Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Calreticulin was recently identified as a hookworm (Necator americanus) allergen, implying secretion, and contact with cells of the immune system, or significant worm attrition in the tissues of the host. As human calreticulin has been shown to bind to and neutralize the haemolytic activity of the complement component C1q, and to be putatively involved in integrin-mediated intracellular signalling events in platelets, it was of interest to determine whether a calreticulin from a successful nematode parasite of humans, with known immune modulatory and antihaemostatic properties, exhibited a capacity to interfere with complement activation and to interact with integrin domains associated with cell signalling in platelets and other leucocytes. We can now report that recombinant calreticulin failed to demonstrate significant calcium binding capacity, which is a hallmark of calreticulins in general and may indicate inappropriate folding following expression in a prokaryote. Nevertheless, recombinant calreticulin retained sufficient molecular architecture to bind to, and inhibit the haemolytic capacity of, human C1q. Furthermore, recombinant calreticulin reacted in surface plasmon resonance analysis (SPR) with peptides corresponding to cytoplasmic signalling domains of the integrins alphaIIb and alpha5, in a calcium independent manner. SPR was also used to ratify the specificity of a polyclonal antibody to hookworm calreticulin, which was then used to assess the stage specificity of expression of the native molecule (in comparison with reverse transcriptase-polymerase chain reaction), to indicate its apparent secretion, and to purify native calreticulin from worm extracts by affinity chromatography. This development will allow the functional tests described above to be repeated for native calreticulin, to ascertain its role in the host-parasite relationship.
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PMID:A calreticulin-like molecule from the human hookworm Necator americanus interacts with C1q and the cytoplasmic signalling domains of some integrins. 1124 Sep 5

A cDNA encoding a putative tissue inhibitor of metalloprotease was cloned from an Ancylostoma caninum adult hookworm cDNA library by immunoscreening with anti-hookworm secretory products antiserum. Ac-TMP (A. caninum tissue inhibitor of metalloproteinase) is encoded by a 480-bp mRNA with a predicted open reading frame of 140 amino acids (molecular weight, 16,100 Da) that contains one potential N-linked glycosylation site and an N-terminal Cys-X-Cys consensus sequence. The open reading frame corresponds to a putative hookworm tissue inhibitor of metalloproteases (TIMP) with 33% identity and 50% similarity to the N-terminal domain of human TIMP-2. Analysis by reverse transcriptase-polymerase chain reaction indicates that transcription of Ac-tmp is restricted to the adult stage. The protein was isolated from A. caninum adult secretory products by reverse-phase high-performance liquid chromatography and identified as one of the most abundant proteins released by the parasite. To our knowledge, this is the first description of a TIMP from a parasitic invertebrate.
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PMID:Molecular cloning and purification of Ac-TMP, a developmentally regulated putative tissue inhibitor of metalloprotease released in relative abundance by adult Ancylostoma hookworms. 1213 14

Antibody against adult Ancylostoma caninum excretory-secretory (ES) products was used to immunoscreen a cDNA expression library leading to the isolation of cDNAs encoding putative hookworm fatty-acid and retinol-binding proteins. Ac-far-1 and Ac-far-2 cDNAs encode open reading frames corresponding to approximately 20kDa proteins with 91 percent amino acid identity. Ac-FAR-1 and Ac-FAR-2 exhibit clear similarities to other FARs of parasitic nematodes, most closely to two of the FAR proteins of Caenorhabditis elegans (Ce-FAR-1 and Ce-FAR-2). By reverse transcriptase polymerase chain reaction (RT-PCR) assay, Ac-far-1 mRNA was detected in both adult and third-stage larvae of A. caninum. However, the respective proteins were detectable by immunoblot only in adult hookworm ES products and adult extracts. Using fluorescence-based binding assays, bacterial recombinant Ac-FAR-1 was found to bind fatty acids and retinol (Vitamin A) with dissociation constants in the micromolar region. Circular dichroism spectra indicated that Ac-FAR-1 possesses a high level of alpha-helix, similar to Ov-FAR-1 from Onchocerca volvulus. This is the first demonstration of a functional FAR secreted by adult hookworms and provides further evidence that FAR proteins secreted by parasitic nematodes are crucial to parasitism.
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PMID:Ac-FAR-1, a 20 kDa fatty acid- and retinol-binding protein secreted by adult Ancylostoma caninum hookworms: gene transcription pattern, ligand binding properties and structural characterisation. 1255 85

Hookworm infection persists as a public health problem in developing nations. Vaccine-based strategies offer the best chance of long-term control. Aspartyl protease inhibitors from parasitic nematodes are highly immunogenic, and have been suggested as potential vaccine antigens. An aspartyl protease inhibitor, API-1, was cloned and characterised from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. Using sequence from the hookworm expressed sequence tag project, specific primers were designed and used to amplify Ac-api-1 from A. caninum infective L3 cDNA by PCR. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create an 874-bp full-length composite sequence of the Ac-api-1 gene. The A. ceylanicum api-1 cDNA of 878 bp was cloned from L3 cDNA using the A. caninum primers. The amino acid sequences of hookworm orthologues were nearly identical, and database searching indicated they belonged to the aspin family, a group of nematode specific aspartyl protease inhibitors that includes the Ascaris pepsin inhibitor PI-3. Ac-api-1 mRNA was detected by reverse transcriptase PCR in eggs, L1, L3 and adult life cycle stages. A polyclonal antiserum against Escherichia coli expressed recombinant Ac-API-1 detected the protein in adult A. caninum excretory/secretory products, but not in those from activated infective larvae. Immunolocalisation experiments using the antiserum indicated that Ac-API-1 is present primarily in the pseudocoelomic fluid in adult hookworms. Soluble, yeast-expressed Ac-API-1 failed to inhibit pepsin or a hookworm gut aspartyl protease in vitro, but inhibited approximately 30% of the proteolytic activity of adult excretory/secretory products. The pseudocoleomic location, presence in all life cycle stages, lack of inhibitory activity against pepsin, and inhibitory activity against excretory/secretory products suggest that Ac-API-1 inhibits an unidentified, putative aspartyl protease secreted by adult hookworms, and may be released as an enzyme-inhibitor complex. The highly immunogenic properties of nematode aspins suggest that Ac-API-1 represents a promising target for a recombinant hookworm vaccine.
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PMID:Cloning and characterisation of an aspartyl protease inhibitor (API-1) from Ancylostoma hookworms. 1572 82

Infective hookworm L3 encounter a host specific signal during invasion that re-activates suspended developmental pathways. Response to this cue is critical for the successful infection and completion of the life cycle in the host. In the free-living nematode Caenorhabditis elegans, recovery from the developmentally arrested dauer stage in response to environmental cues is analogous to the resumption of development in invading hookworm L3. Transforming growth factor beta (TGF-beta) and insulin-like signalling pathways mediate dauer formation and recovery. An insulin-like signalling pathway mediates L3 activation in hookworms. To determine the role of TGF-beta signalling in hookworm infection, an ortholog of the C. elegans TGF-beta signalling molecule daf-7 was cloned and characterised. Sequence from a hookworm expressed sequence tag was used to design specific primers for PCR amplification of Ac-daf-7 from Ancylostoma caninum infective L3 cDNA. Amplicons from the 5' and 3' ends were cloned, sequenced, and combined to create a full-length composite Ac-daf-7 cDNA sequence. The 1,634 nucleotide cDNA encoded a 355 amino acid open reading frame with significant homology to Ce-DAF-7 and other TGF-beta signalling molecules. The deduced amino acid sequence contained seven conserved cysteines characteristic of TGF-beta family members, as well as two additional conserved cysteines found in members of the TGF-beta/activin subfamily. Ac-DAF-7 contains a characteristic C-terminal ligand domain that is predicted to be released from a propeptide by proteolytic cleavage at a tetrabasic cleavage site. Ac-daf-7 mRNA was strongly detected by reverse transcriptase PCR in L3 and serum stimulated L3 cDNA, and weakly in cDNA from L1 and adult life cycle stages. Antiserum against Escherichia coli expressed recombinant Ac-DAF-7 detected the mature protein in L3 and adult soluble extracts, but not in excretory/secretory products from serum stimulated L3 or adults. Increased expression in arrested L3 stages suggests that Ac-daf-7 is important for developmental arrest.
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PMID:Identification of a DAF-7 ortholog from the hookworm Ancylostoma caninum. 1613 66

To elucidate the role of transforming growth factor beta (TGF-beta) signalling in the arrest/reactivation pathway of the Ancylostoma caninum hookworm, two parasite-encoded TGF-beta-like ligands were cloned and characterised. Ac-dbl-1 showed 60% amino acid identity to the Caenorhabditis elegansdbl-1 gene, which regulates growth while Ac-daf-7 showed 46% amino acid identity to Ce-daf-7 which regulates arrested development. Exon/intron organisation of the genes for Ac-dbl-1 and Ac-daf-7 were different from that of the corresponding C. elegans genes with nine and 10 exons, respectively, and introns ranging in size from 56 to 2,556 bp. Based on real-time reverse transcriptase (RT)-PCR, Ac-dbl-1 and Ac-daf-7 were expressed in all stages tested, i.e. egg, first/second stage larvae (L1/L2), infective third stage larvae (iL3), serum-stimulated third stage larvae (ssL3), and male and female adult worms. Expression of Ac-dbl-1 peaked in the adult male stage suggesting a similar role to Ce-dbl-1 in regulating male tail patterning. Ac-daf-7 expression was at a maximum in the arrested iL3 and reactivated ssL3 stages, which differs from that of Ce-daf-7 expression and may be unique to parasitic nematodes that have an obligate requirement to undergo developmental arrest. In support of the PCR results, antibodies to the A. caninum TGF-beta-like ligands detected proteins in iL3, ssL3, and adult worm extracts. Immunofluorescent studies showed that Ac-daf-7 is expressed in the anterior region of the iL3 similar to Ce-daf-7, which is localised to the ASI chemosensory neurons.
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PMID:Cloning and characterisation of genes encoding two transforming growth factor-beta-like ligands from the hookworm, Ancylostoma caninum. 1614 Mar 4

Members of the retrotransposable element (RTE) clade of non-long terminal repeat (LTR) retrotransposon are widely distributed among eukaryote taxa, with representatives known from Caenorhabditis elegans, mammals, mosquitoes, schistosomes, and other taxa. An RTE retrotransposon has not, however, been characterized in detail from a parasitic nematode. Here, we characterize two discrete copies of an RTE-like non-LTR retrotransposon from the genome of the dog hookworm, Ancylostoma caninum. The elements were named dingo-1 and dingo-2. The full-length dingo-1 and dingo-2 elements were 3421 and 3171bp in length, respectively. They exhibited 54% nucleotide sequence identity to one another across their entire length and 40%/58% amino-acid sequence identity/similarity across their open reading frames. dingo-1 and dingo-2 exhibited hallmark structures and sequences of non-LTR retrotransposons of the RTE family including a single open reading frame encoding apurinic-apyrimidinic endonuclease (EN) and reverse transcriptase (RT), in that order. Phylogenetic analyses targeting the RT and the EN domains both confirmed that dingo-1 and dingo-2 were members of the RTE clade and that they were closely related to RTE-1 from C. elegans, to BDDF from Bos taurus and to SR2 from Schistosoma mansoni. Dot blot hybridization indicated that as many as 100-1000 copies of dingo-1 reside within the genome of A. caninum, while detection by RT-PCR of transcripts encoding dingo-like elements suggested that dingo-1 and -2 may be retrotranspositionally active within the genome of A. caninum. The dingo elements are the first retrotransposons to be characterized from a hookworm genome.
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PMID:The dingo non-long terminal repeat retrotransposons from the genome of the hookworm, Ancylostoma caninum. 1644 14

Hookworm infection is still a public health problem in developing countries. Aspartic protease plays an important role in parasite invasion and migration in the host. Aspartic protease gene from Ancylostoma caninum has been reported (Biochem Biophys Res Commun 227:294-302, 1996), but the activity of Acasp in eggs and larvae stage has not been studied. This paper reported the cloning of Acasp, expression in Escherichia coli, and characterization in eggs and larval stage. The mRNA encoding Acasp was detected using reverse transcriptase polymerase chain reaction in L4 and adult worm. A polyclonal antiserum against E. coli expressed recombinant Acasp was produced and used to detect and localize the Acasp in various developmental stages of A. caninum. Results from Western blot and indirect fluorescent immunoassay showed that the Acasp was present in the embryo, larvae, as well as in the adult worms. The recombinant Acasp exhibited the protease activity by the gelatin gel digestion assay.
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PMID:Expression and characterization of aspartic protease gene in eggs and larvae stage of Ancylostoma caninum. 1916 10

Hookworms, bloodfeeding intestinal nematodes, infect nearly one billion people in resource limited countries and are a leading cause of anaemia and malnutrition. Like other nematodes, hookworms lack the capacity to synthesise essential fatty acids de novo and therefore must acquire those from exogenous sources. The cDNA corresponding to a putative Ancylostoma ceylanicum fatty acid and retinol binding protein-1 (AceFAR-1) was amplified from adult hookworm mRNA. Studies using quantitative reverse transcriptase real-time PCR demonstrate that AceFAR-1 transcripts are most abundant in the earliest developmental stages of the parasite, and greater in females than males. Using in vitro assays, the recombinant AceFAR-1 (rAceFAR-1) was shown to bind individual fatty acids with equilibrium dissociation constants in the low micromolar range. The pattern of fatty acid uptake by live adult worms cultured ex vivo was similar to the in vitro binding profile of rAceFAR-1, raising the possibility that the native protein may be involved in acquisition of fatty acids by A. ceylanicum. Animals vaccinated orally with rAceFAR-1 and the mucosal adjuvant cholera toxin exhibited a statistically significant (40-47%) reduction in intestinal worm burden compared with controls immunized with antigen or adjuvant alone. Together, these data suggest a potential role for AceFAR-1 in hookworm biology, making it a potentially valuable target for drug and vaccine development.
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PMID:Characterisation of a fatty acid and retinol binding protein orthologue from the hookworm Ancylostoma ceylanicum. 1959 34

A macrophage migration inhibitory factor (MIF)-like molecule, Tci-MIF-1, was isolated from Teladorsagia circumcincta and subjected to detailed characterization. A cDNA representing Tci-mif-1 was isolated following its identification in third-stage larvae (L3)-enriched cDNA population. Sequencing of the cDNA indicated a 348-bp open reading frame (ORF) with the closest orthologue being a MIF derived from the human hookworm Ancylostoma ceylanicum. Messenger RNA (mRNA) representing the Tci-MIF-1 transcript was detected in eggs, L3 and adult stages of T. circumcincta. The transcript was also present, but to a lesser extent in fourth-stage larvae (L4). Detection of Tci-MIF-1 protein in T. circumcincta developmental stages reflected the transcript levels identified by reverse transcriptase-PCR. Using immunohistochemistry, the Tci-MIF-1 protein was shown to have a diffuse distribution in L3 tissue, and in L4 and adult stages, the protein was localized to the nematode gut. A recombinant version of Tci-MIF-1 was produced, and enzymic assays indicated that this recombinant protein and a somatic extract of L3 possessed dopachrome tautomerase activity as has been observed previously in other MIF-like molecules. Neither native, purified Tci-MIF nor recombinant Tci-MIF-1 dramatically influenced the in vitro migration of sheep monocytes.
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PMID:A macrophage migration inhibitory factor-like tautomerase from Teladorsagia circumcincta (Nematoda: Strongylida). 2059 Nov 21


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