Detection of Ascaris lumbricoides infection by ABA-1 coproantigen ELISA.

Intestinal worms, or soil-transmitted helminths (STHs), affect hundreds of millions of people in all tropical and subtropical regions of the world. The most prevalent STH is Ascaris lumbricoides. Through large-scale deworming programs, World Health Organization aims to reduce morbidity, caused by moderate-to-heavy intensity infections, below 2%. In order to monitor these control programs, stool samples are examined microscopically for the presence of worm eggs. This procedure requires well-trained personnel and is known to show variability between different operators interpreting the slides. We have investigated whether ABA-1, one of the excretory-secretory products of A. lumbricoides can be used as a coproantigen marker for infection with this parasite. Polyclonal antibodies were generated and a coproantigen ELISA was developed. Using this ELISA, it was found that ABA-1 in stool detected Ascaris infection with a sensitivity of 91.5% and a specificity of 95.3%. Our results also demonstrate that there is a correlation between ABA-1 levels in stool and A. lumbricoides DNA detected in stool. Using a threshold of 18.2 ng/g stool the ABA-1 ELISA correctly assigned 68.4% of infected individuals to the moderate-to-heavy intensity infection group, with a specificity of 97.1%. Furthermore, the levels of ABA-1 in stool were shown to rapidly and strongly decrease upon administration of a standard anthelminthic treatment (single oral dose of 400 mg albendazole). In an Ascaris suum infection model in pigs, it was found that ABA-1 remained undetectable until day 28 and was detected at day 42 or 56, concurrent with the appearance of worm eggs in the stool. This report demonstrates that ABA-1 can be considered an Ascaris -specific coproantigen marker that can be used to monitor infection intensity. It also opens the path for development of point-of-care immunoassay-based tests to determine A. lumbricoides infection in stool at the sample collection site.

Shared expression of mucin12 in Ascaris lumbricoides and the human small intestine.

This study focuses on the host-parasite relationship of human Ascaris lumbricoides, which is a parasite of the small intestine and is also one of the commonest parasites worldwide. As part of this investigation, we examined the host-parasite relationship assuming that there is a common antigenicity, shared protein between A. lumbricoides and human small intestinal mucosa, using molecular techniques. We obtained three DNA clones from human colon cDNA library by screening for anti-A. lumbricoides polyclonal antibodies. The transmembrane mucin12 gene was identified after sequencing analysis of these clones. Specific signals of immunostaining with polyclonal anti-mucin12 antibodies were observed in the mucous secretory organs, epidermis, and intestinal canal of A. lumbricoides. These signals disappeared when immunohistochemistry was performed using pre-absorbed polyclonal antibodies with a specific peptide. These results suggest that mucin12 is localized in the mucous secretory organs in the epidermis of A. lumbricoides. Furthermore, we examined the site of mucin12 localization in the host; specific mucin12 signals were observed on the mucosal epithelia present around intestinal crypts and villi of the small intestine. Therefore, we suggest that mucin12 is a protein that shows common antigenicity in both A. lumbricoides and its host. It is presumed that adult A. lumbricoides live in their preferred environment, which is the small intestine, by secreting mucin12 to avoid being attacked by the host immune system.

Early developmental stages of Ascaris lumbricoides featured by high-resolution mass spectrometry.

Ascaris lumbricoides is responsible for a highly disseminated helminth parasitic disease, ascariosis, a relevant parasitosis that responds for great financial burden on the public health system of developing countries. In this work, metabolic fingerprinting using high-resolution mass spectrometry (HRMS) was employed to identify marker molecules from A. lumbricoides in different development stages. We have identified nine biomarkers, such as pheromones and steroidal prohormones in early stages, among other molecules in late development stages, making up four molecules for fertilized eggs, four marker molecules for first larvae (L1) and one marker molecule for third larvae (L3). Therefore, our findings indicate that this approach is suitable for biochemical characterization of A. lumbricoides development stages. Moreover, the straightforward analytical method employed, with almost no sample preparation from a complex matrix (feces) using high-resolution mass spectrometry, suggests that it is possible to seek for an easier and faster way to study animal molding processes.

Comparative analysis of microRNA profiles between adult Ascaris lumbricoides and Ascaris suum.

BACKGROUND: The parasitic nematodes Ascaris lumbricoides and A. suum are of great public health and economic significance, and the two taxa were proposed to represent a single species. miRNAs are known with functions of gene regulations at post-transcriptional level. RESULTS: We herein compared the miRNA profiles of A. lumbricoides and A. suum female adults by Solexa deep sequencing combined with bioinformatics analysis and stem-loop real-time PCR. Using the A. suum genome as the reference genome, we obtained 171 and 494 miRNA candidates from A. lumbricoides and A. suum, respectively. Among which, 74 miRNAs were shared between the two taxa, 97 and 420 miRNAs were A. lumbricoides and A. suum specific. Target and function prediction revealed a significant set of targets which are related to ovarian message protein, vitellogenin and chondroitin proteoglycan of the two nematodes. Enrichment analysis revealed that the percentages of most predicted functions of the miRNA targets were similar, with some taxon specific or taxon enhanced functions, such as different target numbers, specific functions (NADH dehydrogenase and electron carrier functions), etc. CONCLUSIONS: This study characterized comparatively the miRNAs of adult A. lumbricoides and A. suum, and the findings provide additional evidence that A. lumbricoides and A. suum represent a single species. Due to the fast evolution nature of miRNAs and the different parasitic living conditions of humans and pigs, the phenomenon above might indicate a fast evolution of miRNAs of Ascaris in humans and pigs.

Allergenicity of Ascaris lumbricoides tropomyosin and IgE sensitization among asthmatic patients in a tropical environment.

BACKGROUND: Ascaris lumbricoides induces a Th2 response and specific IgE synthesis in humans. This confers antiparasite immunity but could modify the natural history of allergic diseases in the tropics, justifying the study of its allergenic composition. We analyzed the allergenic properties of Ascaris tropomyosin and the frequency of sensitization in subjects exposed to the parasite. METHODS: cDNA was obtained by reverse transcription PCR, cloned into pQE30-UA and purified as a 6× His-tagged protein. Equivalence with its natural counterpart was analyzed by cross-inhibition and liquid chromatography-tandem mass spectrometry. Specific IgE was measured by ELISA in 175 asthmatics and 170 nonasthmatics naturally exposed to the parasite and sensitized to the Ascaris extract. RESULTS: The cDNA encoded 287 residues with high sequence identity with other invertebrate tropomyosins. The 40-kDa protein was recognized by human serum and affinity-purified anti-rBlo t 10 IgE. Specific IgE to tropomyosin could represent approximately 50% of the total IgE response to the extract. Ascaris tropomyosin induced wheal and flare in skin prick tests and histamine release from basophils. Although the prevalence of IgE to Ascaris tropomyosin was higher in asthmatic patients, logistic regression analysis suggested that this result was biased by sensitization to mites. CONCLUSIONS: A. lumbricoides tropomyosin (Asc l 3) is a new allergen that binds specific IgE, induces mediator release from effector cells and is cross-reactive to mite tropomyosins. IgE reactivity to this allergen is very frequent in both asthmatic and normal subjects sensitized to Ascaris extract. The potential role of Ascaris tropomyosin in asthma pathogenesis in tropical regions should be further investigated.

[Larval stages of Ascaris lumbricoides: hyaluronan-binding capacity]. / Estados larvales de Ascaris lumbricoides: capacidad de unión a acido hialurónico.

Hyaluronic acid has important functions in inflammatory and tissue reparation processes. Owing to the varied strategies of the parasites to evade the host's immune response, as well as the multiple functions and physiological importance of hyaluronic acid, the aim was to study the hyaluronan binding capacity by Ascaris lumbricoides larval stages. Larval concentrates were prepared by hatching A. lumbricoides eggs. The larvae were collected by the Baermann method. The test of serum soluble CD44 detection by Agregation Inhibition was modified. All the larval concentrates presented hyaluronan binding capacity. The obtained results allow to suppose the existence of an hyaluronic acid specific receptor in A. lumbricoides. This receptor eventually might compete with the usual receptors of the host. The parasite might use this mechanism to evade the immune response.

Ser/Arg-rich protein-mediated communication between U1 and U2 small nuclear ribonucleoprotein particles.

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.

Regulation of SR protein localization during development.

Ser-Arg-rich (SR) proteins play numerous roles in spliceosome assembly and the regulation of splice-site selection. Whereas considerable attention has focused on the mechanistic details of SR protein activities, little is known concerning how these splicing regulators are controlled by the cell. Here we examined the subcellular localization of precursor mRNA splicing factors during early development of the nematode Ascaris lumbricoides. In the early embryo, before major zygotic gene activation, most SR proteins, along with RNA polymerase II, are localized in the cytoplasm. As development proceeds, we observe a significant decrease in the cytoplasmic levels of these factors and a concomitant increase in nuclear localization. In contrast, trimethylguanosine-capped small nuclear ribonucleoproteins are predominantly localized in the nucleus throughout this period. We previously showed that the phosphorylation state and activity of SR proteins are regulated during A. lumbricoides embryogenesis. These changes correlate with the onset of precursor mRNA splicing and zygotic transcription. Thus, a coordinate change in the subcellular localization of SR proteins and RNA polymerase II occurs at the transition from reliance on maternally deposited factors to embryonic expression. We propose that before zygotic gene activation, SR proteins and RNA polymerase II are stockpiled in the cytoplasm of early embryos, awaiting signals that lead to their activation.

Developmental regulation of SR protein phosphorylation and activity.

Serine/arginine-rich splicing factors (SR proteins) are substrates for serine phosphorylation that can regulate SR protein function. We have observed gross changes in SR protein phosphorylation during early development coincident with major zygotic gene activation in the nematode Ascaris lumbricoides. These differences correlate with large-scale changes in SR protein activity in promoting both trans- and cis-splicing. Importantly, inactive early stage extracts can be made splicing competent on addition of later stage SR proteins. These data suggest that changes in SR protein phosphorylation have a role in the activation of pre-mRNA splicing during early development.

SR proteins are required for nematode trans-splicing in vitro.

SR (ser/arg) proteins have been shown to play roles in numerous aspects of pre-mRNA splicing, including modulation of alternative splicing, commitment of substrates to the splicing pathway, and splice site communication. The last of these, splice site communication, is particularly relevant to trans-splicing in which the 5' and 3' exons originate on separate molecules. The participation of SR proteins in naturally occurring, spliced leader RNA-dependent transsplicing has not been examined. Here, we have isolated SR proteins from an organism that performs both trans- and cis-splicing, the nematode Ascaris lumbricoides. To examine their activity in in vitro splicing reactions, we have also developed and characterized an SR protein-depleted whole-cell extract. When tested in this extract, the nematode SR proteins are required for both trans- and cis-splicing. In addition, the state of phosphorylation of the nematode SR proteins is critical to their activity in vitro. Interestingly, mammalian (HeLa) and A. lumbricoides SR proteins exhibit equivalent activities in cis-splicing, while the nematode SR proteins are much more active in trans-splicing. Thus, it appears that SR proteins purified from an organism that naturally trans-splices its pre-mRNAs promote this reaction to a greater extent than do their mammalian counterparts.

Giant pharyngeal gland nuclei of Ascaris: unusual cytology suggests a novel pathway for transport of ribosomal proteins to the nucleus.

We describe the unusual giant pharyngeal gland nuclei of the parasitic roundworm Ascaris lumbricoides suum and attempt to reconcile the relationships among its uncommon, and in some instances unique, collection of constituents. The nuclei were studied by light and electron microscopy, Feulgen cytophotometry, nuclear size analysis and histochemical methods, including those for detection of DNA, RNA, acidic and basic proteins, lipids and carbohydrates. A highly active nucleus is revealed: an extensive system of intranuclear annulate lamellae (IAL); membrane-bounded, acidophilic bodies which contain non-basic proteins; an abundance of free nucleoli; and an exceedingly large chromocenter containing a core of DNA surrounded by nucleoli (an apparent nucleolar organizing region, NOR). The relationships among the various nuclear constituents suggest that the acidophilic bodies consist of nucleolus-related proteins imported from the cytoplasm to the NOR, and that the annulate lamellae mediate this transport. Incidental findings include rounded cytoplasmic invaginations into the nucleus, thread-like structures with dense cores and surrounding small granules found among concentrations of nucleoli, and bundles of 12 nm filaments closely associated with these nucleoli. The significance of such huge, morphologically complex and highly polyploid nuclei, derived from chromatin-diminished progenitor cells early in development, and the possible interrelationship of these phenomena, remain obscure.

[New studies on natural inhibitors of proteolytic enzymes]. / Novoe o prirodnykh ingibitorakh proteoliticheskikh fermentov.

New data on proteolytic enzyme inhibitors and mechanisms of their interaction with the enzymes are reviewed. In recent years, a number of new inhibitors comprising families earlier unknown have been described such as proteins from the parasitic nematode Ascaris lumbricoides, ecotin from the periplasm of Escherichia coli, proteins PMP-C and PMP-D from locust Locusta migratoria, and hirustasin from the medicinal leech Hirudo medicinalis. At the same time, some proteins that may be assigned to inhibitors on the basis of their structures were found to perform other (not inhibitory) functions. Thus, the family of the Kunitz soybean trypsin inhibitor includes plant storage proteins and proteins whose synthesis is induced by stress factors. Numerous inhibitors interacting with the enzymes by mechanisms other than the substrate-like ones were identified, such as ornithodorin and anticoagulant peptide from tick Ornithodoros moubata (inhibitors of the blood clotting system proteases), an inhibitor from snake (Bothrops jararaca) venom, and ecotin, an inhibitor of serine proteases with an unusually broad specificity range. Special emphasis is placed on enzyme inhibition with propeptides and the mechanism of this process.

Direct analysis of nematode cis- and trans-spliceosomes: a functional role for U5 snRNA in spliced leader addition trans-splicing and the identification of novel Sm snRNPs.

Most nuclear pre-mRNAs in nematodes are processed by both cis- and trans-splicing. In trans-splicing, the 5' terminal exon, the spliced leader sequence (SL), is derived from a trans-splicing specific Sm snRNP, the SL RNP. Because U snRNPs are required cofactors for trans-splicing, and because this processing reaction proceeds via a two-step reaction pathway identical to that of cis-splicing, it has long been assumed that trans-splicing is catalyzed in a complex analogous to the cis-spliceosome. However, similarities or differences between cis- and trans-spliceosomes have not been established. In particular, the role of U5 snRNP in trans-splicing has been unclear. Here, we have used affinity selection to analyze the U snRNA constituents of nematode cis- and trans-spliceosomes. We find that U5 snRNP is an integral component of the trans-spliceosome and, using site-specific crosslinking, we show that U5 snRNP establishes specific Interactions with the SL RNA exon. We also identify two novel Sm snRNPs that are enriched in both cis- and trans-spliceosomes. Finally, we provide evidence that a SL RNP-containing multi-snRNP (SL, U4, U5, and U6 RNPs) may be a functional precursor in trans-spliceosome assembly.

U6 snRNA function in nuclear pre-mRNA splicing: a phosphorothioate interference analysis of the U6 phosphate backbone.

U6 snRNA is essential for and may participate in the catalysis of pre-mRNA splicing. Extensive mutational analyses in several systems have identified nucleotides essential for U6 function in splicing; however, relatively little is known regarding the role of the U6 phosphate backbone. We previously described a mutation in a nematode U6 snRNA that causes it to be used as a splicing substrate within the spliceosome. This unusual reaction has made it possible to apply modification interference analysis to U6 function. Here, we have used phosphorothioate substitution to identify pro-R oxygens throughout the U6 backbone that are necessary for the first and/or second catalytic steps of splicing. Four pro-R oxygens are important for the first step; of these only two appear to be required. One additional pro-R oxygen is uniquely required for the second step. The two pro-R oxygens critical for the first step of splicing are in the helix 1b U2/U6 interaction region and the intramolecular stem-loop of U6, respectively. A comparison of the positions of these two pro-R oxygens with those found to be critical for autocatalytic excision of a group II intron suggests a possible functional similarity between U6 snRNA and domain V of group II introns.

Ribosomal heterogeneity from chromatin diminution in Ascaris lumbricoides.

The genome of Ascaris lumbricoides encodes both germline- and soma-specific proteins homologous to the eukaryotic small ribosomal protein (Rp) S19. The two Ascaris homologs differ by 24 amino acid substitutions and are both components of the small ribosomal subunits. In oocytes, the germline RpS19 homolog (RpS19G) predominates. During chromatin diminution, however, the gene is eliminated from all presomatic cells, and RpS19G is replaced by the product of the somatic gene (RpS19S). Chromatin diminution in A. lumbricoides causes a change in the protein composition of ribosomes during development and represents an alternative means of gene regulation.

Preparation of myosin A from body wall musculature of Ascaris lumbricoides.

Myosin A (Ascaris myosin A) was prepared from the body wall musculature of Ascaris lumbricoides var. suis showing a single peak in analytical ultracentrifuge pattern, and its sedimentation coefficient was about 6 S which was close to the values reported for other myosins. The ATPase activities determined in various media were 3-4 times lower than those of rabbit skeletal myosin A. Nevertheless, the maximum level of the superprecipitation of actomyosin was 4 times higher for Ascaris myosin A than for rabbit skeletal myosin A in the presence of 0.01 mM ATP. It appears that Ascaris myosin A is capable of contracting the actomyosin system in a lower concentration of ATP than that for the usual actomyosin system.