[STRUCTURAL, MOLECULAR AND FUNCTIONAL FEATURES OF NEMATODE SURFACES AND THE POSSIBILITY OF ANTHELMINTIC DRUGS DEVELOPMENT (REVIEW)].

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.

Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles.

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.

Small RNA pathways in the nematode Ascaris in the absence of piRNAs.

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.

Enzyme localization in the anaerobic mitochondria of Ascaris lumbricoides.

Mitochondria from the muscle of the parasitic nematode Ascaris lumbricoides var. suum function anaerobically in electron transport-associated phosphorylations under physiological conditions. These helminth organelles have been fractionated into inner and outer membrane, matrix, and intermembrane space fractions. The distributions of enzyme systems were determined and compared with corresponding distributions reported in mammalian mitochondria. Succinate and pyruvate dehydrogenases as well as NADH oxidase, Mg(++)-dependent ATPase, adenylate kinase, citrate synthase, and cytochrome c reductases were determined to be distributed as in mammalian mitochondria. In contrast with the mammalian systems, fumarase and NAD-linked "malic" enzyme were isolated primarily from the intermembrane space fraction of the worm mitochondria. These enzymes are required for the anaerobic energy-generating system in Ascaris and would be expected to give rise to NADH in the intermembrane space. The need for and possible mechanism of a proton translocation system to obtain energy generation is suggested.

Components of sterol biosynthesis assembled on the oxygen-avid hemoglobin of Ascaris.

The parasitic nematode Ascaris infests a billion people worldwide. Much of its proliferative success is due to prodigious egg production, up to 10(6) sterol-replete eggs per day. Sterol synthesis requires molecular oxygen for squalene epoxidation, yet oxygen is scarce in the intestinal folds the worms inhabit. Ascaris has an oxygen-avid hemoglobin in the perienteric fluid that bathes its reproductive organs. Purified hemoglobin contained tightly bound squalene and functioned as an NADPH-dependent, ferrihemoprotein reductase. All components of the squalene epoxidation reaction--squalene, oxygen, NADPH, and NADPH-dependent reductase--are assembled on the hemoglobin. This molecule may thus function in sterol biosynthesis.

The application of proteomic methods (MALDI-toff MS) for studying protein profiles of some nematodes (dirofilaria and ascaris) for differentiating species.

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.

Dissection of the Ascaris sperm motility machinery identifies key proteins involved in major sperm protein-based amoeboid locomotion.

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.

Biosynthesis of biopterin in Ascaris lumbricoides suum.

In in vivo experiments, radioactivity from [U-14C]GTP was incorporated into biopterin, and, in fact, all carbon atoms of biopterin synthesized in Ascaris lumbricoides suum originated from GTP. Biopterin was also biosynthesized in homogenates of tissue fluid and muscles of Ascaris lumbricoides suum. The enzyme which catalyzes sepiapterin synthesis from D-erythro-7,8-dihydroneopterin-3'-phosphate was found in A. lumbricoides suum extracts and extracted in the 0--30% (NH4)2SO4 fraction from a 40 000 x g supernatant. The enzyme was purified by Sephadex G-200 column and DEAE-cellulose column chromatography. It is suggested that sepiapterin could be an intermediate compound in biopterin biosynthesis.

Ascaris suum hexokinase: purification and possible function in compartmentation of glucose 6-phosphate in muscle.

Hexokinase (EC 2.7.1.1) is present in a soluble and a bound form in homogenates of Ascaris suum muscle. Cellulose acetate electrophoresis, isoelectric focusing, and ion exchange chromatography confirmed the presence of only one molecular form of hexokinase in this muscle. A procedure for purifying hexokinase from Ascaris muscle has been developed utilizing ion-exchange chromatography, ammonium sulfate fractionation and gel filtration. The enzyme is a monomer with a molecular weight of 100 000 as determined by sodium dodecyl sulfate gel filtration. The Stokes' radius, diffusion coefficient, and frictional ratio have been determined. The apparent Michaelis constants for glucose and ATP are 4.7-10(-3) M and 2.2-10(-4) M, respectively. Ascaris hexokinase also exhibits end-product inhibition by glucose 6-phosphate and ADP. It is postulated that the kinetic parameters of the enzyme are the results of its function, that of generating glucose 6-phosphate primarily for glycogen synthesis.

Electron-transfer flavoprotein from anaerobic Ascaris suum mitochondria and its role in NADH-dependent 2-methyl branched-chain enoyl-CoA reduction.

Electron-transfer flavoprotein was purified to apparent homogeneity from mitochondria of the parasitic nematode, Ascaris suum. The native molecular weight of the enzyme was 70,000, as estimated by gel filtration, and it migrated as two bands with apparent subunit molecular weights of 37,000 and 31,500 during sodium dodecylsulfate polyacrylamide gel electrophoresis. The enzyme exhibited an absorption coefficient for the bound FAD of 13.5 mM-1.cm-1 at 436 nm and a protein/flavin (270 nm/436 nm) ratio of 5.6. While the ascarid enzyme is similar to its mammalian counterpart, physiologically it functions in the reverse direction, shuttling reducing power from the electron-transport chain to a soluble 2-methyl branched-chain enoyl CoA reductase. Indeed, when A. suum submitochondrial particles were incubated with NADH, 2-methylcrotonyl-CoA and purified A. suum 2-methyl branched-chain enoyl-CoA reductase, 2-methylbutyryl-CoA formation was proportional to the amount of electron-transfer flavoprotein added.

BSTI, a trypsin inhibitor from skin secretions of Bombina bombina related to protease inhibitors of nematodes.

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.

Distribution of 3H-GABA uptake sites in the nematode Ascaris.

The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, located at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations (Guastella et al., J Comp Neurol 307:584-597, 1991). Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously (Guastella et al., J Comp Neurol 307:584-597, 1991), these results define three classes of GABA-associated neurons in Ascaris: 1) neurons that contain endogenous GABA and possess a GABA uptake system; 2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; 3) neurons that possess a GABA uptake system, but that lack endogenous GABA.

Generation of monoclonal antibodies against a nematode peptide extract: another approach for identifying unknown neuropeptides.

Monoclonal antibodies that cross-react with Ascaris neural antigens were generated in mice immunized with a conjugate made with keyhole limpet hemocyanin (KLH) linked to a crude peptide extract from Caenorhabditis elegans. The response to KLH was suppressed by injection of cyclophosphamide 3 days after immunization with a gamma-aminobutyric acid (GABA)-KLH conjugate. Screening of hybridomas was carried out by enzyme-linked immunosorbent assay and whole mount immunocytochemistry. Two similar clones produced antibodies that recognized a small subset of Ascaris neurons. This result suggests that the monoclonal antibody technique might be useful for identifying new neuropeptides since the antibodies can be used for localization of the neuropeptidelike substances and, potentially, for immunoaffinity chromatography. As a by-product of this experiment, monoclonal antibodies that recognize GABA-like immunoreactivity in whole mounts and plastic sections were also obtained.

Neuropeptide diversity in Ascaris: an immunocytochemical study.

An immunocytochemical method was used for localization of various peptide-like substances in the Ascaris nervous system. Out of 45 antipeptide antisera, 12 demonstrated immunoreactivity in different subsets of neurons; these 12 antisera were raised against luteinizing hormone-releasing hormone (LHRH), Aplysia peptide L11 (L11), Aplysia peptide 12B (12B), small cardioactive peptide B (SCPB), neuropeptide Y (NPY), FMRFamide, gastrin-17, cholecystokinin octapeptide (CCK-8), alpha-melanocyte stimulating hormone (alpha MSH), calcitonin gene related peptide (CGRP), corticotropin releasing factor (CRF), and vasoactive intestinal peptide (VIP). Several peptide-like substances were colocalized to the same neuron. Our results suggest that Ascaris, like other organisms, contains multiple peptidergic systems.

Biophysical transport properties of the cuticle of Ascaris suum.

The transport properties of isolated cuticle from Ascaris suum were studied using standard two-chamber diffusion cells and a number of radiolabeled permeants which varied in molecular size, lipophilicity and electrical charge. The permeability coefficient of the collagen matrix (lipid-extracted cuticle) vs. molecular radius relationship showed the interdependence of molecular size and electrical charge of the permeants with respect to the aqueous pores of the negatively charged matrix. The permeability of neutral solutes decreased monotonically with size. Protonated amines permeated the aqueous pores faster than neutral solutes of comparable size, while the permeation of anions was slower. The average pore size was estimated to be 1.5 nm in radius. A biophysical model which accounted for diffusion of molecules within a fixed electrostatic field of force and for molecular sieving by the pore channels was used in the mechanistic interpretation of the data. The effective permeability coefficient of the non-lipid-extracted cuticle was delineated into the permeability coefficients of the water-filled collagen matrix and the lipoidal component of the cuticle to determine which layer was the rate-controlling barrier. While each solute was capable of penetrating the water-filled collagen matrix, the rate-determining step for the majority of compounds was passive diffusion across the lipid component, which controlled 75-99% of transport. The exception was water, for which transport kinetics was 75% matrix-controlled. In general, permeation across the lipid-filled tissue was more favorable for small lipophilic compounds because of molecular restriction not only in the aqueous pores, but also in the lipid-filled pores.

Metabolism and fate of ecdysteroids in the nematodes Ascaris suum and Parascaris equorum.

When injected with [3H]ecdysone and maintained in vitro, the parasitic nematodes, Ascaris suum and Parascaris equorum each produced a series of polar and relatively apolar metabolites. A. suum metabolised the compound into ecdysonoic acid ([3H]EOIC), ecdysone 25-glucoside ([3H]E25gluc), putative ecdysone 22-phosphate ([3H]E22P) and a series of at least six relatively apolar metabolites. All of these, except ecdysonoic acid, were hydrolysed by a crude enzyme preparation from Helix pomatia, releasing ecdysone. In a similar study, P. equorum produced ecdysone 25-glucoside, putative ecdysone 22-phosphate and a series of relatively apolar compounds all of which were hydrolysed by H. pomatia enzymes, releasing ecdysone. [3H]Ecdysone 25-glucoside was the most abundant single metabolite in both species, and in P. equorum, at least, was released into the culture medium in relatively large amounts. Apolar metabolites were present in worm samples and were the major, if not the only radiolabelled compounds detected in eggs of both species. Data indicated a metabolic relationship between some of the apolar conjugates found in both nematode species and ecdysone 25-glucoside.

Mechanisms of microenvironmental pH regulation in the cuticle of Ascaris suum.

The excretion kinetics of various organic acids by Ascaris suum were quantified to determine if the excretion of these metabolic end-products could generate and maintain a microclimate pH within the aqueous compartment of the cuticle. Ligated and nonligated A. suum were incubated in media buffered with 0.25 or 2.5 mM Hepes (initial pH 7.5) or 0.5 or 5 mM glycine (initial pH 3.25). The concentration of organic acids and the pH of the media were followed for 24 h. Several volatile fatty acids, including acetic, 2-methylbutyric, 2-methylvaleric, n-valeric, and n-butyric, were excreted at relatively high rates. Propionic, n-caproic, 2-methylcaproic, tiglic acid, and the non-volatile organic acids, lactic and succinic, were excreted more slowly. The organic acids were excreted at a constant rate and in apparently fixed molar concentration ratios. The accumulation of organic acids was associated with changes in pH of the medium until a limiting constant pH, in the vicinity of the pKa of the volatile fatty acids, was reached. The rate of organic acid excretion was not affected by initial medium pH, buffer capacity, or parasite ligation. The rate of pH change induced by the excretion of organic acids was also insensitive to whether ligated or nonligated A. suum were used, but was dependent on the initial buffer capacity of the medium. These results suggest that A. suum excrete the end-products of carbohydrate metabolism across the cuticle. The presence of organic acids in the aqueous pores of the cuticle creates and maintains a microclimate pH of about 5.0 +/- 0.3. This pH will influence the transport properties of weak acids and bases and should be considered in the design of delivery systems for anthelmintics.

NADH-dependent tiglyl-CoA reduction in disrupted mitochondria of Ascaris suum.

The incubation of 2-methylcrotonyl-CoA with either succinate or NADH and disrupted Ascaris suum mitochondria results in substantial 2-methylbutyrate formation. Both membrane-bound and soluble components are required and the NADH-dependent reduction is rotenone sensitive, suggesting the involvement of the electron-transport chain. Rat liver mitochondria, incubated under similar conditions, did not catalyze 2-methylbutyrate formation. However, the substitution of A. suum mitochondrial membranes for rat liver membranes stimulated 2-methylbutyrate formation, emphasizing the differences in electron-transport in these two organelles.

Anaerobic metabolism in Ascaris suum: acyl CoA intermediates in isolated mitochondria synthesizing 2-methyl branched-chain fatty acids.

Freshly isolated Ascaris suum mitochondria contained CoASH, acetyl CoA, propionyl CoA, 2-methylcrotonyl CoA, 2-methylbutyryl CoA, 2-methyl-2-pentenoyl CoA, and 2-methylvaleryl CoA, as determined by high-pressure liquid chromatography. Incubation of these mitochondria aerobically in the absence of substrate resulted in the conversion of the branched-chain enoyl CoA's to acetyl CoA and propionyl CoA. With the addition of malate to the incubation medium, succinyl CoA and methylmalonyl CoA accumulated and the levels of propionyl CoA decreased dramatically. However, the branched-chain fatty acids characteristic of A. suum's fermentative metabolism were not formed and it appears that the formation of propionyl CoA may be limiting in these mitochondria. Indeed, the addition of propionate to incubations with malate increased intramitochondrial levels of propionyl CoA and 2-methyl-2-pentenoyl CoA and stimulated significant 2-methylvalerate synthesis. The exclusion of air from these incubations further increased levels of 2-methyl-2-pentenoyl CoA and stimulated 2-methylvalerate synthesis. These studies suggest that in addition to elevated NADH/NAD ratios, elevated enoyl CoA/acyl CoA ratios also are important in the regulation of branched-chain fatty acid synthesis in A. suum mitochondria.

Mechanistic studies in the transcuticular delivery of antiparasitic drugs. II: Ex vivo/in vitro correlation of solute transport by Ascaris suum.

Using live, intact Ascaris suum and a closed perfusion system, the absorption kinetics and tissue distribution of selected radiolabeled permeants were measured to determine the importance of the transcuticular pathway for drug absorption. The data support the conclusions established by previous in vitro transport studies which utilized excised cuticle-hypocuticle tissue preparations. The external surface of A. suum can be breached by drugs and the rate-determining barrier is the lipoidal hypocuticle tissue, provided the permeant is sufficiently small to traverse the aqueous-filled, negatively charged collagen matrix of the cuticle. The ex vivo permeability coefficients of the model permeants for the cuticle-hypocuticle barrier were in good quantitative agreement with the in vitro permeability coefficients. The lipophilic permeants hydrocortisone and p-nitrophenol were preferentially distributed in the gut tissue, whereas the hydrophilic permeant urea was distributed evenly throughout the organism and was extensively metabolized. Ligated and nonligated A. suum showed no significant differences in either uptake kinetics or tissue distribution of the permeants. This indicates that the transcuticular pathway is the major route of drug absorption as compared to oral ingestion.