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R Docampo

Publications and source records attributed to R Docampo.

At least 37 records · Page 2Linked to original sources
PubMed2000

A novel phosphatidylinositol-phospholipase C of Trypanosoma cruzi that is lipid modified and activated during trypomastigote to amastigote differentiation.

The phosphoinositide (PI)-specific phospholipase C gene (TcPI-PLC) of the protozoan parasite Trypanosoma cruzi was cloned, sequenced, expressed in Escherichia coli, and the protein product (TcPI-PLC) was shown to have enzymatic characteristics similar to those of mammalian delta-type PI-PLCs. The TcPI-PLC gene is expressed at high levels in the epimastigote and amastigote stages of the parasite, and its expression is induced during the differentiation of trypomastigotes into amastigotes, where TcPI-PLC associates with the plasma membrane and increases its catalytic activity. In contrast to other PI-PLCs described so far, the deduced amino acid sequence of TcPI-PLC revealed some unique features such as an N-myristoylation consensus sequence at its amino-terminal end, lack of an apparent pleckstrin homology domain and a highly charged linker region between the catalytic X and Y domains. TcPI-PLC is lipid modified in vivo, as demonstrated by metabolic labeling with [(3)H]myristate and [(3)H]palmitate and fatty acid analysis of the immunoprecipitated protein, and may constitute the first example of a new group of PI-PLCs.

Amino Acid Sequence↗
PubMed2000

Regulation of the plasma membrane potential in Pneumocystis carinii.

Many protists use a H(+) gradient across the plasma membrane, the proton motive force, to drive nutrient uptake. This force is generated in part by the plasma membrane potential (DeltaPsi). We investigated the regulation of the DeltaPsi in Pneumocystis carinii using the potentiometric fluorescent dye bisoxonol. The steady state DeltaPsi in a buffer containing Na(+) and K(+) (standard buffer) was found to be -78+/-8 mV. In the absence of Na(+) and K(+) (NMG buffer) or Cl(-) (gluconate buffer), DeltaPsi was not significantly changed suggesting that cation and anion conductances do not play a significant role in the regulation of DeltaPsi in P. carinii. The DeltaPsi was also not affected by inhibitors of the Na(+)/K(+)-ATPase, ouabain (1 mM), and the K(+)/H(+)-ATPase, omeprazole (1 mM). In contrast, inhibitors of the plasma membrane H(+)-ATPase, dicyclohexylcarbodiimide (100 microM), N-ethylmaleimide (100 microM) and diethylstilbestrol (25 microM), significantly depolarized the DeltaPsi to -43+/-7, -56+/-5 and -40+/-12 mV, respectively. The data support that the plasma membrane H(+)-ATPase plays a significant role in the regulation of DeltaPsi in P. carinii.

Cell Membrane↗
PubMed2000

A pyruvate-proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle.

Regulation of intracellular pH (pH(i)) and H(+) efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2', 7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pH(i) in bloodstream and procyclic trypomastigotes was 7.47+/-0.06 and 7. 53+/-0.07 respectively. Differences in the mechanisms for the regulation of pH(i) were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pH(i) at neutral over a wide range of external pH values from 6 to 8, and in the absence of K(+) or Na(+). The H(+)-ATPase inhibitors N, N'-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pH(i) and inhibited its recovery from acidification. The rate of H(+) efflux in these forms was determined to be 62+/-6.5 nmol/min per mg of protein, and was substantially decreased by H(+)-ATPase inhibitors. The data support the presence of an H(+)-ATPase as the major regulator of pH(i) in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pH(i) and recovery from acidification were unaffected by H(+)-ATPase inhibitors, except for DCCD (100 microM). Their steady-state pH(i) was markedly decreased in glucose-free buffer or by >/=10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H(+) efflux in bloodstream trypomastigotes was 534+/-38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499+/-34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4, 4'-di-isothiocyanatodihydrostilbene-2,2'-disulphonic acid and alpha-cyanohydroxycinnamic acid. The pyruvate analogues beta-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pH(i), as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pH(i) supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pH(i) control mechanism in bloodstream forms. Inhibition by H(+)-ATPase inhibitors of residual H(+) efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H(+)-ATPase(s) in control of pH(i) in bloodstream forms.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Intracellular pH in mammalian stages of Trypanosoma cruzi is K+-dependent and regulated by H+-ATPases.

Regulation of intracellular pH (pHi) was investigated in Trypanosoma cruzi amastigotes and trypomastigotes using 2',7'-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein (BCECF). pHi was determined to be 7.33 +/- 0.08 and 7.35 +/- 0.07 in amastigotes and trypomastigotes, respectively, and there were no significant differences in the regulation of pH, between the two stages. Steady-state pHi, recovery of pHi from acidification, and H+-efflux were all decreased markedly by the H+-ATPase inhibitors N,N'-dicyclohexylcarbodi-imide (DCCD), diethylstilbestrol (DES) and N-ethylmaleimide (NEM) supporting a significant role for a plasma membrane H+-ATPase in the regulation of pHi. pHi was maintained at neutrality over a range of external pH (pHe) from 5-8 in parasites suspended in a buffer containing Na+ and K+ (standard buffer) but was acidified at low pHe in the absence of these cations (choline buffer). The pHi of trypomastigotes decreased significantly when they transformed into amastigotes. The rate of recovery of pHi by acidified parasites was similar in Na+-free buffer and standard buffer but was slower in the absence of K+ (K+-free or choline buffer) and parasites suspended in choline buffer were acidic by 0.25 pH units as compared with controls. Ba2+ and Cs+ decreased the pHi of parasites suspended in standard but not choline buffer suggesting the presence of an inward directed K+ channel. The pHi of amastigotes and trypomastigotes suspended in Cl(-)-free buffer was decreased by 0.13 and 0.2 pH units, respectively, supporting the presence of a chloride conductive channel. No evidence of pH regulation via a Na+/H+ or Cl-/HCO3- exchanger was found. These results are consistent with the presence of a plasma membrane H+-ATPase that regulates pHi and is supported by K+ and Cl- channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2000

Calcium mobilization by arachidonic acid in trypanosomatids.

A recent report (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A, and arachidonic acid, Biochem J 1998:336:659-66) provided evidence that calcium entry in Trypanosoma brucei bloodstream trypomastigotes is regulated via a signaling pathway involving phospholipase A2-mediated generation of arachidonic acid and stimulation of a plasma membrane-located calcium channel. Here we show that Ca2+ influx in T. brucei procyclic trypomastigotes, Leishmania donovani promastigotes and T. cruzi amastigotes was also stimulated in a dose-dependent manner (50-400 nM) by the amphiphilic peptide melittin. This effect was blocked by the phospholipase A, inhibitor 3-(4-octadecyl)-benzoylacrylic acid. The unsaturated fatty acid arachidonic acid, in the range of 10-75 microM, induced Ca2+ entry by a mechanism sensitive to LaCl3. However, both melittin and arachidonic acid induced an increase in [Ca2+]i in T. brucei procyclic trypomastigotes incubated in Ca2+-free medium implying Ca2+ mobilization from intracellular stores. This hypothesis was supported by experiments showing that arachidonic acid promoted Ca2+ release from the acidocalcisomes of these cells. The results showing changes in mitochondrial membrane potential, release of acridine orange and Ca2+ from the acidocalcisomes and Ca2+ transport across the plasma membrane suggest that in addition to the possible stimulation of a Ca2+ channel-mediated process, arachidonic acid, in the range of concentrations used here, have other nonspecific effects on the trypanosomatids membranes.

Acridine Orange↗
PubMed2000

The fine structure of acidocalcisomes in Trypanosoma cruzi.

Trypanosoma cruzi survives in vertebrate and invertebrate hosts and has developed mechanisms that allow it to adapt to changes in the microenvironment such as temperature, pH, and ionic composition. Most of its calcium is concentrated in an organelle named the acidocalcisome, which is acidified by a (V-H+)-adenosine triphosphatase and has H+/Ca2+ counter-transportation for calcium uptake. In this work, acidocalcisomes were examined using different transmission electron microscopy techniques. In thin sections of different stages, acidocalcisomes presented a circular shape with an electron-dense inclusion containing P3-, Ca2+, Na+, Mg2+, K+, and Zn2+. They could be distinguished from gold-labeled albumin-containing reservosomes in whole epimastigotes, and a morphometric analysis showed higher amounts of these organelles in amastigotes as compared with epimastigotes and trypomastigotes. It is possible that this variation in the amount of acidocalcisomes in the different evolutive stages could reflect adaptation mechanisms used by the parasite to survive and multiply in different environmental conditions.

Animals↗
PubMed2000

Sulphur-containing derivatives structurally related to fenoxycarb are potent growth inhibitors against the intracellular form of Trypanosoma cruzi.

Sulphur-containing derivatives structurally related to the insect growth regulator fenoxycarb were shown to be extremely active antiproliferative agents against the amastigote form of Trypanosoma cruzi in in vitro assays. All of these drugs had previously been proved to be remarkably potent growth inhibitors against the epimastigote form of the parasite.

Animals↗
PubMed1999

Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs.

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.

Animals↗
PubMed1999

A plant-like vacuolar H(+)-pyrophosphatase in Plasmodium falciparum.

Inorganic pyrophosphate promoted the acidification of a subcellular compartment in cell homogenates of Plasmodium falciparum trophozoites. The proton gradient driven by pyrophosphate was collapsed by addition of NH(4)Cl or the K(+)/H(+) exchanger nigericin and eliminated by the pyrophosphate analog aminomethylenediphosphonate. Pyrophosphatase activity was dependent upon K(+), and partially inhibited by Na(+). The presence of a plant-like vacuolar H(+)-translocating pyrophosphatase (V-H(+)-PPase) was confirmed using antibodies raised against conserved peptide sequences of the enzyme, which cross reacted with a protein band of 76.5 kDa. Immunofluorescence microscopy using these antibodies showed a general fluorescence over the whole parasites and intracellular bright spots suggesting a vesicular and plasma membrane localization. Together, these results indicate the presence in P. falciparum of a V-H(+)-PPase of similar characteristics to those of the enzyme from plants.

Animals↗
PubMed1999

Mercaptopyridine-N-oxide, an NADH-fumarate reductase inhibitor, blocks Trypanosoma cruzi growth in culture and in infected myoblasts.

The enzyme NADH-fumarate reductase is not found in mammalian cells but it is present in several parasitic protozoa including Trypanosoma cruzi, the parasite that causes Chagas' disease. This study shows that the drug 2-mercaptopyridine-N-oxide (MPNO) inhibits NADH-fumarate reductase purified from T. cruzi (ID50 = 35 microM). When added to intact cells, MPNO inhibited the growth of T. cruzi epimastigotes in culture (ID50 = 0.08 microM) as well as the infection of mammalian myoblasts by T. cruzi trypomastigotes (ID50 = 20 microM). At a concentration of 2.4 microM, MPNO also inhibited the growth of amastigotes (intracellular dividing forms) in cultured mammalian myoblasts. Supplementation of culture media with 5 mM succinate, the product of fumarate reductase, partially protected against the inhibition of the growth of epimastigotes by MPNO. Moreover, MPNO inhibited the accumulation of succinate in cultures of epimastigotes, as measured by high performance liquid chromatography. Although MPNO may have other intracellular targets in addition to fumarate reductase, these results support the hypothesis that compounds which inhibit the enzyme fumarate reductase may be potential chemotherapeutic agents against Chagas' disease.

Animals↗
PubMed1999

Presence of a vacuolar H+-pyrophosphatase in promastigotes of Leishmania donovani and its localization to a different compartment from the vacuolar H+-ATPase.

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized promastigotes of Leishmania donovani, as measured by Acridine Orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH4Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions, and inhibited by NaF, the pyrophosphate analogues imidodiphosphate and aminomethylenediphosphonate (AMDP), dicyclohexylcarbodiimide, and the thiol reagents p-hydroxymercuribenzoate and N-ethylmaleimide, all at concentrations similar to those that inhibit the plant vacuolar proton-pumping pyrophosphatase (H+-PPase). The proton translocation activity had a pH optimum in the range 7.0-7.5, and was unaffected by bafilomycin A1 (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 microM) and KNO3 (200 mM). AMDP-sensitive pyrophosphate hydrolysis was also detected in promastigotes, and potassium ions also stimulated this activity. Sodium ions disrupted pH gradients established in the presence of ATP but not in the presence of pyrophosphate, and sequential addition of ATP and pyrophosphate resulted in partially additive Acridine Orange accumulation, suggesting that the vacuolar H+-PPase is in a different intracellular compartment from the vacuolar H+-ATPase and Na+/H+ exchanger of L. donovani promastigotes. Separation of promastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity but lacked ATPase activity and markers for mitochondria, glycosomes and lysosomes. The organelles in this fraction appeared by electron microscopy to consist of electron-dense vacuoles. In summary, these results indicate that, in contrast to plant vacuoles, vacuolar H+-PPase and vacuolar ATPase activities are present in different compartments in L. donovani promastigotes.

Acridine Orange↗
PubMed1999

Acidocalcisome: A novel Ca2+ storage compartment in trypanosomatids and apicomplexan parasites.

Acidocalcisomes are novel acidic Ca2+ storage organelles found in trypanosomatids and apicomplexan parasites, abundant in the intracellular stages of these parasites, and characterized by their high electron density, and high content of phosphorus, Ca2+, Mg2+, Na+ and Zn2+. A number of energy-utilizing pumps and exchangers have been found in these organelles, which underlines their importance in the homeostasis of different elements, as discussed here by Roberto Docampo and Silvia Moreno.

Animals↗
PubMed1999

Characterization of a vacuolar pyrophosphatase in Trypanosoma brucei and its localization to acidocalcisomes.

Inorganic pyrophosphate promoted the acidification of an intracellular compartment in permeabilized procyclic trypomastigotes of Trypanosoma brucei, as measured by acridine orange uptake. The proton gradient generated by pyrophosphate was collapsed by addition of nigericin or NH(4)Cl. Pyrophosphate-driven proton translocation was stimulated by potassium ions and inhibited by KF, by the pyrophosphate analogs imidodiphosphate and aminomethylenediphosphonate (AMDP), and by the thiol reagent p-hydroxymercuribenzoate at concentrations similar to those that inhibit the plant vacuolar H(+)-pyrophosphatase (PPase). The proton translocation activity had a pH optimum around 7.5 and was partially inhibited by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (10 microM) and unaffected by bafilomycin A(1) (40 nM), concanamycin A (5 nM), sodium o-vanadate (500 microM), oligomycin (1 microM), N-ethylmaleimide (100 microM), and KNO(3). AMDP-sensitive pyrophosphate hydrolysis was detected in both procyclic and bloodstream trypomastigotes. Measurements of acridine orange uptake in permeabilized procyclic trypomastigotes in the presence of different substrates and inhibitors suggested the presence of H(+)-ATPase, H(+)-PPase, and (ADP-dependent) H(+)/Na(+) antiport activity in the same compartment. Separation of bloodstream and procyclic trypomastigote extracts on Percoll gradients yielded fractions that contained H(+)-PPase (both stages) and H(+)/Na(+) exchanger (procyclics) activities but lacked markers for mitochondria, glycosomes, and lysosomes. The organelles in these fractions were identified by electron microscopy and X-ray microanalysis as acidocalcisomes (electron-dense vacuoles). These results provide further evidence for the unique nature of acidocalcisomes in comparison with other, previously described, organelles.

Adenosine Diphosphate↗
PubMed1998

Growth inhibitory effect of juvenile hormone analogues on epimastigotes of Trypanosoma cruzi.

Several compounds, structurally related to the insect growth regulator Fenoxycarb, exhibited interesting inhibition action to control proliferation of Trypanosoma cruzi, the parasite responsible for Chagas' disease. Some of these drugs were shown to be potent growth inhibitors of this parasite. All of these drugs had previously presented juvenoid activity on several non-related bug species such as Tenebrio molitor, Galleria mellonella, Dysdercus cingulatos, and Pyrrhocoris apterus.

Animals↗
PubMed1998

Presence of a plant-like proton-pumping pyrophosphatase in acidocalcisomes of Trypanosoma cruzi.

The vacuolar-type proton-translocating pyrophosphatase (V-H+-PPase) is an enzyme previously described in detail only in plants. This paper demonstrates its presence in the trypanosomatid Trypanosoma cruzi. Pyrophosphate promoted organellar acidification in permeabilized amastigotes, epimastigotes, and trypomastigotes of T. cruzi. This activity was stimulated by K+ ions and was inhibited by Na+ ions and pyrophosphate analogs, as is the plant activity. Separation of epimastigote extracts on Percoll gradients yielded a dense fraction that contained H+-PPase activity measured both by proton uptake and phosphate release but lacked markers for mitochondria, lysosomes, glycosomes, cytosol, and plasma membrane. Antiserum raised against specific sequences of the plant V-H+-PPase cross-reacted with a T. cruzi protein, which was also detectable in the dense Percoll fraction. The organelles in this fraction appeared by electron microscopy to consist mainly of acidocalcisomes (acidic calcium storage organelles). This identification was confirmed by x-ray microanalysis. Immunofluorescence and immunoelectron microscopy indicated that the V-H+-PPase was located in the plasma membrane and acidocalcisomes of the three different forms of the parasite. Pyrophosphate was able to drive calcium uptake in permeabilized T. cruzi. This uptake depended upon a proton gradient and was reversed by a specific V-H+-PPase inhibitor. Our results imply that the phylogenetic distribution of V-H+-PPases is much wider than previously perceived but that the enzyme has a unique subcellular location in trypanosomes.

Acridine Orange↗
PubMed1998

Functional expression of a vacuolar-type H+-ATPase in the plasma membrane and intracellular vacuoles of Trypanosoma cruzi.

Acid-loaded Trypanosoma cruzi amastigotes and trypomastigotes regained normal cytoplasmic pH (pHi), as measured in cells loaded with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), by a process that was sensitive to bafilomycin A1 at concentrations comparable to those that inhibited vacuolar (V) H+-ATPases from different sources. Steady-state pHi was also decreased by similar concentrations of bafilomycin A1 in a concentration-dependent manner. The efflux of H+ equivalents from amastigotes and trypomastigotes was measured by following changes in the fluorescence of extracellular BCECF. Basal H+ extrusion in the presence of glucose was 15.4+/-2.8 (S.D.) nmol of H+/min per 10(8) amastigotes and 6. 37+/-0.8 nmol of H+/min per 10(8) trypomastigotes. Bafilomycin A1 treatment significantly decreased the efflux of H+ equivalents by amastigotes (8.9+/-2.2 nmol of H+/min per 10(8) cells), but not by trypomastigotes (5.1+/-1.7 nmol of H+/min per 10(8) cells). The localization of the V-H+-ATPase of T. cruzi was investigated by immunocytochemistry. Confocal and electron microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the V-H+-ATPase of different stages of T. cruzi is also located in the plasma membrane. However, no labelling was detected in the plasma membrane lining the flagellar pocket of the different developmental stages. Surface localization of the V-H+-ATPase was confirmed by experiments involving the biotinylation of cell surface proteins and immunoprecipitation with antibodies against the V-H+-ATPase. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of amastigotes and with an important role for intracellular acidic compartments in the maintenance of pHi in different stages of T. cruzi.

Animals↗
PubMed1998

Ecto-protein tyrosine phosphatase activity in Trypanosoma cruzi infective stages.

Live T. cruzi trypomastigotes and amastigotes possess ecto-protein tyrosine phosphatase activity as indicated by the ability of intact cells to catalyze dephosphorylation of tyrosine phosphorylated myelin basic protein, [32P]TyrRaytide, phosphotyrosine, or the phosphotyrosine analog p-nitrophenylphosphate (p-NPP). The dephosphorylation of myelin basic protein (MBP) and p-NPP was inhibited by sodium o-vanadate, zinc chloride and NaF, while dephosphorylation of [32P]TyrRaytide was insensitive to zinc chloride but sensitive to o-vanadate and NaF. In contrast, live cells were not able to dephosphorylate serine or threonine phosphorylated peptides ([32P]Kemptide) or proteins ([32P]RCM-lysozyme and [32P]MBP).

Animals↗