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Biomedical subjects

A Loukas

Publications and source records attributed to A Loukas.

At least 19 recordsLinked to original sources
PubMed2006

Saposin-like proteins from the intestine of the blood-feeding hookworm, Ancylostoma caninum.

Hookworms feed on blood, utilizing haemoglobin for nutrition, growth and reproduction. The haemoglobin digestion cascade has been partially elucidated, but the process immediately preceding this event, haemolysis, has received considerably less attention. We have cloned and expressed Ancylostoma caninum mRNAs encoding 2 proteins belonging to the saposin-like protein (SAPLIP) family, termed Ac-slp-1 and Ac-slp-2. The open reading frames of SLP-1 and SLP-2 were used to identify expressed sequence tags encoding SAPLIPs from the 4 major clades of animal parasitic nematodes. Both Ac-slp-1 and slp-2 mRNAs were shown to be expressed in all life stages assessed, with slp-1 predominantly being expressed in third-stage larvae (L3) before and after activation with dog serum. Recombinant SLP-1 and SLP-2 were expressed in insect cells and used to raise specific antisera in mice. These antisera were used as probes in fluorescence microscopy to localize the anatomic expression sites of both proteins to small, punctate organelles or vesicles within the intestinal cells of adult worms; weak staining was detected on the microvillar brush border of the intestine. Using transmission electron microscopy, both proteins were localized to similar vesicles in the intestinal cells of the L3. Recombinant proteins contained C-terminal purification tags that potentially precluded dimerization and possibly interfered with the subsequent detection of haemolytic activity. Their expression in the gut of the L3 and adult stages suggests a role for these hookworm SAPLIPs in the lysis of host cells during tissue migration and/or feeding.

Amino Acid Sequence↗
PubMed2006

A survey of the intestinal transcriptomes of the hookworms, Necator americanus and Ancylostoma caninum, using tissues isolated by laser microdissection microscopy.

The gastrointestinal tracts of multi-cellular blood-feeding parasites are targets for vaccines and drugs. Recently, recombinant vaccines that interrupt the digestion of blood in the hookworm gut have shown efficacy, so we explored the intestinal transcriptomes of the human and canine hookworms, Necator americanus and Ancylostoma caninum, respectively. We used Laser Microdissection Microscopy to dissect gut tissue from the parasites, extracted the RNA and generated cDNA libraries. A total of 480 expressed sequence tags were sequenced from each library and assembled into contigs, accounting for 268 N. americanus genes and 276 A. caninum genes. Only 17% of N. americanus and 36% of A. caninum contigs were assigned Gene Ontology classifications. Twenty-six (9.8%) N. americanus and 18 (6.5%) A. caninum contigs did not have homologues in any databases including dbEST-of these novel clones, seven N. americanus and three A. caninum contigs had Open Reading Frames with predicted secretory signal peptides. The most abundant transcripts corresponded to mRNAs encoding cholesterol-and fatty acid-binding proteins, C-type lectins, Activation-Associated Secretory Proteins, and proteases of different mechanistic classes, particularly astacin-like metallopeptidases. Expressed sequence tags corresponding to known and potential recombinant vaccines were identified and these included homologues of proteases, anti-clotting factors, defensins and integral membrane proteins involved in cell adhesion.

Amino Acid Sequence↗
PubMed2006

Transcriptome profiling of lung schistosomula,in vitro cultured schistosomula and adult Schistosoma japonicum.

The schistosomulum is the main target of vaccine-induced protective immunity; however, most studies have utilized schistosomula produced by mechanical transformation of infective larvae followed by in vitro culture rather than larvae isolated directly from the lungs of infected mammals. Using transmission electron microscopy, we demonstrated that there was little difference in the ultrastructure of Schistosoma japonicum schistosomula obtained by the two methods. However, significant differences in gene expression profiles were apparent when we used an oligonucleotide microarray to compare the gene expression profiles of schistosomula obtained in vivo from lung tissue with those maintained in vitro, and with adult worms of S. japonicum. It is likely that host environmental factors, which cannot be reliably reproduced in vitro, do influence the growth, development and overall biology of schistosomes. Thus caution is urged when using in vitro-cultured schistosomes and mechanically transformed/cultured schistosomula in molecular, biochemical and immunological studies.

Animals↗
PubMed2004

Minors' tobacco possession law violations and intentions to smoke: implications for tobacco control.

OBJECTIVES: To test: (1) whether citation under the Minors in Possession (MIP) law, vicarious citation (knowing someone who was cited), and threat of driving licence suspension are associated with decreased intentions to smoke next year; and (2) whether the policy is differentially enforced. SUBJECTS: 28,249 white, Hispanic, and African American students in grades 6-12 (11-18 years old) participated in the study. METHOD: The 86 item anonymous Texas Youth Tobacco Survey was completed by students attending 37 schools in 14 east and central Texas communities. RESULTS: Hierarchical linear modelling showed that MIP citation was unrelated to the future smoking intentions of most youth. However, there was a negative association between citation and smoking intentions for ever daily smoking youth at four schools. Threat of licence suspension was associated with a lower likelihood of future smoking intentions among ever daily smoking youth and vicarious citation did not deter youth from future smoking. African American and Hispanic youth had a higher probability of being cited than their peers. CONCLUSIONS: Threat of driving licence suspension has the intended effect upon youth who are/were committed smokers and MIP citation has the intended effect upon committed smokers at only four schools. However, differential enforcement of the law based on ethnicity may be occurring. Before drawing firm conclusions, current findings must be replicated with longitudinal data to determine the consequences of citation on subsequent tobacco use.

Adolescent↗
PubMed2003

Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules.

Cathepsin D aspartic proteases of hookworms were recently implicated in the host-specific digestion of haemoglobin by adult parasites. Ac-APR-1 from the dog hookworm, Ancylostoma caninum and Na-APR-1 from the human hookworm, Necator americanus, were shown to be expressed in the infective larval stage (L3) as well as adult worms. We now show that both proteases degraded skin macromolecules and serum proteins, some of which were cleaved more readily from permissive definitive hosts as opposed to non-permissive hosts. Na-APR-1 degraded human collagens more efficiently than did Ac-APR-1, and Ac-APR-1 degraded canine serum albumin more efficiently than did Na-APR-1. On the other hand, both enzymes degraded human serum proteins (albumin and fibrinogen) with approximately equal efficiency under the conditions of our assays in vitro. Molecular models of these 2 orthologous, aspartic proteases showed that, despite having active site clefts with identical primary sequences, residues in the S3 pocket adopted different conformations, likely accounting for different substrate preferences reported previously. Antisera raised to both proteases partially inhibited (16-26%) migration of hookworm L3 through hamster skin in vitro, further implying a connective tissue invasive role for these enzymes in addition to digestion of serum and erythrocyte proteins for nutrition.

Ancylostoma↗
PubMed2001

Hemoglobin-degrading, aspartic proteases of blood-feeding parasites: substrate specificity revealed by homology models.

Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4' were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH(2)]Leu-Tyr-Tyr-Ser- NH(2) (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4' residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.

Amino Acid Sequence↗
PubMed2001

A cDNA encoding Tc-MUC-5, a mucin from Toxocara canis larvae identified by expression screening.

Toxocara canis is an ascarid nematode parasite of canids. Larvae infect a wide range of accidental hosts including humans, in whom they are the aetiologic agent of visceral and ocular Larva migrans. The labile surface coat of T. canis larvae consists of a family of mucin glycoproteins termed TES-120, for which the cDNAs have recently been cloned. In this paper, we describe the identification of a novel cDNA (Tc-muc-5) encoding an apomucin by expression screening of a cDNA library with antiserum raised to T. canis excretory/secretory products, and compare the predicted Tc-MUC-5 protein with those of other T. canis mucins (Tc-MUC-1-Tc-MUC-4) that include the TES-120 surface coat glycoproteins. Tc-MUC-5 has both a larger open reading frame and a more divergent sequence than the other T. canis mucins. It contains a putative signal peptide followed by two six-cysteine (SXC) domains, an extended threonine-rich central mucin core domain and two C-terminal SXC domains. Amino acid composition analysis of secreted TES-120 glycoproteins revealed a distinct lack of lysine residues; while this finding is in agreement with the primary sequences of Tc-MUC-1-Tc-MUC-4, Tc-MUC-5 is conspicuous by its relative abundance of lysines (6.7%), suggesting that this protein is not part of the TES-120 family of surface coat proteins.

Amino Acid Sequence↗
PubMed2001

Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum.

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have named this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box. Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes.

Amino Acid Sequence↗
PubMed2001

Schistosoma japonicum cathepsin D aspartic protease cleaves human IgG and other serum components.

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

Amino Acid Sequence↗
PubMed2001

Parental alcoholism and co-occurring antisocial behavior: prospective relationships to externalizing behavior problems in their young sons.

The hypothesis that parental alcoholism and co-occurring antisocial behavior would be indirectly linked to child externalizing behavior problems through child lack of control, current levels of parent depression, family conflict, and parent-child conflict was tested using manifest variable regression analysis. Participants were a community sample of 125 families with an alcoholic father and 83 ecologically matched but nonsubstance abusing families involved in the first 2 waves of an ongoing longitudinal study (with 3 years between each wave). All families had a biological son who was 3-5 years old at study onset. Results revealed that child lack of control mediated the relation between paternal alcoholism and the son's subsequent externalizing behavior problems. Family conflict was a significant mediator of maternal and paternal lifetime antisocial behavior effects and father-son conflict mediated paternal lifetime antisocial behavior effects. Study implications are discussed within the context of parental socialization of antisocial behavior.

Alcoholism↗
PubMed2001

Immune responses in hookworm infections.

Hookworms infect perhaps one-fifth of the entire human population, yet little is known about their interaction with our immune system. The two major species are Necator americanus, which is adapted to tropical conditions, and Ancylostoma duodenale, which predominates in more temperate zones. While having many common features, they also differ in several key aspects of their biology. Host immune responses are triggered by larval invasion of the skin, larval migration through the circulation and lungs, and worm establishment in the intestine, where adult worms feed on blood and mucosa while injecting various molecules that facilitate feeding and modulate host protective responses. Despite repeated exposure, protective immunity does not seem to develop in humans, so that infections occur in all age groups (depending on exposure patterns) and tend to be prolonged. Responses to both larval and adult worms have a characteristic T-helper type 2 profile, with activated mast cells in the gut mucosa, elevated levels of circulating immunoglobulin E, and eosinophilia in the peripheral blood and local tissues, features also characteristic of type I hypersensitivity reactions. The longevity of adult hookworms is determined probably more by parasite genetics than by host immunity. However, many of the proteins released by the parasites seem to have immunomodulatory activity, presumably for self-protection. Advances in molecular biotechnology enable the identification and characterization of increasing numbers of these parasite molecules and should enhance our detailed understanding of the protective and pathogenetic mechanisms in hookworm infections.

Ancylostomatoidea↗
PubMed2001

Receptor for Fc on the surfaces of schistosomes.

Schistosoma mansoni masks its surface with adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatibility complex class I, and beta(2)-microglobulin (beta(2)m), presumably as a means of avoiding host immune responses. How this is accomplished has not been explained. To identify surface receptors for host proteins, we biotinylated the tegument of live S. mansoni adults and mechanically transformed schistosomula and then removed the parasite surface with detergent. Incubation of biotinylated schistosome surface extracts with human immunoglobulin G (IgG) Fc-Sepharose resulted in purification of a 97-kDa protein that was subsequently identified as paramyosin (Pmy), using antiserum specific for recombinant Pmy. Fc also bound recombinant S. mansoni Pmy and native S. japonicum Pmy. Antiserum to Pmy decreased the binding of Pmy to Fc-Sepharose, and no proteins bound after removal of Pmy from extracts. Fluoresceinated human Fc bound to the surface, vestigial penetration glands, and nascent oral cavity of mechanically transformed schistosomula, and rabbit anti-Pmy Fab fragments ablated the binding of Fc to the schistosome surface. Pmy coprecipitated with host IgG from parasite surface extracts, indicating that complexes formed on the parasite surface as well as in vitro. Binding of Pmy to Fc was not inhibited by soluble protein A, suggesting that Pmy does not bind to the region between the CH2 and CH3 domains used by many other Fc-binding proteins. beta(2)m did not bind to the schistosome Fc receptor (Pmy), a finding that contradicts reports from earlier workers but did bind to a heteromultimer of labeled schistosomula surface proteins. This is the first report of the molecular identity of a schistosome Fc receptor; moreover it demonstrates an additional aspect of the unusual and multifunctional properties of Pmy from schistosomes and other parasitic flatworms.

Animals↗
PubMed2000

A family of secreted mucins from the parasitic nematode Toxocara canis bears diverse mucin domains but shares similar flanking six-cysteine repeat motifs.

Infective larvae of the parasitic nematode Toxocara canis secrete a family of mucin-like glycoproteins, which are implicated in parasite immune evasion. Analysis of T. canis expressed sequence tags identified a family of four mRNAs encoding distinct apomucins (Tc-muc-1-4), one of which had been previously identified in the TES-120 family of glycoproteins secreted by this parasite. The protein products of all four cDNAs contain signal peptides, a repetitive serine/threonine-rich tract, and varying numbers of 36-amino acid six-cysteine (SXC) domains. SXC domains are found in many nematode proteins and show similarity to cnidarian (sea anemone) toxins. Antibodies to the SXC domains of Tc-MUC-1 and Tc-MUC-3 recognize differently migrating members of TES-120. TES-120 proteins separated by chromatographic methods showed distinct amino acid composition, mass, and sequence information by both Edman degradation and matrix-assisted laser desorption ionization/time of flight mass spectrometry on peptide fragments. Tc-MUC-1, -2, and -3 were shown to be secreted mucins with real masses of 39.7, 47.8, and 45.0 kDa in contrast to their predicted peptide masses of 15.7, 16.2, and 26.0 kDa, respectively. The presence of SXC domains in all mucin products supports the suggestion that the SXC motif is required for mucin assembly or export. Homology modeling indicates that the six-cysteine domains of the T. canis mucins adopt a similar fold to the sea anemone potassium channel-blocking toxin BgK, forming three disulfide bonds within each subunit.

Amino Acid Sequence↗
PubMed2000

Sjalpha elements, short interspersed element-like retroposons bearing a hammerhead ribozyme motif from the genome of the oriental blood fluke Schistosoma japonicum.

Smalpha is a short interspersed element (SINE)-like retroposon that occurs in high copy number of the genome of the human blood fluke Schistosoma mansoni. The sequence of the consensus Smalpha element includes the hallmark features of SINE-like elements including a promoter region for RNA polymerase III, an AT-rich stretch at its 3'-terminus, a short length of 500 bp or less, and short direct repeat sequences flanking the insertion site. Interestingly, the sequence of Smalpha also encodes an active ribozyme bearing a hammerhead domain. Contrary to the recent findings of Ferbeyre et al. (Mol. Cell. Biol. 18 (1998) 3880-8) that indicated that Smalpha-like elements were absent from the genome of the Oriental blood fluke Schistosoma japonicum, we report here that the genome of S. japonicum does contain a family of Smalpha-like retroposons, elements that we have named the Sjalpha family. Like Smalpha, Sjalpha elements are SINE-like in structure and sequence, are present at high copy number interspersed throughout the S. japonicum genome, and contain an ostensibly functional, hammerhead ribozyme motif. The presence of these elements in all species of Schistosoma so far examined suggests that the hammerhead domain was acquired by vertical transmission from a common schistosome ancestor.

Animals↗
PubMed2000

Toxocara canis: genes expressed by the arrested infective larval stage of a parasitic nematode.

Toxocara canis is a widely distributed nematode parasite which reaches maturity in dogs. However, eggs voided by canid animals are infective to a very wide range of paratenic hosts including humans. In noncanid hosts, infective larvae emerge from the eggs and invade the soft tissues, often entering the brain and musculature. Such larvae may remain for many months or years in these tissues without further growth or differentiation, and yet appear to evade inflammatory reactions or other modes of immune attack. To understand the ability of T. canis larvae to survive in the immunocompetent host, we have undertaken a molecular analysis of the major genes expressed at this stage. By a combination of protein sequencing, gene identification, and expressed sequence tag (EST) analysis we have characterised a range of potentially important gene products from this parasite. Some of these are homologues of prominent mammalian proteins such as C-type lectins (represented by the secreted products TES-32 and TES-70), and mucins (TES-120), and additional products show strong similarities to known cysteine proteases, phosphatidylethanolamine-binding proteins and other ligands. A number of these proteins include a conspicuous 36-amino acid motif containing six cysteines. This domain (termed NC6 or SXC) appears to be an evolutionarily mobile module, which in T. canis is combined with a spectrum of diverse functional domains in different genes. In addition, we have identified a set of novel gene sequences that show no resemblance to any genes encoded by the free-living nematode C. elegans. Four of these are designated abundant novel transcripts, and collectively these account for nearly 20% of the cDNA isolated from the arrested infective stage. Such parasite-specific genes expressed at a high level by a stage that shows remarkable endurance may represent critical products necessary for the success of the parasitic mode of life.

Amino Acid Sequence↗
PubMed2000

Helminth C-type lectins and host-parasite interactions.

C-type lectins (C-TLs) are a family of carbohydrate-binding proteins intimately involved in diverse processes including vertebrate immune cell signalling and trafficking, activation of innate immunity in both vertebrates and invertebrates, and venom-induced haemostasis. Helminth C-TLs sharing sequence and structural similarity with mammalian immune cell lectins have recently been identified from nematode parasites, suggesting clear roles for these proteins at the host-parasite interface, notably in immune evasion. Here, Alex Loukas and Rick Maizels review the status of helminth lectin research and suggest ways in which parasitic worms might utilize C-TLs during their life history.

Amino Acid Sequence↗