RESUMEN

Small RNA pathways play key and diverse regulatory roles in C. elegans, but our understanding of their conservation and contributions in other nematodes is limited. We analyzed small RNA pathways in the divergent parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that associate with secondary 5'-triphosphate 22-24G-RNAs. These small RNAs target repetitive sequences or mature mRNAs and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Even in the absence of a piRNA pathway, Ascaris CSR-1 may still function to "license" as well as fine-tune or repress gene expression. Ascaris ALG-4 and its associated 26G-RNAs target and likely repress specific mRNAs during testis meiosis. Ascaris WAGO small RNAs demonstrate target plasticity changing their targets between repeats and mRNAs during development. We provide a unique and comprehensive view of mRNA and small RNA expression throughout spermatogenesis. Overall, our study illustrates the conservation, divergence, dynamics, and flexibility of small RNA pathways in nematodes.

Asunto(s)

RESUMEN

INTRODUCTION: Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-toff MS) is a reliable method for diagnosing a number of bacterial and fungal infections. It is also effective as a method of rapid diagnosis of several parasitic agents. We used MALDI-toff MS to study the protein profiles of four nematodes: Dirofilaria repens, Dirofilaria. immitis, Ascaris suum and Ascaris lumbricoides. METHODS: We studied the protein profiles of dirofilaria (five of each species: D. repens and D. immitis) and ascaris (five of each species: A. suum and A. lumbricoides), using a proteomic analysis based on MALDI-toff MS. RESULTS: Analysis of protein extracts of dirofilaria and ascaris showed spectra with high-intensity peaks in the range of 2-20 kDa. The quality of the spectra (clear graphical reflection of mass/charge to luminous intensity, consistent in repeated analyzes) and the intensity of the spectral peaks were consistent in all samples of the same species. The spectra profiles of D. repens and D. immitis differed in eight major peaks which makes it possible to differentiate species according to the protein profile. The spectra profiles obtained from A. suum and A. lumbricoides proteins differed slightly in 3 major peaks in both species and were discovered in m/z 13000; 13400 and 14400. The protein peaks in diapason 3000 kD-7300 kD specific for all genus ascaris are constant. CONCLUSIONS: MALDI-toff MS-based proteomic analysis can serve as an effective taxonomic tool for parasitological studies.

Asunto(s)

RESUMEN

Helminthiases caused by parasitic nematodes are widespread in different regions of the world. The main adaptation for overcoming adverse conditions is a barrier properties of the cuticle surface structure, which differs from the membrane teguments of trematodes and cestodes. Different types of nematodes have specific structural and biochemical adaptations at different stages of their life cycle. While creating specific areas of habitat and nutrition, some types of parasites change the morphology and functioning of the host tissues. Ascaris suum and Caenorabditis elegans were widely used as model organisms in the study of genetics, biochemistry of nematodes. Studying of biochemistry and molecular biology of structural components of nematode surfaces is important for development of effective and safe anthelmintic drugs. The differences in the structure and functioning of transport enzymes of parasites and humans will help to create effective specific inhibitors and anthelmintic remedies. An important point of application of anthelmintic drugs can serve as inorganic ions transport proteins in the membranes of the surfaces. Glycolipids of cuticle contribute to the evasion from the host immune system, protecting the surface proteins from degradation by proteases. Study of helminth surfaces makes an important contribution to the development of anthelmintic drugs and vaccines, for helminthiasis treat.

Asunto(s)

RESUMEN

In mature gametes and during the oocyte-to-embryo transition, transcription is generally silenced and gene expression is post-transcriptionally regulated. However, we recently discovered that major transcription can occur immediately after fertilization, prior to pronuclear fusion, and in the first cell division of the oocyte-to-embryo transition in the nematode Ascaris suum. We postulate that the balance between transcriptional and post-transcriptional regulation during the oocyte-to-embryo transition may largely be determined by cell cycle length and thus the time available for the genome to be transcribed.

Asunto(s)

RESUMEN

Nematode sperm offer a unique perspective for investigating amoeboid cell motility. These cells display the hallmark features of amoeboid movement but power their locomotion with a cytoskeleton composed of major sperm protein (MSP) filaments in place of the familiar actin cytoskeleton found in other crawling cells. Thus, properties of sperm can be compared to those of actin-rich cells to identify the shared features that are essential to motility. Sperm are simple cells in which cytoskeletal dynamics are tightly coupled to protrusion of the leading edge and retraction of the cell body. These features have facilitated reconstitution of both protrusion and retraction in cell-free extracts and enabled identification of accessory components in the motility apparatus as well as elucidation of the mechanical basis of movement. Six MSP accessory proteins have been isolated including four components of the sperm cytoskeleton and two enzymes that play key roles in regulating cytoskeletal dynamics and locomotion. Analysis of this versatile in vitro motility system has identified motor-independent mechanisms for protrusion and retraction that are based on changes in filament-packing density. These changes result in expansion and contraction of the MSP-filament network that generate the forces for movement. We discuss how the mechanisms of motility that operate in nematode sperm may contribute generally to the movement of crawling cells.

Asunto(s)

RESUMEN

Eukaryotic cells express several classes of small RNAs that regulate gene expression and ensure genome maintenance. Endogenous siRNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs) mainly control gene and transposon expression in the germline, while microRNAs (miRNAs) generally function in post-transcriptional gene silencing in both somatic and germline cells. To provide an evolutionary and developmental perspective on small RNA pathways in nematodes, we identified and characterized known and novel small RNA classes through gametogenesis and embryo development in the parasitic nematode Ascaris suum and compared them with known small RNAs of Caenorhabditis elegans. piRNAs, Piwi-clade Argonautes, and other proteins associated with the piRNA pathway have been lost in Ascaris. miRNAs are synthesized immediately after fertilization in utero, before pronuclear fusion, and before the first cleavage of the zygote. This is the earliest expression of small RNAs ever described at a developmental stage long thought to be transcriptionally quiescent. A comparison of the two classes of Ascaris endo-siRNAs, 22G-RNAs and 26G-RNAs, to those in C. elegans, suggests great diversification and plasticity in the use of small RNA pathways during spermatogenesis in different nematodes. Our data reveal conserved characteristics of nematode small RNAs as well as features unique to Ascaris that illustrate significant flexibility in the use of small RNAs pathways, some of which are likely an adaptation to Ascaris' life cycle and parasitism. The transcriptome assembly has been submitted to NCBI Transcriptome Shotgun Assembly Sequence Database(http://www.ncbi.nlm.nih.gov/genbank/TSA.html) under accession numbers JI163767­JI182837 and JI210738­JI257410.

Asunto(s)

RESUMEN

The major sperm protein (MSP) motility system in nematode sperm is best known for propelling the movement of mature sperm, where it has taken over the role usually played by actin in amoeboid cell motility. However, MSP filaments also drive the extension of filopodia, transient organelles composed of a core bundle of MSP filaments, that form in the late in sperm development but are not found on crawling cells. We have reconstituted filopodial extension in vitro whereby thin bundles of MSP filaments, each enveloped by a membrane sheath at their growing end, elongated at rates up to 17 microm/min. These bundles often exceeded 500 microm in length but were comprised of filaments only 1 microm long. The reconstituted filopodia assembled in the same cell-free sperm extracts that produced MSP fibers, robust meshworks of filaments that exhibit the same organization and dynamics as the lamellipodial filament system that propels sperm movement. The filopodia and fibers that assembled in vitro both had a membranous structure at their growing end, shared four MSP accessory proteins, and responded identically to agents that alter MSP-based motility by modulating protein phosphorylation. However, filopodia grew three- to four-fold faster than fibers. The reconstitution of filopodial extension shows that, like the actin cytoskeleton, MSP filaments can adopt two architectures, bundles and meshworks, each capable of pushing against membranes to generate protrusion. The reconstitution of both forms of motility in the same in vitro system provides a promising avenue for understanding how the forces for membrane protrusion are produced.

Asunto(s)

RESUMEN

A spliced leader contributes the mature 5'ends of many mRNAs in trans-splicing organisms. Trans-spliced metazoan mRNAs acquire an m3(2,2,7)GpppN cap from the added spliced leader exon. The presence of these caps, along with the typical m7GpppN cap on non-trans-spliced mRNAs, requires that cellular mRNA cap-binding proteins and mRNA metabolism deal with different cap structures. We have developed and used an in vitro system to examine mRNA degradation and decapping activities in nematode embryo extracts. The predominant pathway of mRNA decay is a 3' to 5' pathway with exoribonuclease degradation of the RNA followed by hydrolysis of resulting mRNA cap by a scavenger (DcpS-like) decapping activity. Direct decapping of mRNA by a Dcp1/Dcp2-like activity does occur, but is approximately 15-fold less active than the 3' to 5' pathway. The DcpS-like activity in nematode embryo extracts hydrolyzes both m7GpppG and m3(2,2,7)GpppG dinucleoside triphosphates. The Dcp1/Dcp2-like activity in extracts also hydrolyzes these two cap structures at the 5' ends of RNAs. Interestingly, recombinant nematode DcpS differs from its human ortholog in its substrate length requirement and in its capacity to hydrolyze m3(2,2,7)GpppG.

Asunto(s)

RESUMEN

Monomeric invertabrate hemoglobins with high oxygen affinity usually contain a tyrosine in the distal region of the heme. This feature has stimulated investigations revealing that one of the properties resulting from the presence of the distal tyrosines is a decreased off rate on the binding of oxygen, thus developing the high affinity. Despite that fact that the pK value of the tyrosine group differs significantly from the groups it replaces little attention has been paid to the pH dependence of the binding of oxygen to the high affinity hemoglobins. Such a pH dependence has been reported on two of the monomeric hemoglobins with relatively low oxygen affinity and one monomeric hemoglobin of intermediate affinity. The pH data of these hemoglobins has been analysed with a linked function model involving the hydrogen ion. pK values required for the low-affinity hemoglobins vary from 4.5 to 7.5. When applied to the high-affinity hemoglobins, the linked function model provides reasonable values for the binding parameters. These pK values vary from 3.0 to 9.0.

Asunto(s)

RESUMEN

We report the ligand dependence of the conformer distribution in the distal heme pocket of Ascaris suum hemoglobin (Hb) studied by resonance Raman spectroscopy. The heme-bound CO is used as a spectroscopic antenna to probe the original distribution of conformers in the dioxygen derivative of Ascaris Hb, by utilizing sol-gel encapsulation. The first step is to encapsulate the dioxygen derivative in the porous sol-gel and let the gel age, thus trapping the equilibrium conformational distribution of Ascaris dioxygen Hb. In the second step, the dioxygen ligand is replaced by CO. The sol-gel environment impedes any large scale movements, drastically slowing down the conformational relaxation triggered by the ligation change, essentially "locking in" the initial quaternary and even tertiary structure of the protein. Studying the Fe-CO frequencies of the latter sample allows evaluation of the distribution of the distal heme pocket conformers that was originally associated with the dioxygen derivative. Extending the study to the Ascaris mutants allows for examination of the effect of specific residues in the distal pocket on the conformational distribution. The choice of mutants was largely based on the anticipated variation in hydrogen bonding patterns. The results show that the sol-gel encapsulation can slow or prevent re-equilibration within the distal heme pocket of Ascaris Hb and that the distribution of distal heme pocket conformers for the CO derivative of Ascaris Hb in the sol-gel is highly dependent on the history of the sample. Additionally, we report a detailed study of the CO complex of the mutants in solution for assignment of the various heme pocket conformers, and we present a comparison of the sol-gel data with solution data. The results support a picture in which the dioxygen derivative biases the population strongly toward a tightly packed configuration that favors the network of strong hydrogen bonding interactions, and suggest that Ascaris Hb is uniquely designed for dioxygen capture.

Asunto(s)

RESUMEN

The electronic structures of Ascaris trypsin inhibitor in solution are obtained by the first-principles, all-electron, ab initio calculation using the self-consistent cluster-embedding (SCCE) method. The inhibitor, made up of 62 amino acid residues with 912 atoms, has two three-dimensional solution structures: 1ata and 1atb. The calculated ground-state energy of structure 1atb is lower than that of structure 1ata by 6.12 eV. The active sites are determined and explained: only structure 1atb has a N terminal at residue ARG+31. This shows that the structure 1atb is the stable and active form of the inhibitor, which is in agreement with the experimental results. The calculation reveals that some parts of the inhibitor can be easily changed while the inhibitor's biological activity may be kept. This kind of information may be helpful in fighting viruses such as AIDS, SARS, and flu, since these viruses have higher variability. The calculation offers an independent theoretical estimate of the precision of structure determination.

Asunto(s)

RESUMEN

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.

Asunto(s)

RESUMEN

Organisms co-habiting with bacteria have developed efficient bactericidal agents to control their microbe-rich environment. The Ascaris nematode lives in its final development stages in the gut of its host and is believed to feed on bacteria. Ascaris suum survive in pig intestine while A. lumbricoides is the principal species in humans. Here we show that A. suum and A. lumbricoides both produce linear (cecropin P1) and cysteine-rich (ASABF) peptides with activity against either gram-negative or gram-positive bacteria, respectively. Thus nematodes rely in part on a peptide-based antibacterial system for digestion of bacteria, which may also confer protection against infection. Cecropin P1 was previously isolated from pig intestine but we can now conclude that was due to contaminating nematodes.

Asunto(s)

RESUMEN

Carbohydrates substituted with phosphocholine (PC) and phosphoethanolamine (PE) were released from zwitterionic glycosphingolipids of the pig parasitic nematode Ascaris suum by treatment with endoglycoceramidase. Individual glycans were obtained by HPLC on porous graphitic carbon followed by high-pH anion-exchange chromatography. In addition to the known pentasaccharides Gal alpha 3GalNAc beta 4[PC6]GlcNAc beta 3Man beta 4Glc and Gal alpha 3GalNAc beta 4[PC6]GlcNAc beta 3[PE6]Man beta 4Glc, the corresponding tri- and tetra-saccharides, as well as components with elongated structures, could be identified by matrix-assisted laser-desorption ionization-time-of-flight MS, methylation analysis, 1H- and 13C-NMR spectroscopy, exoglycosidase cleavage and electrospray ionization ion-trap MS. The extended components comprised novel structural motifs such as di-substituted alpha-galactose carrying two beta-linked galactosyl residues, which were found to bear, in part, further fucose, galactose, N -acetylgalactosamine and/or N -acetylglucosamine moieties. Furthermore, additional fucosylation of the PC-substituted N -acetylglucosamine and a non-terminal fucosyl motif were detected. In conclusion, this study contributes significant new information on the glycome of nematodes.

Asunto(s)

RESUMEN

The pharmacological profiles of the 5-hydroxytryptamine (5-HT) receptors on Ascaris suum pharyngeal and somatic body wall muscles were investigated. The mechanisms involved following activation of these receptors were also studied. 5-HT activated and maintained pumping in isolated pharynxes with an EC-50 value of 44+/-1.7 microM. The 5-HT agonists, tryptamine, sumatriptan 8-OH-DPAT and 5-carboxyamidotryptamine all failed to stimulate pumping. The 5-HT2 antagonist, ketanserin, initially excited and then inhibited pumping while the 5-HT3 antagonist, ondansetron, had no effect. 5-HT and 5-HT agonists, 8-OH-DPAT, 5-carboxyamidotryptamine, alpha-methyl-5-HT and tryptamine all inhibited ACh-induced contractions of a somatic body wall muscle strip. Ketanserin partially blocked the inhibitory effect of alpha-methyl-5-HT and ACh-induced contractions while the 5-HT uptake blocker, fluoxetine, potentiated the effect of 5-HT on ACh-induced contractions. Basal levels of cAMP, 1540+/-232 pmol/mg, in pharyngeal muscle and 1721+/-134 pmol/mg, somatic body wall muscle, were both increased by forskolin. 5-HT had no effect on pharyngeal muscle cAMP levels but raised cAMP levels in somatic body wall muscle, e.g. 100 micron 5-HT, raised the level to 2851+/-212 pmol/mg and 1000 microM raised levels to 4578+/-1234 pmol/mg. 5-HT, 1000 microM, increased inositol phosphate levels in pharyngeal muscle. These results provide some evidence for a 5-HT2-like receptor on pharyngeal muscle. In contrast, the situation on somatic body wall muscle is more confusing since the pharmacological profile partly indicates a 5-HT2-like receptor but this receptor is linked to a rise in cAMP levels. Further studies are required to resolve the position but they must be based on the rational design of ligands specifically for nematode 5-HT receptors and not simply using ligands developed for the classification of mammalian 5-HT receptors. Such a design must take into account data from molecular biology studies of nematode 5-HT receptors.

Asunto(s)

Asunto(s)

Asunto(s)

RESUMEN

From skin secretions of the European frog Bombina bombina, a new peptide has been isolated that contains 60 amino acids, including 10 cysteine residues. Its sequence was determined by automated Edman degradation and confirmed by analysis of the cDNA encoding the precursor. A search in the databanks demonstrated that the pattern of cysteine residues in this skin peptide is similar to the ones found in protease inhibitors from Ascaris and in a segment of human von Willebrand factor. The 3D structure of the trypsin inhibitor from Ascaris suum could be used as a template to build a model of the amphibian peptide. In addition, we have demonstrated that this constituent of skin secretion is indeed an inhibitor of trypsin and thrombin, with K(i) values in the range of 0.1 to 1 microM. The new peptide was thus named BSTI for Bombina skin trypsin/thrombin inhibitor.

Asunto(s)

RESUMEN

The hemoglobin from Ascaris suum, a parasitic nematode, has a spontaneous dissociation rate for the dioxygen ligand that is 3 orders of magnitude less than for mammalian myoglobins or hemoglobins. In this hemoglobin, the distal histidine is replaced with a glutamine which is capable of forming a stabilizing hydrogen bond to the bound dioxygen. A single hydrogen bond from a glutamine is, under typical circumstances, not sufficient to account for the low off rate for oxygen. Several studies point to a second hydrogen bond to the heme-bound dioxygen originating from tyrosine B10 as the source of this unusual reactivity. In this study ultraviolet (UV) resonance Raman spectroscopy is used to directly observe the formation of this hydrogen bond upon oxygen binding. The study reveals that both oxygen and carbon monoxide induce similar conformational changes in the globin upon binding to the heme; however, in the case of oxygen, a strong hydrogen bond involving a tyrosine is also observed. Similar studies on the QE7L mutant of this Hb suggest that the glutamine plays a role in stabilizing a rigid tertiary structure associated with the distal heme pocket. This conformation maintains the tyrosine in an orientation conducive to hydrogen bond formation with a heme-bound dioxygen ligand.

Asunto(s)

RESUMEN

The perienteric hemoglobin of the parasitic nematode Ascaris has an exceptionally high affinity for oxygen. It is an octameric protein containing two similar heme-binding domains per subunit, but recombinant constructs expressing a single, monomeric heme-binding domain (domain 1; D1) retain full oxygen avidity. We have solved the crystal structure of D1 at 2.2 A resolution. Analysis of the structure reveals a characteristic globin fold and illuminates molecular features involved in oxygen avidity of Ascaris perienteric hemoglobin. A strong hydrogen bond between tyrosine at position 10 in the B helix (tyrosine-B10) and the distal oxygen of the ligand, combined with a weak hydrogen bond between glutamine-E7 and the proximal oxygen, grips the ligand in the binding pocket. A third hydrogen bond between these two amino acids appears to stabilize the structure. The B helix of D1 is displaced laterally by 2.5 A when compared with sperm whale myoglobin. This shifts the tyrosine-B10 hydroxyl far enough from liganded oxygen to form a strong hydrogen bond without steric hindrance. Changes in the F helix compared with myoglobin contribute to a tilted heme that may also be important for oxygen affinity.

Asunto(s)