Ornithine decarboxylase from Leishmania mexicana promastigotes: interaction with pyridoxal 5'-phosphate and alpha-difluoromethylornithine

The catalytic properties of ornithine decarboxylase (ODC) from Leishmania mexicana as well as the interaction with its cofactor pyridoxal 5'-phosphate (PLP) and the irreversible inhibitor alpha-difluoromethylornithine (DFMO) have been studied using partially purified preparations of the enzyme obtained from parasite promastigotes. Leishmania extracts prepared in the presence of saturating concentrations of PLP yielded an enzyme considerably more resistant to heat inactivation and with a three-fold higher activity than the ODC obtained without the addition of cofactor. The complete removal of PLP by treatment with hydroxylamine yielded the apoenzyme which shows an absolute requirement for PLP to recover its enzymatic activity. The Km values for L-ornithine and PLP were 0.7 mM and 25 microM, respectively, while Ki for DFMO was 0.2 mM. The restoration of ODC activity from apoenzyme and cofactor seems to involve time and temperature-dependent activation processes. L. mexicana ODC has an apparent molecular mass of 240 +/- 20 kDa.     

Ornithine decarboxylase and polyamines in familial adenomatous polyposis

The effects of concanavalin A (Con A) on liver cytokine gene expression was studied in mice. The CD4 mRNA expression in normal liver suggests the presence of CD4+ T cells. The administration of Con A induced interleukin (IL)-1beta, IL-2 and IL-2 receptor mRNAs, which implies lymphocyte activation in the liver. Interferon-gamma and tumor necrosis factor-alpha mRNA expressions were increased gradually. The present results showed that Con A induced liver cytokine genes. This cytokine gene induction might have been the result of lymphocyte activation in the liver.     

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.     

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.     

Ornithine decarboxylase activity during development of cerebellar granule neurons

Ornithine decarboxylase (ODC), the key enzyme for polyamine biosynthesis, dramatically decreases in activity during normal cerebellar development, in parallel with the progressive differentiation of granule neurons. We have studied whether a similar pattern is displayed by cerebellar granule neurons during survival and differentiation in culture. We report that when granule cells were kept in vitro under trophic conditions (high K+ concentration), ODC activity progressively decreased in parallel with neuronal differentiation. Under nontrophic conditions (cultures kept in low K+ concentration), the enzymatic activity dropped quickly in parallel with an increased apoptotic elimination of cells. Cultures kept in high K+ but chronically exposed to 10 mM lithium showed both an increased rate of apoptotic cell death at 2 and 4 days in vitro and a quicker drop of ODC activity and immunocytochemical staining. A short chronic treatment of rat pups with lithium also resulted in transient decrease of cerebellar ODC activity and increased programmed cell death, as revealed by in situ detection of apoptotic granule neurons. The present data indicate that a sustained ODC activity is associated with the phase of survival and differentiation of granule neurons and that, conversely, conditions that favor their apoptotic elimination are accompanied by a down-regulation of the enzymatic activity.     

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.     

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.     

Ornithine decarboxylase inactivation by putrescine: involvement of lipid peroxidation

Effect of polyamines on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced reduction of lipid peroxidation was studied. Putrescine protected this lowering of lipid peroxidation in a concentration-dependent manner, but spermidine or spermine could not do so. Putrescine also inhibited the TPA-induced ornithine decarboxylase (ODC) activity and lowered the free sulfhydryl content of TPA exposed mouse skin. These observations indicate that putrescine inactivates ODC probably by lowering SH groups through lipid peroxidation.     

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.     

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.     

Ornithine decarboxylase induction by glucocorticoids in brain and liver of adrenalectomized rats

Ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, was measured in the brain and the liver of adrenalectomized rats after an acute s.c. treatment with glucocorticoids. The effects of corticosterone and dexamethasone were compared in three brain areas, the cerebral cortex, hippocampus, and cerebellum. These structures have similar concentrations of cytosolic glucocorticoid receptor, as measured by an in vitro exchange assay using a specific glucocorticoid ligand, [3H]RU 26988, but contain different amounts of mineralocorticoid receptor. Corticosterone and dexamethasone increased ODC activity in the liver and brain areas in a dose-dependent manner, dexamethasone being more active than corticosterone in all tissues. Moreover, estradiol, progesterone, and testosterone were inactive. Aldosterone, at high doses, increased brain ODC activity. Glucocorticoids, selected for their weak binding, or lack of binding to the mineralocorticoid receptor, were tested and found to be highly active in inducing brain and liver ODC, thus showing that ODC induction by steroids is specific for glucocorticoids. These results are among the first to suggest biochemically a central action of glucocorticoids following an acute treatment and confirm that the brain is a glucocorticoid target organ.     

Leishmania donovani attachment stimulates PKC-mediated oxidative events in bone marrow-derived macrophages

We investigated activation signaling events in bone marrow-derived macrophages after infection with Leishmania donovani, an intracellular parasite of macrophages. Leishmania donovani infection caused a general suppression of activation parameters like O2- and NO production. However, conditions which allow parasite attachment and prevent entry resulted in triggering of O2- and NO production and stimulation of O2 consumption. Optimal NO and O2- production occurred when bone marrow-derived macrophages and Leishmania ratio was 1:100. The activation signal for O2- production was initiated 15 min after parasite attachment, whereas augmentation of NO production started 6 h after attachment Activation of O2- and NO generation by L. donovani attachment was inhibited by staurosporine as well as by prolonged treatment of phorbol myristate acetate suggesting a protein kinase C-dependent mechanism. Translocation studies showed that protein kinase C activity in cell membrane fraction rapidly and transiently increased following parasite attachment. No such protein kinase C translocation event occurred in L. donovani infected bone marrow-derived macrophages. Phorbol myristate acetate was found to stimulate membrane translocation of protein kinase C in parasite attached cells whereas it was impaired in infected cells. However, both attachment and infection induced a similar shift of phorbol receptors from cytosolic to membrane fraction indicating that in infected cells the translocation of protein kinase C protein was not impaired but the activity of the membrane associated enzyme was somehow inhibited. These results suggest that although internalization of intracellular parasites like L. donovani caused inhibition of nitrite and superoxide release, mere attachment on macrophage surface resulted in an activation of protein kinase C-mediated downstream oxidative events.     

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.     

Defective galactofuranose addition in lipophosphoglycan biosynthesis in a mutant of Leishmania donovani

A mutant cell line of Leishmania donovani (R2D2), previously selected for resistance to the cytotoxic lectin ricin agglutinin, was found to be totally deficient in the synthesis and expression of lipophosphoglycan, a dominant surface virulence factor. The metabolic defect in R2D2 parasites responsible for its lipophosphoglycan (LPG-) phenotype was investigated in this study. Following metabolic labeling of R2D2 parasites with either [3H]galactose or [3H]mannose, the main glycosylphosphatidylinositide product that accumulated was Glc-PO4-Man-Man-GlcN-lyso-1-O-alkylphosphatidylinositol (PI). The metabolic defect was further defined using a cell-free glycosylation system. When membrane preparations from wild-type cells were incubated with UDP-[3H]galactose and unlabeled GDP-mannose in the absence of exogenous acceptors, radiolabeled lipophosphoglycan was synthesized. The addition of exogenous Man-Man-GlcN-PI or Galf-Man-Man-GN-PI stimulated lipophosphoglycan synthesis in vitro. In contrast, when membrane preparations from R2D2 cells were incubated with exogenous Man-Man-GlcN-PI as an acceptor or in the absence of exogenous acceptor, the truncated glycosylphosphatidylinositide Glc-PO4-Man-Man-GlcN-PI was the main radioactive product synthesized. However, when exogenous Galf-Man-Man-GN-PI was added to the R2D2 in vitro system, radioactive lipophosphoglycan was synthesized. Collectively, these results indicate that the mutant R2D2 cells are unable to complete the assembly of the glycan core of LPG because of a defect in the synthesis of the "activated" galactofuranosyl donor or the lack of a functional galactofuranosyltransferase.     

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.     

Leishmania donovani: acquired resistance to visceral leishmaniasis in the golden hamster

Multiple rough endoplasmic cisternae were found in "C" cells of the adult rat, at interphase. They are considered to be normal constituents of "C" cells. Their morphological relation to rough endoplasmic reticulum and their close proximity to mitochondria, Golgi dictyosomes and secretory granules suggest that they may have a role in the secretory activity of this endocrine cells.