Purification of N-acetyl-beta-D-hexosaminidase from Trichomonas vaginalis.
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Animals↗
Biomedical subjects
F Lawrence
Publications and source records attributed to F Lawrence.
Ultrastructural localization of sulfated and unsulfated keratan sulfate in normal and macular corneal dystrophy type I.
Keratan sulfate (KS) proteoglycans are of importance for the maintenance of corneal transparency as evidenced in the condition macular corneal dystrophy type I (MCD I), a disorder involving the absence of KS sulfation, in which the cornea becomes opaque. In this transmission electron microscope study quantitative immuno- and histochemical methods have been used to examine a normal and MCD I cornea. The monoclonal antibody, 5-D-4, has been used to localize sulfated KS and the lectin Erythrina cristagalli agglutinin (ECA) to localize poly N -acetyllactosamine (unsulfated KS). In normal cornea high levels of sulfated KS were detected in the stroma, Bowman's layer, and Descemet's membrane and low levels in the keratocytes, epithelium and endothelium. Furthermore, in normal cornea, negligible levels of labeling were found for N -acetyllactosamine (unsulfated KS). In the MCD I cornea sulfated KS was not detected anywhere, but a specific distribution of N -acetyllactosamine (unsulfated KS) was evident: deposits found in the stroma, keratocytes, and endothelium labeled heavily as did the disrupted posterior region of Descemet's membrane. However, the actual cytoplasm of cells and the undisrupted regions of stroma revealed low levels of labeling. In conclusion, little or no unsulfated KS is present in normal cornea, but in MCD I cornea the abnormal unsulfated KS was localized in deposits and did not associate with the collagen fibrils of the corneal stroma. This study has also shown that ECA is an effective probe for unsulfated KS.
Adult↗
Characterization of prenylated protein methyltransferase in Leishmania.
Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-l-[methyl-(3)H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans, trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, but N-acetyl-S-trans, trans-geranyl-l-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5'-[gamma-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-l-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5'-[gamma-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.
Animals↗
Proton-ATPase activities involved in the uptake of an S-adenosylmethionine analogue.
Characteristics of the transport of sinefungin (SF) were studied in Leishmania donovani promastigotes grown in vitro in a semi-defined medium. The uptake is time and pH dependent, temperature sensitive, saturable and independent of the growth phase. Metabolic inhibitors decrease the influx, indicating that sinefungin uptake is an energy requiring process. The presence of Na+ is unnecessary for activity. The uptake is sensitive to valinomycin and nigericin and to the H+-ATPases inhibitors such as N'N'-dicyclohexylcarbodiimide, bafilomycin A and oligomycin. Sulfhydryl group(s) are involved in carrier activity. Use of SF analogues shows, stereospecificity of the transporter, recognition of the 6'-amino group and to a lesser degree of the 9'-amino group of the lateral chain, whereas the 9'-carboxyl group of the lateral chain is not implicated in the recognition. Adenosine and ornithine do not interfere with the uptake. No significant amount of SF is tightly bound to macromolecules. In a SF-resistant clone, though the uptake of SF is reduced (the apparent Vmax is 276 pmoles mg protein(-1) 30 min(-1) compared with 2061 pmoles mg protein(-1) 30 min(-1) for the wild-type clone), the apparent affinity for SF is similar to that of wild-type cells (Km 0.7 and 0.6 microM respectively). This lower uptake activity is not the reflection of an increased efflux of the drug. In these resistant cells, the susceptibility of SF uptake to variation of the external pH, as well as to azide, NaF, and valinomycin are decreased, that to nigericin is lost.
Adenosine↗
Uptake into leishmania donovani promastigotes and antileishmanial action of an organometallic complex derived from pentamidine.
Iridium (Ir)-(COD)-pentamidine tetraphenylborate (CAS 225-75-4) was selected from a primary screening to be evaluated in vitro on three Leishmania (L.) strains comparatively to pentamidine used as reference compound. The IC50 values obtained from in vitro evaluation on promastigotes of L. major CRE 26, L. donovani DD8 and L. donovani LV9 were 3.9, 23.5, and 3.3 mumol/l for Ir-(COD)-pentamidine tetraphenylborate and 1.6, 7.7, and 3.9 mumol/l for pentamidine isethionate, respectively. Cytotoxicity on mouse peritoneal macrophages led to determine a chemotherapeutic index of 1.7 for Ir-(COD)-pentamidine tetraphenylborate and 4 for pentamidine. Considering L. donovani DD8, the uptake of iridium complex by the promastigotes was shown to be saturable with a Km value of 17.4 mumol/l and Vmax of 1.3 nmol/mg protein/2 h. After 2 and 4 h incubation of treated promastigotes in drug free medium the absence of Ir-complex efflux is in favour of intracellular drug binding. As a matter of fact iridium complex was shown to bind ribosomal subunits in vitro, with no effect on macromolecular biosynthesis.
Animals↗
Effects of pentamidine on polyamine level and biosynthesis in wild-type, pentamidine-treated, and pentamidine-resistant Leishmania.
Polyamine biosynthesis was studied in wild-type promastigotes of Leishmania donovani and Leishmania amazonensis treated with pentamidine and in the parasites resistant to this drug. Treatment of wild-type clones with low pentamidine concentrations for 24 hr provoked a strong decrease in arginine, ornithine, and putrescine pools, while the level of intracellular spermidine remained unchanged. In these cells, the activity of the enzyme ornithine decarboxylase was found to be decreased. Compared to wild-type cells, resistant clones had a lower level of putrescine, higher pools of arginine and ornithine, and a similar spermidine content. Analysis by Western blot and DFMO-binding showed reduced amount of ornithine decarboxylase. Furthermore, in the resistant cells, the kinetic parameters of the enzyme spermidine synthase were markedly changed, showing increased affinity to putrescine and decreased affinity to pentamidine. Thus, it seems that polyamine biosynthesis pathway is a target of pentamidine in Leishmania and is altered in resistant clones.
Animals↗
Biochemical alterations in paromomycin-treated Leishmania donovani promastigotes.
Paromomycin is used for the treatment of leishmaniasis in humans, but little is known about its mechanism of action. Investigating the effect of this antibiotic on promastigotes of Leishmania donovani, we showed that inhibition of the multiplication of these parasites could be related to its effect on RNA synthesis and to modifications of membranous polar lipids and membrane fluidity, leading to altered membrane permeability.
Amebicides↗
Altered transport properties of pentamidine-resistant Leishmania donovani and L. amazonensis promastigotes.
Pentamidine-resistant clones of Leishmania donovani and L. amazonensis promastigotes were developed by increase of the drug pressure in the culture medium and characterized. The resistant clones could grow in 40 and 20 microM pentamidine as determined for L. donovani and L. amazonensis, respectively, with 50% inhibitory concentrations (IC50 values) being 140 and 60 microM, which were 18 and 75 times higher than those recorded for the parental clones, respectively. Biochemical analysis of the clones showed that the acquired pentamidine resistance was specific (no cross-resistance to unrelated drugs and no reversibility with verapamil) and stable in vitro and in vivo. Pentamidine resistance is related to decreased drug uptake and highly increased efflux in both clones of Leishmania spp., accompanied by an alteration in polyamine carriers. Furthermore, a modification of the uptake of pyrimidine nucleosides and several amino acids by these resistant clones indicates alterations in the surface membrane.
Amino Acids↗
In vitro sensitivity of Leishmania donovani to organometallic derivatives of pentamidine.
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Animals↗
Pentamidine uptake in Leishmania donovani and Leishmania amazonensis promastigotes and axenic amastigotes.
A transport system for pentamidine in Leishmania donovani and Leishmania amazonensis promastigotes and axenic amastigotes has been identified and characterized. Pentamidine is not metabolized by these parasites. Its uptake process is saturable, carrier-mediated and energy-dependent. This drug does not inhibit purine or pyrimidine uptake, whereas it inhibits uptake of several amino acids non-competitively and that of putrescine and spermidine competitively. The results suggest that pentamidine shares polyamine-carrier systems in these parasites.
Animals↗
Effects of single P-element insertions on bristle number and viability in Drosophila melanogaster.
Single P-element mutagenesis was used to construct 1094 lines with P[lArB] inserts on all three major chromosomes in an isogenic background previously free of P elements. The effects of insertions on bristle number and on viability were assessed by comparison to 392 control lines. The variance and effects of P-element inserts on bristle number and viability were larger than those inferred from spontaneous mutations. The distributions of effects on bristle number were symmetrical and highly leptokurtic, such that a few inserts with large effects caused most of the increase in variance. The distribution of effects on viability were negatively skewed and platykurtic. On average, the effects of P-element insertions on bristle number were partly recessive and on viability were completely recessive. P-element inserts with large effects on bristle number tended to have reduced viability, but the correlation between the absolute value of the effects on bristle number and on viability was not strong. Fifty P-element inserts tagging quantitative trait loci (QTLs) with large effects on bristle number were mapped cytogenetically. Two P-element-induced scabrous alleles and five extramacrochaetae alleles were generated. Single P-element mutagenesis is a powerful method for identifying QTLs at the level of genetic locus.
Analysis of Variance↗
Sinefungin shares AdoMet-uptake system to enter Leishmania donovani promastigotes.
The involvement of a carrier for sinefungin (SF) uptake in Leishmania donovani promastigotes is indicated by saturation kinetics, competition studies and SF accumulation against a 270-fold concentration gradient across the cell membrane. Whether SF uptake occurs via nucleoside- or AdoMet-carrier systems was investigated by competition experiments and comparison of the uptake of various molecules in wild-type and SF-resistant cells. Results show that SF did not inhibit purine or pyrimidine uptake whereas it competitively inhibited AdoMet uptake. Furthermore, the uptake of nucleosides in SF-resistant cells is similar to that in wild-type cells, whereas uptake of SF and AdoMet is lower.
Adenosine↗
Ribosomes of Leishmania are a target for the aminoglycosides.
Ribosomes of Leishmania, a parasitic protozoan (member of the order of Kinetoplastidae), were purified on a sucrose density gradient. Two different types of ribosomes were isolated from the promastigotes: cytoplasmic (88S and 91S from L. tropica and L. donovani, respectively) and mitochondrial (75S in both species). Both types of ribosome dissociated into their subunits at low Mg2+ concentration (1-2 mM) as follows: 67S and 49S for the 91S cytoplasmic ribosomes of L. donovani and 61S and 43S for the 88S cytoplasmic ribosome of L. tropica; 55S and 34S for L. tropica and 60S and 39S for L. donovani mitochondrial ribosomes, respectively. Paromomycin (aminosidine), an aminoglycoside aminocyclitol antibiotic, interacted with the ribosomes to promote the association of the subunits. Under similar experimental conditions, spermidine and pentamidine were inactive.
Animals↗
Expression of the major surface glycoprotein of Leishmania, gp63, in wild-type and sinefungin-resistant promastigotes.
In this study, we have surveyed gp63 expression in sinefungin-(SF)-resistant and wild-type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3-4-fold and 1.5-2-fold increase of gp63 protein in SF-resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild-type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF-resistant compared to wild-type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF-resistant and wild-type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF-resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF-resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post-transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF-resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF-resistant compared to wild-type L. donovani promastigotes.
Adenosine↗
Characterization of sinefungin-resistant Leishmania donovani promastigotes.
Promastigotes resistant to sinefungin (SF), a nucleoside antibiotic that is structurally related to S-adenosylmethionine (AdoMet), were obtained starting from two cloned strains of Leishmania donovani. The resistance was induced by increasing the drug pressure gradually until promastigotes capable of growing in the presence of concentrations 10,000 times higher than the 50% growth-inhibitory (IC50) values for the control cells were obtained. The resistance to SF of both clones was specific and stable in the absence of drug pressure. High-performance liquid chromatographic (HPLC) analyses indicated highly reduced levels of SF in the two resistant clones. However, the intracellular SF concentration in these resistant cells was much higher than the IC50 values for wild-type cells. In one clone, the decreased drug uptake was coupled to a decrease in the affinity of two protein methylases for SF, whereas in the other clone the biosynthesis of polyamine precursors was modified. This study demonstrates that resistance to a drug molecule with pleiotropic targets can be developed through various mechanisms by different strains.
Adenosine↗
Leishmania donovani: antagonistic effect of S-adenosyl methionine on ultrastructural changes and growth inhibition induced by sinefungin.
Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent in vitro and in vivo (Bachrach et al. 1980, FEBS Letters 121, 287-291; Neal et al. 1985, Transactions of the Royal Society of Tropical Medicine and Hygiene 79, 85-122). It was previously shown that this molecule is a competitive inhibitor of AdoMet for transmethylases (Paolantonacci et al. 1986, Molecular and Biochemical Parasitology 21, 47-54; Avila et al. 1987, Molecular and Biochemical Parasitology 26, 69-76) and that it induces shape changes of Leishmania donovani promastigotes as observed by light microscopy (Lawrence and Robert-Gero 1990; Bulletin de la Societé Française de Parasitologie 8, 13-18). In the present work the effect of the antibiotic on the ultrastructure was analyzed by electron microscopy. The main changes induced at sublethal concentrations (0.26 microM sinefungin for 16 hr) were progressive rounding, decreased motility, enlargement of the flagellar pocket, and shortening and loss of the external part of the flagellum. The comparison with control cells showed shorter Golgi saccules and fragmentation of the trans-Golgi network into vesicles, indicating a stimulated Golgi apparatus activity. This result, associated with the enlarged flagellar pocket, suggests an unbalanced cytoplasmic exchange between exocytosis and endocytosis. These effects are quite different from those induced by tunicamycin (Dagger et al. 1984, Biology of the Cell 50; 173-180) or paromomycin. In addition, other nucleoside and nonnucleoside growth inhibitors failed to induce similar changes. AdoMet antagonized the sinefungin-induced shape changes and ultrastructural modifications but had no effect with respect to other growth inhibitors. This suggests that the sinefungin activity at the cellular level is specifically related to competition with AdoMet. A comparative study of N-methylation and carboxylmethylation of proteins in sinefungin-treated promastigotes showed that the antibiotic preferentially inhibits the latter, catalyzed by protein-O-methyltransferases. These enzymes are known to regulate the function of various proteins involved in secretion. Overall the results suggest that one of the main targets of sinefungin in exponentially growing cells is the protein carboxylmethylation involved in membrane transport.
Adenosine↗
Total synthesis of uracil analogues of sinefungin.
Analogues of sinefungin derivatives 18a and 18b have been prepared from uridine and L-aspartic acid. The key step in the synthesis was the coupling of the radical derived from 14 with the unsaturated amide 13. The latter was produced from the known N-hydroxy-2-thiopyridone ester of L-aspartic acid 12 with the olefin 11. Thus, the essential carbon skeleton was constructed by way of two radical coupling reactions. These analogues as well as 1a and 1b synthesized previously were tested for their antileishmanial effect in vivo and for their inhibitory activity of protein carboxymethylase (protein methylase II). The replacement of the adenine moiety by uracil or dihydrouracil considerably decreases the antiparasitic activity and the affinity for protein methylase II. The synthetic (S)-sinefungin was as active as the natural one. Interestingly, the C-6' epimer 1b was 50% less active in vitro than the natural sinefungin, but both had identical affinities for the target enzyme.
Adenosine↗
Synthesis and biological activity of sinefungin analogues.
A series of nucleosides (2-4) that derive from adenosine by chain extension at the 5'-end have been synthesized starting from the known phosphonate 7. The latter was first combined with 4-pentenal to give 8, which underwent chemical manipulations to provide triacetate 11, which was found suitable for the adenylation step. Further transformations, among them the Hofmann degradation of the amide group of compound 13, and final deprotection gave nucleosides 2-4. They were considered as analogues of sinefungin (1) and tested for their antileishmanial activity together with compounds 5 and 6, which were obtained independently. All the modifications with respect to sinefungin resulted in nearly complete loss of growth inhibitory activity. These results indicate that the 9' terminal amino and carboxyl groups are necessary for the activity and that the presence of the amino group at C-6' is not sufficient to maintain the antileishmanial effect. Some of the analogues however could antagonize or reverse the inhibitory activity of sinefungin (1).
Adenosine↗