Trypanothione Reductase Activity is Prominent in Metacyclic Promastigotes and Axenic Amastigotes of Leishmania amazonesis. Evaluation of its Potential as a Therapeutic Target

The activity of trypanothione reductase in Leishmania amazonensis was evaluated and it was demonstrated that TR is expressed in the soluble fractions of infective promastigotes and amastigotes, while non-infective promastigotes expressed the enzyme at basal levels. This data allows an association of enzyme activity and the infective capacity of the parasite. We have also previously demonstrated that amidine compounds (N, N′-diphenyl-4-methoxy-benzamidine and pentamidine) were active against this parasite. Here, experiments concerning the effect of these compounds on TR activity, showed that both compounds significantly inhibited the enzyme. However, against glutathione reductase, only pentamidine showed a significant inhibitory action, suggesting an association with the toxic effects of this drug used in the clinic for the treatment of leishmaniasis.     

O2''-Methylinosine, a constituent of the ribosomal RNA of Crithidia fasciculata.

A novel nucleoside, O2'-methylinosine (Im), has been identified as a constituent of the ribosomal RNA of Crithidia fasciculata, a hemoflaggelate protozoan. The nucleoside is released as part of an alkali-stable dinucleotide, Im-Up, by alkaline hydrolysis of Crithidia rRNA, and as a 5'-nucleotide, pIm, by snake venom hydrolysis of the same RNA. The Im-containing derivatives isolated from Crithidia rRNA were characterized by comparison with marker compounds prepared by chemical deamination of the corresponding adenosine analogues. O2'-Methylinosine prepared from either natural Im-Up or natural pIm had the same ultraviolet absorption spectra and chromatographic properties as marker Im. Characterization of the base and sugar components of Im as hypoxanthine and 2-O-methylribose, respectively, provided final confimration of structure. Control experiments have eliminated the possibility that Im arises from O2'-methyladenosine (Am), a known constituent of ribosomal RNA, by chemical or enzymatic deamination during hydrolysis of Crithidia rRNA.     

The Effect of Tunicamycin on the Glucose Uptake,Growth, and Cellular Adhesion in the Protozoan Parasite Crithidia fasciculata

Crithidia fasciculata represents a very interesting model organism to study biochemical, cellular, and genetic processes unique to members of the family of the Trypanosomatidae. Thus, C. fasciculata parasitizes several species of insects and has been widely used to test new therapeutic strategies against parasitic infections. By using tunicamycin, a potent inhibitor of glycosylation in asparaginyl residues of glycoproteins (N-glycosylation), we demonstrate that N-glycosylation in C. fasciculata cells is involved in modulating glucose uptake, dramatically impacting growth, and cell adhesion. C. fasciculata treated with tunicamycin was severely affected in their ability to replicate and to adhere to polystyrene substrates and losing their ability to aggregate into small and large groups. Moreover, under tunicamycin treatment, the parasites were considerably shorter and rounder and displayed alterations in cytoplasmic vesicles formation. Furthermore, glucose uptake was significantly impaired in a tunicamycin dose-dependent manner; however, no cytotoxic effect was observed. Interestingly, this effect was reversible. Thus, when tunicamycin was removed from the culture media, the parasites recovered its growth rate, cell adhesion properties, and glucose uptake. Collectively, these results suggest that changes in the tunicamycin-dependent glycosylation levels can influence glucose uptake, cell growth, and adhesion in the protozoan parasite C. fasciculata.     

Effect of Temperature and Osmolarity on Growth of Crithidia fasciculata,C. hutneri,C. luciliae thermophila,and Herpetomonas samuelpessoai1

Crithidia fasciculata (Anopheles, Culex, and Nöller strains), C. hutneri, C. luciliae thermophila, and Herpetomonas samuelpessoai were grown in a defined medium with different values of osmolarity at different temperatures. C. fasciculata (all strains) grew best between 300 to 500 mOsm; H. samuelpessoai, 400–500 mOsm; and C. hutneri and C. luciliae thermophila, 500–800 mOsm. At higher temperatures better growth was obtained at the upper osmolarities.     

Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan.

In a previous study from this laboratory, presumptive ribosomal ribonucleic acid (RNA) species were identified in the total cellular RNA directly extracted from intact cells of the trypanosomatid protozoan Crithidia fasciculata (M. W. Gray, Can. J. Biochem. 57:914-926, 1979). The results suggested that the C. fasciculata ribosome might be unusual in containing three novel, low-molecular-weight ribosomal RNA components, designated e, f, and g (apparent chain lengths 240, 195, and 135 nucleotides, respectively), in addition to analogs of eucaryotic 5S (species h) and 5.8S (species i) ribosomal RNAs. In the present study, all of the presumptive ribosomal RNAs were indeed found to be associated with purified C. fasciculata ribosomes, and their localization was investigated in subunits produced under different conditions of ribosome dissociation. When ribosomes were dissociated in a high-potassium (880 mM K+, 12.5 mM Mg2+) medium, species e to i were all found in the large ribosomal subunit, which also contained an additional, transfer RNA-sized component (species j). However, when subunits were prepared in a low-magnesium (60 mM K+, 0.1 mM Mg2+) medium, two of the novel species (e and g) did not remain with the large subunit, but were released, apparently as free RNAs. Control experiments have eliminated the possibility that the small RNAs are generated by quantitative and highly specific (albeit artifactual) ribonuclease cleavage of large ribosomal RNAs during isolation. In terms of RNA composition and dissociation properties, therefore, the ribosome of C. fasciculata is the most "atypical" eucaryotic ribosome yet described. These observations raise interesting questions about the function and evolutionary origin of C. fasciculata ribosomes and about the organization and expression of ribosomal RNA genes in this organism.     

Changes in the Shape of Leishmania major Promastigotes in Response to Hexoses, Proline, and Hypo-osmotic Stress

Leishmania major promastigotes in late-log phase are generally long and slender, and remain so during a 1 h incubation in buffer without exogenous substrate. When glucose, 2-deoxyglucose, fructose, mannose, or proline are added, the cells become shorter and more rounded. The shape change in response to glucose is complete within 20 min and is reversible upon incubating the cells without substrate. Galactose, 3-O-methylglucose, 6-deoxyglucose, sucrose, maltose, ribose, glycerol, alanine, glutamate or aspartate do not cause the shape change. Decreasing the osmolarity of the medium causes a rounding of the cells similar to that observed in the presence of glucose, and increasing the osmolarity inhibits the shape change in response to glucose. Inhibitors of glucose transport and 2nd messenger analogs do not affect the shape change.     

Fluorescein-Conjugated Bandeiraea simplicifolia Lectin as a Marker of Endodermal, Yolk Sac, and Trophoblastic Differentiation in the Mouse Embryo

Abstract. The Bandeiraea simplicifolia lectin I (BSA-I) conjugated to fluorescein isothiocyanate was used as a histochemical reagent to study the mouse embryos from fertilization to early somitogenesis. No lectin binding could be detected on the embryonic cells in the preimplantation embryo. Lectin labeled intensely the zona pellucida. In the implanting embryos lectin binding was detected along the subtrophectodermal and Reichert's membrane, in the cytoplasm of the parietal and visceral endoderm, and the trophoblastic giant cells, but not in the ectodermal cells. Studies on explanted blastocyts cultured in vitro disclosed that the cytoplasmic BSA-I binding sites in trophoblastic cells develop gradually. In the 9-day somitic embryo BSA-I reacted with epithelial cells of the yolk sac, but not with the mesenchymal cells. A continuity between the lectin-reactive endoderm and the foregut epithelium could be demonstrated. These data indicated that BSA-I lectin can be used as a histochemical probe for endodermal (yolk sac) and trophoblastic differentiation in the peri-implantational mouse embryo.     

Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases

Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

IL-5-Induced Eosinophils Suppress the Growth of Leishmania amazonensis In Vivo and Kill Promastigotes In Vitro in Response to Either IL-4 or IFN-gamma

In IL-5 transgenic mice (C3H/HeN-TgN(IL-5)-Imeg), in which 50% of peripheral blood leukocytes are eosinophils, the development of infection by Leishmania amazonensis was clearly suppressed. To determine mechanistically how this protozoan parasite is killed, we performed in vitro killing experiments. Either IL-4 or IFN-gamma effectively stimulated eosinophils to kill Leishmania amazonensis promastigotes, and most of the killing was inhibited by catalase but not by the NO inhibitor L-N5-(1-iminoethyl)-ornithine, suggesting that hydrogen peroxide is responsible for the killing of L. amazonensis by eosinophils. There was no significant degranulation of eosinophils in the culture, because eosinophil peroxidase was not detected in culture supernatants when L. amazonensis promastigotes were killed by activated eosinophils. Such resistance was also observed in BALB/c mice, which are highly susceptible to L. amazonensis. Expression plasmids for IL-4, IL-5, and IFN-gamma were transferred into muscle by electroporation in vivo starting 1 week before infection. Expression plasmid for IL-5 was most effective in slowing the development of infection among three expression plasmids. Expression plasmid for IL-4 was slightly effective and that for IFN-gamma had no effect on the progress of disease. These results suggest that IL-5 gene transfer into muscle by electroporation is useful as a supplementary protection method against L. amazonensis infection.     

An Insight into the Structural Requirements and Pharmacophore Identification of Carbonic Anhydrase Inhibitors to Combat Oxidative Stress at High Altitudes: An In-Silico Approach

Carbonic anhydrases (CA) inhibitory action could be linked to the treatment of a number of ailments, including cancer, osteoporosis, glaucoma, and several neurological problems. For the development of effective CA inhibitors, a variety of heterocyclic rings have been investigated. Furthermore, at high altitudes, oxygen pressure drops, resulting in the formation of reactive oxygen and nitrogen species, and CA inhibitors having role in combating this oxidative stress. Acetazolamide contains thiadiazole ring, which has aroused researchers’ interest because of its CA inhibitory action. In the present study, we used a number of drug design tools, such as pharmacophore modeling, 3D QSAR, docking, and virtual screening on twenty-seven 1,3,4-thiadiazole derivatives that have been described as potential CA inhibitors in the literature. An atom-based 3D-QSAR analysis was carried out to determine the contribution of individual atoms to model generation, while a pharmacophore mapping investigation was carried out to find the common unique pharmacophoric properties required for biological activity. The coefficient of determination for both the training and test sets were statistically significant in the generated model. The best QSAR model was chosen based on the values of R² (0.8757) and Q² (0.7888). A molecular docking study was also conducted against the most potent analogue 4m, which has the highest SP docking score (−5.217) (PDB ID: 6g3v). The virtual screening revealed a number of promising compounds. The screened compound ZINC77699643 interacted with the amino acid residues, Pro201 and Thr199, in the virtual screening study (PDB ID: 6g3v). These interactions demonstrated the significance of the CA inhibitory activity of the compound. Furthermore, ADME study revealed useful information regarding compound’s drug-like properties. Therefore, the findings of the present investigation could aid in the development of more potent CA inhibitors, which could benefit the treatment of oxidative stress at high altitudes.     

Insight into the secondary structure of non-native proteins bound to a molecular chaperone alpha-crystallin. An isotope-edited infrared spectroscopic study

alpha-Crystallin, the major lens protein, acts as a molecular chaperone by preventing the aggregation of proteins damaged by heat and other stress conditions. To characterize the backbone conformation of protein folding intermediates that are recognized by the chaperone, we prepared the uniformly (13)C-labeled alphaA-crystallin. The labeling greatly reduced the overlapping between the conformation-sensitive amide I bands of alpha-crystallin and unlabeled substrate proteins. This procedure has allowed us to gain insight into the secondary structure of alpha-crystallin-bound species, an understanding which has previously been unattainable. Analysis of the infrared spectra of two substrate proteins (gamma- and beta(L)-crystallins) indicates that heat-destabilized conformers captured by alpha-crystallin are characterized by a high proportion of native-like secondary structure. In contrast to the chaperone-bound species, the same proteins subjected to heat treatment in the absence of alpha-crystallin preserve very little native secondary structure. These data show that alpha-crystallin specifically recognizes very early intermediates on the denaturation pathway of proteins. These aggregation-prone species are characterized by native-like secondary structure but compromised tertiary interactions. The experimental approach described in this study can be further applied to probe the backbone conformation of proteins bound to chaperones other than alpha-crystallin.     

The preparation of large-scale synchronous cultures of the trypanosomatid, Crithidia fasciculata,by cell-size selection: changes in respiration and adenylate charge through the cell-cycle.

Large-scale synchronous cultures of Crithidia fasciculata were prepared by a sedimentation-velocity-size-election method using a non-somotic gradient in a zonal rotor. Synchrony indices of up to 0-68 were obtained (average length of cell-cycle, 5-1h). Dry weight, protein and RNA increased continuously so as to double in one cell-cycle. Rates of oxygen uptake/ml culture increased overall so as to double over a cell-cycle, but rose to maxima at five periods in the cell-cycle. KCN gave a similar degree of inhibition throughout, and so did not alter the periodicity or amplitude of the oscillations. Total adenylates doubled over a cycle, and complex changes in pool sizes of ATP, ADP and AMP were temporally interrelated and were correlated with changes of oxygen uptake rates rather than with changes in biosynthetic requirements. Adenylate charge varied between 0-47 and 0-66 Discontinuous respiratory activity of mitochondria through the cell-cycle and possible mechanisms for its control are discussed with reference to previous data.     

Contribution of proteomics of Leishmania spp. to the understanding of differentiation, drug resistance mechanisms, vaccine and drug development

Leishmania spp., protozoan parasites with a digenetic life cycle, cause a spectrum of diseases in humans. Recently several Leishmania spp. have been sequenced which significantly boosted the number and quality of proteomic studies conducted. Here a historic review will summarize work of the pre-genomic era and then focus on studies after genome information became available. Firstly works comparing the different life cycle stages, in order to identify stage specific proteins, will be discussed. Identifying post-translational modifications by proteomics especially phosphorylation events will be discussed. Further the contribution of proteomics to the understanding of the molecular mechanism of drug resistance and the investigation of immunogenic proteins for the identification of vaccine candidates will be summarized. Approaches of how potentially secreted proteins were identified are discussed. So far 30-35% of the total predicted proteome of Leishmania spp. have been identified. This comprises mainly the abundant proteins, therefore the last section will look into technological approaches on how this coverage may be increased and what the gel-free and gel-based proteomics have to offer will be compared.     

An Evaluation of Molecular Diagnostic Tools for the Detection and Differentiation of Human-Pathogenic Cryptosporidium spp.

ABSTRACT: The performance of 10 commonly used genotyping tools in the detection and differentiation of 7 human-pathogenic Cryptosporidium spp. ( C. hominis, C. parvum, C. meleagridis, C. felis, C. canis, C. muris and Cryptosporidium pig genotype I) was evaluated. All 3 SU rRNA gene-based tools could amplify the DNA of 7 Cryptosporidium spp. efficiently. However, the tools based on the antigens TRAP-C1, TRAP-C2 and COWP genes, the housekeeping genes HSP70 and DHFR, or a genomic sequence, failed to detect the DNA of C. felis, C. canis, Cryptosporidium pig genotype I, and C. metris. With the exception of 1 tool based on the TRAP-C2 gene, the PCR-RFLP or the PCR sequencing tools evaluated in this study could differentiate C. hominis, C. parvum and C. meleagridis from each other, and 2 SSU rRNA genebased tools could differentiate all 7 Cryptosporidium spp. Thus, a thorough understanding of the strength and weakness of each technique is needed when using molecular diagnostic tool in epidemiological investigations of human cryptosporidiosis.     

Kinetoplast-associated DNA topoisomerase in Crithidia fasciculata: crosslinking of mitochondrial topoisomerase II to both minicircles and maxicircles in cells treated with the topoisomerase inhibitor VP16.

The mitochondrial DNA of the trypanosomatid Crithidia fasciculata consists of thousands of copies of a 2.5 kb minicircle and a small number of 37kb maxicircles catenated into a single enormous network. Treatment of C. fasciculata with the type II DNA topoisomerase inhibitor VP16 produces cleavable complexes of a type II DNA topiosomerase with both minicircles and maxicircles. A combined Southern and Western blot analysis of the cleaved DNA species released from the network by SDS treatment has identified topollmt, the kinetoplast-associated topisomerase, in covalent complexes with linear forms of minicircle and maxicircle DNAs. These results directly implicate topollmt in the topological reactions required for the duplication of the kinetoplast network.     

An Approach to the Chemotaxonomic Differentiation of Two European Dog's Mercury Species: Mercurialis annua L. and M. perennis L.

Mercurialis annua and M. perennis are medicinal plants used in complementary medicine. In the present work, analytical methods to allow a chemotaxonomic differentiation of M. annua and M. perennis by means of chemical marker compounds were established. In addition to previously published compounds, the exclusive presence of pyridine‐3‐carbonitrile and nicotinamide in CH₂Cl₂ extracts obtained from the herbal parts of M. annua was demonstrated by GC/MS. Notably, pyridine‐3‐carbonitrile was identified for the first time as a natural product. Further chromatographic separation of the CH₂Cl₂ extracts via polyamide yielded a MeOH fraction exhibiting a broad spectrum of side‐chain saturated n‐alkylresorcinols. While the n‐alkylresorcinol pattern was similar for both plant species, some specific differences were observed for particular n‐alkylresorcinol homologs. Finally, the investigation of H₂O extracts by LC/MS/MS revealed the presence of depside constituents. Whereas, in M. perennis, a mixture of mercurialis acid (=(2R)‐[(E)‐caffeoyl]‐2‐oxoglutarate) and phaselic acid (=(E)‐caffeoyl‐2‐malate) could be detected, in M. annua solely phaselic acid was found. By comparison with synthesized enantiomerically pure (2R)‐ and (2S)‐phaselic acids, the configuration of the depside could be determined as (2S) in M. annua and as (2R) in M. perennis.     

Carbonic anhydrase inhibition. Insight into the characteristics of zonisamide, topiramate, and the sulfamide cognate of topiramate

Some useful therapeutic agents inhibit certain carbonic anhydrase (CA) isozymes to varying degrees. We have conducted enzyme kinetics studies in a 4-nitrophenyl acetate (4-NPA) hydrolysis assay with the marketed antiepileptic drugs topiramate (1) and zonisamide (2) to determine if their full inhibition of human CA-II and CA-I requires extended preincubation conditions. We found that neither 1 nor 2 requires appreciable preincubation with either enzyme to manifest full inhibitory activity. We also examined the sulfamide cognate of topiramate (3) to characterize its CA inhibitory activity, and confirmed that it is a very weak inhibitor, unlike 1 or 2. In a CO(2) hydration assay, 3 behaved as a very weak, partial inhibitor of CA-II and CA-I. We conclude that topiramate (1), zonisamide (2), and sulfamide 3 do not require extended exposure to human CA-I or CA-II to manifest full inhibitory activity (4-NPA assay).