Kinetoplast DNA minicircles of Leishmania donovani express a protein product.
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We describe an unprecedented finding of an open reading frame present in the variable region in one of the minicircle sequence classes of a human pathogenic strain of Leishmania donovani (MHOM/IN/90/RMRI 68) which is transcribed and translated. The encoded protein showed homologies to known transport proteins. (+info)
Developmental regulation of spliced leader RNA gene in Leishmania donovani amastigotes is mediated by specific polyadenylation.
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Leishmania cycles between the insect vector and its mammalian host undergoing several important changes mediated by the stage-specific expression of a number of genes. Using a genomic differential screening approach, we isolated differentially expressed cosmid clones carrying several copies of the mini-exon gene. We report that the spliced leader (SL) RNA, essential for the maturation of all pre-mRNAs by trans-splicing, is developmentally regulated in Leishmania donovani amastigotes and that this regulation is rapidly induced upon parasite growth under acidic conditions. Stage-specific regulation of the SL RNA is associated with the expression of a larger approximately 170-nucleotide transcript that bears an additional 15-nucleotide sequence at its 3'-end and is polyadenylated in contrast to the mature SL RNA. The poly(A)+ SL RNA represents 12-16% of the total SL transcript synthesized in amastigotes and is 2.5-3-fold more stable than the poly(A)- transcript. The poly(A)+ SL transcript is synthesized specifically from one class of the genomic mini-exon copies. Polyadenylation of the SL RNA may control the levels of the SL mature transcript under amastigote growth and may represent an additional step in the gene regulation process during parasite differentiation. (+info)
Immunoglobulin subclass distribution and diagnostic value of Leishmania donovani antigen-specific immunoglobulin G3 in Indian kala-azar patients.
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Visceral leishmaniasis, or kala-azar, a fatal tropical disease, remains problematic, as early diagnosis is difficult and treatment often results in drug resistance and relapse. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA), using leishmanial membrane antigenic extracts (LAg) to detect specific antibody responses in 25 untreated Indian visceral leishmaniasis patients. To investigate the pathogenetic significance of isotype markers in kala-azar, relative levels of specific immunoglobulin G (IgG), IgM, IgA, IgE, and IgG subclasses were analyzed under clinically established diseased conditions. Since LAg showed higher sensitivity for specific IgG than lysate, the immunoglobulin isotype responses were evaluated, with LAg as antigen. Compared to 60 controls, which included patients with malaria, tuberculosis, leprosy, and typhoid and healthy subjects, visceral leishmaniasis patients showed significantly higher IgG (100% sensitivity, 85% specificity), IgM (48% sensitivity, 100% specificity), and IgE (44% sensitivity, 98.3% specificity) responses. Low levels of IgA in visceral leishmaniasis patients contrasted with a 13-fold-higher reactivity in sera from patients with leprosy. Among IgG subclasses, IgG1, -3, and -4 responses were significantly higher in visceral leishmaniasis patients than in the controls. IgG2 response, however, was significantly higher (twofold) in leprosy than even visceral leishmaniasis patients. The rank orders for sensitivity (IgG = IgG1 = IgG3 = IgG4 > IgG2 > IgM > IgE > IgA) and specificity (IgM = IgG3 > IgE > IgG4 > IgG2 > IgG > IgG1 > IgA) for LAg-specific antibody responses suggest the potentiality of IgG3 as a diagnostic marker for visceral leishmaniasis. (+info)
Molecular cloning and expression of adenosine kinase from Leishmania donovani: identification of unconventional P-loop motif.
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The unique catalytic characteristics of adenosine kinase (Adk) and its stage-specific differential activity pattern have made this enzyme a prospective target for chemotherapeutic manipulation in the purine-auxotrophic parasitic protozoan Leishmania donovani. However, nothing is known about the structure of the parasite Adk. We report here the cloning of its gene and the characterization of the gene product. The encoded protein, consisting of 345 amino acid residues with a calculated molecular mass of 37173 Da, shares limited but significant similarity with sugar kinases and inosine-guanosine kinase of microbial origin, supporting the notion that these enzymes might have the same ancestral origin. The identity of the parasite enzyme with the corresponding enzyme from two other sources so far described was only 40%. Furthermore, 5' RNA mapping studies indicated that the Adk gene transcript is matured post-transcriptionally with the trans-splicing of the mini-exon (spliced leader) occurring at nt -160 from the predicted translation initiation site. The biochemical properties of the recombinant enzyme were similar to those of the enzyme isolated from leishmanial cells. The intrinsic tryptophan fluorescence of the enzyme was substrate-sensitive. On the basis of a multiple protein-alignment sequence comparison and ATP-induced fluorescence quenching in the presence or the absence of KI and acrylamide, the docking site for ATP has been provisionally identified and shown to have marked divergence from the consensus P-loop motif reported for ATP- or GTP-binding proteins from other sources. (+info)
Indolylquinoline derivatives are cytotoxic to Leishmania donovani promastigotes and amastigotes in vitro and are effective in treating murine visceral leishmaniasis.
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A wide variety of biologically active compounds contain indole and quinoline nuclei. Some novel indolylquinoline derivatives were synthesized from indole by Friedel-Crafts acylation reaction. Out of the four derivatives tested, 2-(2''-acetamidobenzyl)-3-(3'-indolylquinoline) (C) had no effect on the promastigotes or amastigotes of Leishmania donovani in vitro. The remaining three analogues, 2-(2''-dichloroacetamidobenzyl)-3-(3'-indolylquinoline) (A), 2-(2''-chloroacetamidobenzyl)-3-(3'-indolylquinoline) (B), and 2-(2''-aminobenzyl)-3-(3'-indolylquinoline) (D), inhibited the growth of L. donovani promastigotes in vitro and were cytotoxic to both the promastigote and amastigote forms of the parasite. These three derivatives were also effective in eliminating L. donovani amastigotes from BALB/c mouse peritoneal macrophages in vitro. One indolylquinoline derivative [A] was used to treat established visceral leishmaniasis in BALB/c mice. This compound was significantly more effective than sodium antimony gluconate (SAG) in reducing the splenic parasite load at a much lower concentration (5% of SAG). Our results suggest that indolylquinoline derivatives may be exploited as antileishmanial agents. (+info)
Treatment of experimental leishmaniasis with the immunomodulators imiquimod and S-28463: efficacy and mode of action.
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There is a need for new, effective, and less toxic treatments for leishmaniasis, an infectious disease caused by Leishmania protozoa and is a major cause of suffering and morbidity in much of the developing world. Imiquimod, an immune-response modifier, has recently been approved by the Food and Drug Administration for the treatment of genital warts caused by human papillomaviruses. Imiquimod initiates a local immune reaction, including the stimulation of macrophages, resulting in resolution of human papillomavirus infection and regression of the viral lesion. Since imiquimod activates a number of immune cells, including macrophages, which are the only host cells of Leishmania species, an investigation was done to determine whether it induces leishmanicidal properties in infected macrophages in vitro and in vivo in a mouse model. Imiquimod and a related compound, S-28463, effectively stimulated leishmanicidal activity in macrophages; moreover, imiquimod stimulated signal transduction associated with inducing nitric oxide synthesis in macrophages. (+info)
Verification of differential gene transcription using virtual northern blotting.
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We introduce here an alternative to conventional northern blotting that requires only minute amounts of RNA. This has been achieved by modification of methods currently used for the mapping of mRNA 5'-terminal ends. The terminal desoxynucleotidyl transferase-mediated G-tailing, cap finder, ligation-anchored and RNA ligase-mediated approaches followed by polymerase chain reaction protocols all produced high quality cDNAs in large amounts. These cDNAs could be separated by electrophoresis to obtain virtual northern blots that could replace conventional northern blots. All the essential information, including transcript length and the expression pattern, are preserved in these cDNAs, even if the transcripts are long or GC-rich. In addition, minute amounts of material (less than 100 cells) are sufficient to produce more than 100 virtual northern blots, making this approach extremely versatile. (+info)
Antisense oligonucleotides have been considered as inhibitors of growth of intracellular parasites such as Leishmania, but only limited inhibition has been observed in vitro. We have encapsulated an antisense oligonucleotide, complementary to the Leishmania universal miniexon sequence, in cationic liposomes. Low concentrations (4 microM) of encapsulated oligonucleotides specifically reduced the amastigote burden within cultured macrophages by 80%. This result illustrates the importance of effective delivery for efficient antiparasitic activity of antisense oligonucleotides. (+info)