Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.     

Eosinophil chemotactic activity in Leishmania amazonensis promastigotes

AIM: To study the effects of chronic administration of SPD on the density and turnover of striatal D1 and D2 dopamine (DA) receptors. METHODS: Receptor density was monitored by radio-receptor binding assay. The receptor recovery and turnover were studied after irreversible inactivation by N-ethoxycarbonyl-2-ethoxy-1, 2-dihydro-quinoline (EEDQ). RESULTS: Chronic SPD treatment (sc, 20 mg.kg-1.d-1 x 21 d) upregulated both striatal D1 and D2 receptor density. As compared to vehicle-treated rats, SPD increased D1 and D2 receptors by 41.5% and 43.7%, respectively SPD also altered the turnover of both D1 and D2 receptors. The degradation rate constant (k = 0.0082.h-1) and the synthesis rate (r = 2.65 pmol.h-1/g protein) of D2 receptors in SPD-treated rats were significantly increased vs vehicle-treated rats (k = 0.0049.h-1; r = 1.10 pmol.h-1/g protein). The degradation rate constant (k = 0.0059.h-1) and the synthesis rate (r = 3.1 pmol.h-1/g protein) of D1 receptors was also increased in SPD-treated rats vs vehicle-treated rats (k = 0.0048.h-1; r = 1.8 pmol.h-1/g protein), but the alteration of degradation rate constant missed significance (P > 0.05). As a result, receptor recovery following EEDQ was accelerated. The half time for D1 and D2 receptors recovery in SPD group were 117.5 h and 84.5 h, respectively, shorter than 144.4 h and 141.4 h in vehicle-treated rats. CONCLUSION: Chronic SPD treatment upregulated D1 and D2 receptors, and accelerated DA receptor turnover and recovery mainly by increasing receptor synthesis.     

Phenotypic characterization of Leishmania mexicana pentamidine-resistant promastigotes. Modulation of the resistance during in-vitro developmental life cycle

Two clones of Leishmania mexicana resistant to 5 microM (LmR5CL2) and 20 microM (LmR20CL1) pentamidine, derived from a parental wild-type clone (LmWTCL3) were selected in vitro using a continuous drug pressure protocol. Both resistant clones expressed a cross-resistance to diminazene aceturate. No differences in their in-vitro infectivity for mouse peritoneal macrophages between wild-type and pentamidine-resistant promastigotes were observed. During these experiments, promastigotes of LmR20CL1 derived from intramacrophagic amastigote forms reverted to the pentamidine-sensitive phenotype, unlike the lower resistant ones. In the same way, when a complete developmental sequence of L. mexicana was achieved in axenic cultures, LmR20CL1 promastigotes derived from axenically growing amastigotes expressed an IC50 value close to the wild-type one, whereas resulting LmR5CL2 promastigotes remained pentamidine resistant. This modulation of the chemoresistance during the developmental life cycle could be significant in the transmission of drug-resistant strains by Phlebotominae as well as in basic research to follow drug resistance during the in-vitro and in-vivo life cycle of Leishmania.     

Experimental infection of domestic sheep with culture-derived Leishmania donovani promastigotes

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.     

Is PGL-1 also present in Leishmania donovani promastigotes?

We report a 14 year old boy who presented as a neonate with functional pulmonary atresia due to Uhl's disease with emphasis on the later detection of restrictive right ventricular physiology.     

Efficacies of KY62 against Leishmania amazonensis and Leishmania donovani in experimental murine cutaneous leishmaniasis and visceral leishmaniasis

Carcass measurements of 12th-rib fat thickness (CARCFAT), longissimus muscle area (CARCLMA), and weight (CARCWT) on 2,028 Brangus and Brangus-sired fed steers and heifers, as well as yearling weights (YWT) and ultrasound measures of 12th-rib fat thickness (USFAT) and longissimus muscle area (USLMA) on 3,583 Brangus bulls and heifers were analyzed to estimate genetic parameters. Data were analyzed using a six-trait animal model and an average information REML algorithm. The model included fixed effects for contemporary group and breed of dam, covariates for age at slaughter or measurement, and random animal and residual effects. Heritabilities for CARCFAT, CARCLMA, CARCWT, USFAT, USLMA, and YWT were .27+/-.05, .39+/-.05, .59+/-.06, .11+/-.03, .29+/-.04, and .40+/-.04, respectively. Genetic correlations between CARCFAT and USFAT, CARCLMA and USLMA, and CARCWT and YWT were .69+/-.18, .66+/-.14, and .61+/-.11, respectively. The favorable and moderately strong genetic correlations between carcass measurements and similar yearling breeding-animal ultrasound measurements indicate that such measurements of 12th-rib fat and longissimus muscle area are useful in predicting genetic values for carcass leanness and longissimus muscle area. Selection using yearling ultrasound measurements of breeding cattle should result in predictable genetic improvement for carcass characteristics. Inclusion of yearling ultrasound measurements for fat thickness and longissimus muscle area should enhance national cattle evaluation programs.     

Specificity of the glucose transport system in Leishmania tropica promastigotes

The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q10 of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (alpha-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3-0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature--they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident; therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Induction of complement-sensitivity in Leishmania amazonensis metacyclic promastigotes by protease treatment but not by specific antibodies

Acute appendicitis, particularly pelvic, may cause a suppurative process that spontaneously drains to the bladder and an appendicovesical fistula may result. The pelvic appendix and the bladder may occasionally become fused in an inflammatory and necrotic focus, directly creating a fistula. A case of vesicointestinal fistula secondary to appendiceal pelvic peritonitis is described and the cases reported in the literature are reviewed.     

Macrophage mannosyl fucosyl receptor: its role in invasion of virulent and avirulent L. donovani promastigotes

The interaction of leishmania parasites with macrophages is known to be receptor mediated. Previous study from this laboratory (J. Parasitol. 82:632, 1996) showed the significant involvement of LPG and gp63 receptors in the recognition of virulent strains onto the macrophages. The role of carbohydrate receptors the other major receptors besides LPG and gp63 receptors, in the recognition of both virulent (strains AG83 and GE1) and avirulent (strain UR6) leishmania onto the host macrophages has been the major focus of the present investigation. Various neoglycoproteins were used as efficient ligands to preblock the carbohydrate receptors on the macrophage surface. Similarly, various sugar specific lectins were used to preblock the corresponding carbohydrate ligands on the parasite surface. When these preblocked macrophages or parasites were used to study their mode of recognition, it was obvious from the findings that avirulent leishmania promastigotes possibly use the mannosyl fucosyl receptors (MFR) more avidly for their initial attachment and subsequent internalization into the macrophages whereas the virulent leishmania exhibits limited use of this receptor. When a macrophage-like cell line (J774), lacking in MFR, was purposely selected to test the previous findings, as expected, the attachment of avirulent promastigotes (UR6) onto the cell line was found to be negligible when compared to the peritoneal macrophages. Thus, it appears that avirulent leishmania promastigotes probably utilize MFR significantly for their initial recognition and subsequent internalization by macrophages.     

Reduced expression of lipophosphoglycan (LPG) and kinetoplastid membrane protein (KMP)-11 in Leishmania donovani promastigotes in axenic culture

The expression of surface lipophosphoglycan (LPG) and the lipophosphoglycan-associated kinetoplastid membrane protein (KMP)-11 was studied in the strain AG83 of Leishmania donovani in axenic culture. The expression of LPG and KMP-11 decreased along with parasite virulence as a function of the time of the subculture.     

Membrane characterization of amastigote-like forms of Leishmania donovani

During in vitro transformation, Leishmania donovani promastigotes converted into amastigote-like forms and underwent several changes in membrane parameters. They exhibited significantly increased microviscosity comparable to true amastigotes. Activities of several functionally important membrane bound enzymes were also altered, thereby indicating a change in their orientation. Peanut agglutinin was found to be specific for agglutination of stationary phase promastigotes whereas wheat-germ agglutinin was specific for the amastigote-like forms as well as for pure amastigotes, implying the presence of specific glycoconjugates on the parasite surface.     

Genetic analysis of purine metabolism in Leishmania donovani

To dissect the contributions of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), adenine phosphoribosyltransferase (APRT), and adenosine kinase (AK) to purine salvage in Leishmania donovani, null mutants genetically deficient in HGPRT and/or APRT were generated by targeted gene replacement in wild type cells and preexisting mutant strains lacking either APRT or AK activity. These knockouts were obtained either by double targeted gene replacement or by single gene replacement followed by negative selection for loss-of-heterozygosity. Genotypes were confirmed by Southern blotting and the resultant phenotypes evaluated by enzymatic assay, resistance to cytotoxic drugs, ability to incorporate radiolabeled purine bases, and growth on various purine sources. All mutant strains could propagate in defined growth medium containing any single purine source and could metabolize exogenous [3H]hypoxanthine to the nucleotide level. The surprising ability of mutant L. donovani lacking HGPRT, APRT, and/or AK to incorporate and grow in hypoxanthine could be attributed to the ability of the parasite xanthine phosphoribosyltransferase enzyme to salvage hypoxanthine. These genetic studies indicate that HGPRT, APRT, and AK, individually or in any combination, are not essential for the survival and growth of the promastigote stage of L. donovani and intimate an important, if not crucial, role for xanthine phosphoribosyltransferase in purine salvage.     

Effect of new diamidines against Leishmania donovani infection

The impact of interamidine distance on antileishmanial activity of new aryldiamidines have been evaluated against amastigotes of L. donovani in hamster. Of the 20 compounds tested, only four (2,8-diamidino-9,10-dihydrodibenzoxepin; 2,7-diamidinoxanthone; 2,7-diamidinothioxanthone and 2,7-diamidinoxanthene) showed significant inhibition (more than 80%) of multiplication of amastigotes in spleen. The interamidine distance in the structure appears to have bearing on antileishmanial activity. The observations made are likely to evoke new understanding on the structure activity relationship of diarylamidines.     

Ornithine decarboxylase gene deletion mutants of Leishmania donovani

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of Deltaodc cells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The Deltaodc L. donovani exhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the Deltaodc strain could grow in medium supplemented with either 1,3-diaminopropane or 1, 5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.     

Inducible expression of suicide genes in Leishmania donovani amastigotes

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. The Leishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani 3'-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitro and in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.     

Survival strategies of Leishmania donovani in mammalian host macrophages

Microbes have evolved a variety of strategies to survive inside their host cells. Some have the molecular machinery to survive in the hostile environment of phagolysosomes; others escape the phagosome to the more cozy environment of the cell cytoplasm; others inhibit the phagosome fusion with hydrolase-enriched endocytic organelles. This is the case for the promastigote form of the protozoan parasite Leishmania donovani which resides in a phagosome displaying poor fusogenic properties towards endosomes and lysosomes. Recent results indicate that the lipophosphoglycan (LPG), the major cell surface molecule of Leishmania, is involved in the inhibition of phagosome maturation. Further studies in our laboratories are addressing the molecular mechanisms of action of LPG to modulate phagosome fusion properties and its effect on the biogenesis of phagolysosomes.     

Fate of the promastigotes of Leishmania tropica major and L. gymnodactyli in the body of Phlebotomus papatasi under conditions of a mixed infection

We have shown that 8S and U2 fractions of low molecular weight nuclear RNA lmwn RNA) have tow stable conformational states with different electrophoretic mobilities. We have shown also that fractions UIa and UIb are conformational isomers. Fractions 8S and U2 are present only in one conformational state during electrophoresis, while UI RNA is always represented by both conformations. The conformation of lmwn RNAs is dependent upon conditions of isolation of nuclei and of extraction of RNA. It may change during storage of water solution of RNA at -20 degrees. Conformational changes discovered are not the result of degradation. Only one of two stable conformations of any lmwn RNA may be physiologically active, as it is the case of 5S rRNA and tRNAS. One ought to keep this in mind while studying the functions of low molecular weight nuclear RNAs.     

Leishmania donovani heat shock protein 100. Characterization and function in amastigote stage differentiation

We report the cloning and molecular analysis of the Leishmania donovani clpB gene. The protein-coding region is highly conserved compared with its L. major homologue, while 5'- and 3'-flanking DNA sequences display considerable divergence. The encoded mRNA has an unusually long 5'-leader sequence typical for RNAs, which are translated preferentially under heat stress. The gene product, a 100-kDa heat shock protein, Hsp100, becomes abundant only during sustained heat stress, but not under common chemical stresses. Hsp100 associates into trimeric complexes and is found mostly in a cytoplasmic, possibly membrane-associated, localization as determined by immune electron microscopy. Hsp100 shows immediate early expression kinetics during axenic amastigote development. In its absence, expression of at least one amastigote stage-specific protein family is impaired.     

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.