalian cells, neither of the nucleoside transporters in C. luciliae were inhibited by nitrobenzylthioinosine, dilazep, or dipyridamole, potent inhibitors of nucleoside transport in mammalian cells, whilst p-chloromercuribenzoate sulphonate inhibited both nucleoside transporters in C. luciliae.