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Query: UNIPROT:P06889 (Mol)
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Two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis were purified using species-specific monoclonal antibodies and characterized. The molecular weights of the proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were approximately 70,000 and approximately 72,000, respectively. The 70 kDa protein, which appears as a diffuse band on silver staining, was resolved into a doublet by Western blotting with monoclonal antibody. Though of similar molecular weight and amino acid composition, the two proteins were shown to be distinct by peptide mapping and Western blotting of the purified material. The two proteins are recognized specifically by human visceral leishmaniasis serum and not by serum from cutaneous leishmaniasis or Chagas' disease. These proteins will be useful in developing a direct serodiagnostic assay for visceral leishmaniasis.
Mol Biochem Parasitol 1988 Jan 01
PMID:Purification of two Leishmania donovani membrane proteins recognized by sera from patients with visceral leishmaniasis. 334 99

Extraction of whole promastigotes with a mixture of hexane-isopropanol yielded two carbohydrate-lipid fractions immunologically active against immune sera from patients with cutaneous leishmaniasis (CL): CLF-1 and CLF-2. Thin layer chromatography (TLC) separated both fractions into eight bands labeled A-H. Four of these bands, Rf 0.19, 0.25, 0.39 and 0.48 (A, B, C and E, respectively) were recognized by antibody from patients with CL in a solid phase radioimmunoassay. Antigens were also detected by autoradiography after immunoblotting of TLC. Compound A could be labeled biosynthetically with [3H]oleic acid, [14C]galactose, [14C]mannose, [14C]glucose and [32P]phosphate. B incorporated [14C]galactose, [14C]mannose, [14C]glucose and [14C]myo-inositol. C was labeled with [14C]galactose and [14C]mannose, while E incorporated [14C]glucose, [14C]mannose, [3H]oleic acid and [14C]myoinositol. Two antigens (A and B) could be also labeled on the surface of living promastigotes using galactose oxidase and [3H]sodium borohydride. Experimental data showed that CLF-1 and CLF-2, both carbohydrate-containing fractions, had different chromatographic patterns from excreted factor (EF), a polysaccharide antigen from Leishmania. The present study characterizes glycolipid molecules from L. major promastigotes, able to stimulate the immune system from patients with CL.
Mol Biochem Parasitol 1988 Jan 01
PMID:Leishmania major: glycolipid antigens recognized by immune human sera. 334 1

There is a need for accurate, rapid and early diagnosis of leishmaniasis, which would distinguish between the benign and severe forms of the disease. We have used a monoclonal antibody directed to a polymorphic, species-specific antigen in diagnostic assays for leishmaniasis. The dot-blot enzyme-linked immunosorbent assay described here can detect as few as 300 culture promastigotes and 20,000 amastigotes of Leishmania major with no cross-reaction with other species and no background from skin macrophages or other cells. This level of sensitivity is sufficient to detect parasite antigen aspirated in a few microliters of liquid from small lesions in mice. This assay could form the basis for a sensitive, rapid and inexpensive dip-stick test for large-scale use for the diagnosis and epidemiology of cutaneous leishmaniasis.
Mol Biol Med 1987 Dec
PMID:Leishmania major: a very sensitive dot-blot ELISA for detection of parasites in cutaneous lesions. 343 21

This study describes the characterisation of externally oriented surface peptides of both morphological forms of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar). Using 125I surface labelling techniques and peptide extraction in the detergents Triton X-100 and Triton X-114, a major iodinable promastigote peptide at 63 kDa or 65 kDa (depending on detergent used) was identified. This peptide was demonstrated to be the immunodominant membrane peptide of L. donovani and was strongly recognised by human sera from parasitologically confirmed cases of kala-azar. This peptide was not demonstrated on the surface of tissue amastigotes, although in vitro translations of poly(A+) RNA from both promastigotes and amastigotes demonstrated that both forms possessed mRNA that directs the synthesis of a 63 kDa peptide. It is suggested therefore that in amastigotes this peptide may be a processed antigen. We also report the isolation of a recombinant cDNA clone in the bacteriophage vector lambda gt10 which encodes a 63 kDa polypeptide that is recognised by human kala-azar sera. It is proposed that this surface peptide could be used in a specific immunodiagnostic test for leishmaniasis.
Mol Biochem Parasitol 1987 Apr
PMID:Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes. 360 Jun 97

Molecular analysis of kinetoplast deoxyribonucleic acid (kDNA) minicircles has permitted the genotypic characterization of pathogenic isolates of Leishmania species. The apparent size in agarose gels of unit-length minicircles released by EcoRI digestion of kDNA networks is not conserved during speciation in this genus since the minicircles of strains and clones of L. major are smaller (710 base pairs, bp) than those found in certain strains of L. mexicana subspecies (820 bp), L. donovani (825, 865 bp) or L. tropica (900, 930 bp). EcoRI-cut minicircles within any one species of Leishmania are heterogeneous in mobility during electrophoresis in acrylamide gels. Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of closely related clones and strains within a given species. Southern blot hybridization reveals that overall minicircle sequence homology is conserved among clones and strains of one species (L. major or L. tropica) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of Leishmania isolates. The analysis of kDNA from two L. tropica strains isolated at 14 year intervals from a patient with leishmaniasis recidivans has shown that the two strains are closely related, suggesting that the individual suffered the cutaneous disease as a result of a resurgence of the same parasite which caused the initial infection. The differences in the properties of kDNA from the L. tropica and L. major strains studied support the taxonomic separation of L. tropica and L. major into distinct species.
Mol Biochem Parasitol 1984 Jun
PMID:Identification of species, strains and clones of Leishmania by characterization of kinetoplast DNA minicircles. 609 Aug 98

Cloning by limit dilution of an isolate of Leishmania tropica (LRC-L137) that is infective for mice resulted in 7 stable clones, only one of which was infective in BALB/c mice. Three of the non-infective clones that were examined for survival in BALB/c macrophages in vitro seemed to be killed more readily, suggesting failure to establish in macrophages as the basis for non-infectivity in vivo. Promastigotes from three non-infective clones and one infective clone were biosynthetically labelled or surface radioiodinated, and the detergent lysates were analyzed by 2-dimensional gel electrophoresis. The pattern of the radiolabelled cytoplasmic and membrane proteins of promastigotes from all L. tropica clones was similar, with minor differences. All clones as well as the uncloned population bound to the same extent to a series of lectins with galactose and N-acetylgalactosamine as specificities. They also bound in a solid-phase radioimmunoassay to 9 monoclonal antibodies raised against the uncloned L. tropica (LRC-L137). The genetic characterization of four L. tropica clones was attempted by analysis of their isolated kinetoplast DNA. The clones from two schizodemes since they possess kinetoplast DNAs which exhibit similar restriction endonuclease fingerprints and show extensive DNA sequence homology, suggesting that the four clones are closely related and that the non-infective variants may be derived from the infective presumptive parental clone L137-7-121. Further characterization of the clones of L. tropica should allow a better understanding of the genetic basis of parasite virulence in cutaneous leishmaniasis.
Mol Biochem Parasitol 1983 Feb
PMID:Isolation and characterization of infective and non-infective clones of Leishmania tropica. 685 10

Ultrastructural analysis has been carried out on three Leishmania isolates which are proven causal agents of human cutaneous Leishmaniasis, L. tropica major, L. aethiopica and a unidentified species, Leishmania SP48. No significant differences in submicroscopic morphology have been found in thin-sectioned organisms from the three isolates. Extensive plate cristae have been observed within the mitochondria and connections between the rim of the kinetoplast nucleoid and the inner mitochondrial membrane noted. Kinetoplast DNA (kDNA) has been isolated from these isolates and from L. tarentolae and examined by protein monolayer spreading and darkfield electronmicroscopy. The basic molecular arrangement of isolated kDNA in the form of 5 micrometers networks of 0.28--0.3 micrometer mini-circles with long looped DNA in the interior and at the periphery of networks is similar in all isolates. Minor differences between L. aethiopica and SP48 compared with L. tropica major have been observed. The kDNAs of L. aethiopica and SP48 are identical morphologically. Buoyant density analysis has shown that kDNA from L. aethiopica and SP48 have identical values and these are different from the values for L. tropica major. The finding of similar buoyant densities for kDNA from L. tropica major and L. tarentolae also imply a sequence homology by this criteria which is refuted by the results given in the following paper. The results given in this and the following paper (Arnot, D.E. and Barker, D.C.(1981) Mol. Biochem. Parasitol. 3, 47--56 indicate that the unknown Leishmania SP48 is very closely related to, if not identical with, L. aethiopica. This finding is consistent with the clinical and ecological facts known for the organism SP48.
Mol Biochem Parasitol 1981 May
PMID:Biochemical identification of cutaneous leishmanias by analysis of kinetoplast DNA. I. Ultrastructural and buoyant density analysis. 725 45

Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans. During their life cycle, Leishmania protozoans exist as flagellated promastigotes in the sand fly vector and as nonmotile amastigotes in the mammalian hosts. The promastigote-to-amastigote transformation occurs in the phagolysosomal compartment of the macrophage cell and is a critical step for the establishment of the infection. To study this cytodifferentiation process, we differentially screened an amastigote cDNA library with life cycle stage-specific cDNA probes and isolated seven cDNAs representing amastigote-specific transcripts. Five of these were closely related (A2 series) and recognized, by Northern (RNA) blot analyses, a 3.5-kb transcript in amastigotes and in amastigote-infected macrophages. Expression of the amastigote-specific A2 gene was induced in promastigotes when they were transferred from culture medium at 26 degrees C and pH 7.4 to medium at 37 degrees C and pH 4.5, conditions which mimic the macrophage phagolysosomal environment. A2 genes are clustered in tandem arrays, and a 6-kb fragment corresponding to a unit of the cluster was cloned and partially sequenced. An open reading frame found within the A2-transcribed region potentially encoded a 22-kDa protein containing repetitive sequences. The recombinant A2 protein produced in Escherichia coli cells was specifically recognized by immune serum from a patient with visceral leishmaniasis. The A2 protein repetitive element has strong homology with an S antigen of Plasmodium falciparum, the protozoan parasite responsible for malaria. Both the A2 protein of Leishmania donovani and the S antigen of P. falciparum are stage specific and developmentally expressed in mammalian hosts.
Mol Cell Biol 1994 May
PMID:Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene. 754 21

Visceral leishmaniasis has been found to be associated with severe anemia and premature lysis of erythrocytes. Peroxidative damage of red cells has been noted in several hemolytic anemias. Present study shows enhanced formation of methemoglobin in hamsters infected with Leishmania donovani. Increased formation of malonyldialdehyde and diene conjugate has been noted in the erythrocytes of the infected animals with the progress of anemia. Results showed decreased activities of protective enzymes like superoxide dismutase, catalase and glutathione reductase against peroxidative attack. An increase in the membrane cholesterol/phospholipid ratio and a decrease in membrane fluidity of erythrocytes were observed under the diseased condition. Densitometric scan after SDS-PAGE of red cell membrane of the infected animals revealed significant degradation of band 3 and band 4.1 proteins. The results suggest that alteration in the membrane may lead to reduced life span of the red cells in experimental visceral leishmaniasis.
Mol Cell Biochem 1995 May 24
PMID:Lipid peroxidation of erythrocytes during anemia of the hamsters infected with Leishmania donovani. 756 50

Monoclonal antibodies (MAbs) were generated against Leishmania major promastigote lipophosphoglycan (LPG) to use as tools in defining functional epitopes of this major cell surface glycoconjugate. Epitope mapping of four MAbs, designated 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2, revealed that the phosphorylated oligosaccharide repeat unit PO4-6[Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, P3, is a highly immunogenic epitope which has previously been demonstrated, by chemical analyses, to be a repeat unit specific to L. major. Two antibodies, 4A2-A2 and 5E10-F2, also recognised the repeat unit PO4-6[Ara(beta 1-2)Gal(beta 1-3)]Gal(beta 1-4)Man alpha 1-, 4Pa, with less affinity than P3, while 2G11-A3 recognised P4a with greater affinity than for P3. The L. major metacyclic-specific antibody 3F12 only recognised repeat units terminating with arabinose residues. In particular, 3F12 recognised P4a, which is upregulated in metacyclic LPG compared to the procyclic form of the molecule. The oligosaccharides P3, P4a and P5a are specific to L. major LPG. The epitopes of 4A2-A2, 2G11-A3, 5E6-D10 and 5E10-F2 were found on the cell surface and in the flagellar pocket of both procyclic and metacyclic V121 promastigotes, but were only detected at very low levels on amastigotes. The repeat unit P3 is able to inhibit attachment of procyclic promastigotes to the midgut of the sandfly vector, but neither Fab fragments of the four antibodies nor purified P3 could inhibit attachment of metacyclic promastigotes to the macrophage cell line J774. It was also shown that human sera from patients with cutaneous leishmaniasis recognised purified P3. The data suggests that while P3 is an immunogen in the natural course of infection of the human host, P3 plays no role in attachment and internalisation of promastigotes into the macrophages of the mammalian host.
Mol Biochem Parasitol 1994 Aug
PMID:Epitope mapping of monoclonal antibodies directed against lipophosphoglycan of Leishmania major promastigotes. 780 69


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