[Identification and cytochemical study of an extranuclear basic protein in the spermatozoa of decapod crustaceans]. / Mise en evidence et etude cytochimique d'une proteine basique extranucleaire dans les spermatozoides des crustaces decapodes.

Extranuclear basic proteins have been detected in the capsule of the spermatozoa of three species of decapod crustaceans (Nephrops norvegicus L., Macrura; Eupagurus bernhardus L., Anomura; Carcinus maenas Penn., Brachyura). Their properties have been studied by cytochemical methods. Their position inside the capsule of the spermatozoon has been specified with the aid of the electron microscope. Present in a constant fashion in the three species cited, their relative importance is very variable. In contrast to the refringent cone of the spermatozoon of Ascaris, which contains an acid protein, ascaradine, the capsule of the spermatozoon of the three decapod crustaceans studied contains basic proteins which we propose to designate by the general term "decapodine".

AF1, a sequenced bioactive neuropeptide isolated from the nematode Ascaris suum.

An FMRFamide-like neuropeptide, named AF1, was isolated from head extracts of the nematode Ascaris suum using five steps of HPLC. AF1 is a heptapeptide with the amino acid sequence Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2. Synthetic AF1 (10(-9) to 10(-7) M) rapidly and reversibly abolished slow membrane potential oscillations of identified ventral and dorsal inhibitory motoneurons and selectively reduced their input resistances. Synaptic transmission was not blocked. In intact Ascaris, AF1 inhibited locomotory movements. This study indicates a potential physiological role for an endogenous neuropeptide in nematodes.

Studies on the subunits of myosin from muscle layer of Ascaris lumbricoides suum.

1. A purified preparation of Ascaris myosin was obtained from the muscle layer of Ascaris lumbricoides suum, using gel filtration and ion-exchange chromatography. 2. Ascaris myosin whether purified or unpurified, had almost the same ability for ATP-splitting and superprecipitation. 3. Ascaris myosin and rabbit skeletal myosin were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A significant difference in the number of light chains between both myosins was found. Ascaris myosin was found to have one heavy chain and two distinct light chain components (LC1-A and LC2-A), having molecular weights of 18000 and 16000, respectively. These light chains correspond in molecular weight to the light chain 2 (LC2-S) and light chain 3 (LC3-S) in rabbit skeletal myosin. 4. LC1-A could be liberated from the Ascaris myosin molecule reacted with 5,5'-dithio-bis(2-nirobenzoic acid( Nbs2) with recovery of ATPase activity by addition of dithiothreitol. These properties are equivalent to those of the LC2-S in rabbit skeletal myosin, although Ascaris myosin when treated with Nbs2-urea lost its ATPase activity.

GABA-immunoreactive neurons in the nematode Ascaris.

gamma-Aminobutyric acid (GABA) immunoreactive neurons in the cephalic, somatic, and caudal regions of the Ascaris nervous system were visualized with serial section and whole-mount GABA immunocytochemistry. In the ventral and dorsal nerve cords, GABA-like immunoreactivity (GLIR) is localized to the neurites and cell bodies of identified inhibitory motor neurons and to two fibers, one in each cord, that arise from neurons in the nerve ring. GLIR is absent from identified excitatory motor neurons and from ventral cord interneurons. In neurons containing GLIR, immunoreactivity was present throughout the cell, which argues against an exclusive localization of GABA at conventional synapses. In whole mounts, ten GABA-immunoreactive neurons were present in the cephalic region. These include four nerve ring-associated cells (the RME-like cells), two bilaterally symmetrical pairs of lateral ganglia neurons (the amphid-GABA and deirid-GABA cells) and one bilaterally symmetrical pair of ventral ganglion cells (the VG-GABA cells). In sections, the RME-like cells and the VG-GABA cells were consistently stained through the cephalic region. However, anti-GABA staining of the lateral ganglia cells in sections was light, thus suggesting that they contain less GLIR than the other more intensely stained GABA-immunoreactive neurons. In the caudal region, a single GABA-immunoreactive neuron was present in the dorsal rectal ganglion. Our data suggest that these ten cephalic neurons, and a single dorsal rectal ganglion neuron, use GABA as a neurotransmitter.

A versatile dot-ELISA method with femtomole sensitivity for detecting small peptides.

Several protocols for conjugating peptides in situ to a protein carrier on paper, nitrocellulose, or nylon membranes were explored for their usefulness in dot-ELISA detection of the peptides. The most sensitive method in which peptide diluted in bovine serum albumin is applied to nitrocellulose, then fixed with glutaraldehyde, can detect several peptides, ranging from 4 to 38 amino acids in length, at the level of 2-10 fmol. Both immunohistochemical grade antisera and monoclonal antibodies have been used successfully. The method may be a useful alternative to radioimmunoassay since there is no requirement for radiolabelled peptide, or (for quantitation) for known quantities of unlabelled peptide. The method has been used to monitor, semiquantitatively, the fractionation of FMRFamide-like or CCK-like peptides from the nematode Ascaris, and to detect peptide-like immunoreactivities in tissue extracts.

Identity and tissue localization of free and conjugated ecdysteroids in adults of Dirofilaria immitis and Ascaris suum.

Adult males and females of the dog heartworm, Dirofilaria immitis, and of the swine parasite, Ascaris suum, were extracted, the free and polar conjugated ecdysteroid fractions separated and the latter hydrolysed enzymically. The ecdysteroids released by hydrolysis of the conjugates and the free hormones were analysed by radioimmunoassay, high-performance liquid chromatography on reversed phase and adsorption columns monitoring fractions by radioimmunoassay, and by gas-liquid chromatography/mass spectrometry (selected ion monitoring). In both species, males and females contained free and polar conjugated ecdysteroids, with evidence for the presence primarily of ecdysone and 20-hydroxyecdysone together with smaller amounts of 20,26-dihydroxyecdysone. Males and females of both species were then dissected into body fluid, reproductive system, gut and remaining body wall compartments, the ecdysteroids extracted, fractionated and analysed by radioimmunoassay and high-performance liquid chromatography monitoring fractions by radioimmunoassay. The results for both sexes in the two species were similar and indicated that ecdysteroids were not detectable in body fluids and that free ecdysteroids occurred in the reproductive system and the body wall, whereas polar conjugated ecdysteroids were detected in the reproductive system and the gut; a minor portion of the free ecdysteroids in A. suum was also apparently present in the gut. Further localization of the ecdysteroids in the body wall of A. suum females suggested that negligible immunoreactivity was associated with the circumpharyngeal nerve ring. The possible significance of the results is discussed.

Biotin as a probe of the surface of Ascaris suum developmental stages.

Sulfo-NHS-biotin (aqueous soluble) and NHS-biotin (organic soluble) labeled similar SDS-2ME (sodium dodecyl sulfate/beta-mercaptoethanol) soluble cuticular proteins of second stage larvae (L2) and third stage Ascaris suum larvae (L3). Comparable analysis of biotin-labeled fourth stage larvae (L4), young adults, and mature adult Ascaris suum revealed strong labeling of several SDS-2ME soluble cuticular proteins with NHS-biotin, while sulfo-NHS-biotin appeared to strongly label a single SDS-2ME soluble cuticular protein. Both biotin probes labeled only cuticular proteins, since no evidence of internal labeling was observed in any developmental stage examined by either electroblot analysis or by electron microscopy. Our data suggest a greater cuticular permeability to the organic soluble biotin reagent in the later developmental stages (greater than L3) of A. suum than to the aqueous soluble biotin reagent, and may indicate the presence of a hydrophobic barrier in the cuticle of the later stages of the parasite.

Electrophoretic analyses of myofibrillar proteins from the body wall muscles of Ascaris suum.

A myofibrillar protein extract has been isolated from the muscle of Ascaris suum. Two-dimensional electrophoresis of this extract revealed that the myosin light chain 1 (ALC1) migrates as 3 components with approximate isoelectric points in the range of 5.3-5.6. The most acidic component of ALC1 appeared to be phosphorylated when the myofibrillar extract was incubated for 10 s with catalytic subunit of cAMP dependent protein kinase and [gamma-32P] ATP. The myosin light chain 2 (ALC2) migrated as a single component in isoelectric focusing with an approximate isoelectric point of 5.5 Actin was resolved into 2 components with identical molecular weight but isoelectric points differing by approximatley 0.2 pH units. A protein was tentatively identified in the myofibrillar extract as tropomyosin. It migrated as a single band with an approximate isoelectric point of 5.0 and a molecular weight of 39 000. None of the troponin components could be identified in the myofibrillar extract. It is postulated that muscle contraction in A. suum muscle could be controlled by phosphorylation of myosin.

Tissue and ultrastructural localization of 5-hydroxytryptamine (serotonin) in the tissues of Ascaris suum with energy dispersive X-ray spectrometry of immunoreactive structures.

Muscle, hypodermis and gastrointestinal epithelial cells from adult female Ascaris lumbricoides var. suum were found to contain serotonin based upon glyoxylic acid induced histofluorescence and indirect immunolabeling with an antiserotonin monoclonal antibody conjugated to protein A-colloidal gold. Histofluorescence indicated that muscle-hypodermis and intestinal epithelial cells contained significant concentrations of 5-hydroxytryptamine while fluorescence was absent in the nerve cord and cuticle. Immunolabeling at the ultrastructural level indicated that serotonin was sequestered in electron-opaque patches, dense vesicles and mitochondria of the muscle-hypodermis and intestinal tissue. Perfusion of whole worms and isolated tissues with 10(4) M-serotonin further indicated: (1) immunolabeled patches and dense vesicles were often associated with cytoskeletal elements, (2) serotonin did not appear to enter the intestinal or muscle cells by endocytosis, (3) immunolabeled patches examined with energy dispersive X-ray spectrometry (X-ray microanalysis) were found to contain iron at concentrations approximately double that of the surrounding cytoplasm.

Trypsin inhibitors from Ascaris: the reactive P1 site of the inhibitors (a correction) and location of the inhibitors and host trypsin in cross-sections of Ascaris.

Ascaris trypsin inhibitors 1, 2, and 3 have arginine at their reactive P1 site. This corrects an earlier report that lysine is the reactive P1 site residue in Ascaris trypsin inhibitor 1 (Peanasky et al., 1974, Bayer Symposium V: Proteinase Inhibitors, pp. 649-666). The present work illustrates that the residue modification method of Fritz et al. (1969, Z. Physiol. Chem., 350, 933-944) may not be reliably interpreted when trypsin inhibitors have an unusually high lysine content (greater than 12% of the molecular weight of the inhibitor). Thus the following procedure is recommended: treat the inhibitor with maleic anhydride first and second with butanedione reagent; then remove the maleyl groups in an acid environment and determine the activity of the inhibitor. Immunoperoxidase staining shows that antibody to Ascaris trypsin inhibitor 1 binds to body wall muscle, intestine, eggs and sperm in cross-sections of Ascaris. Antibody to TLCK-porcine trypsin binds to the same tissues and at the same sites as the antibody to Ascaris trypsin inhibitor 1. This is the first demonstration that a protein that originated in the host has been found in the parasite, Ascaris. Analyses of homogenates and of extracts of separated tissues always show an excess of free trypsin inhibitor and no evidence of active trypsin. The host protein is present inside the parasite, probably as the trypsin-inhibitor complex.

Analysis of the cuticular collagens of Ascaris suum.

The nematode cuticle is an extracellular structure composed mainly of collagens, with an insoluble epicuticle on the surface. The extracted collagens from adult Ascaris suum can be separated by SDS-PAGE into three major groups of polypeptides with apparent molecular masses of 34, 60-70 and 120-140 kDa. Densitometric evaluation of the polypeptide bands indicated that the three groups are present in the ratio of 1:2:6. Rotary shadowing of reduced, extracted molecules showed fibers 45 nm in length. This length is in excellent agreement with the calculated total length of amino acids in (Gly-X-Y) regions deduced from the collagen gene sequence of Caenorhabditis elegans and A. suum. It is proposed that the three groups of collagen polypeptides found in SDS-PAGE correspond to collagen monomers, dimers and trimers, and that the molecules in the dimeric and trimeric forms are cross-linked via non-reducible bonds.

The cuticular biology in developmental stages of Ascaris suum.

The cuticles from distinct developmental stages of Ascaris suum were isolated by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with SDS and 2-mercaptoethanol (2ME) and analyzed by PAGE. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by bacterial collagenase, suggesting that these proteins are collagen-like structural elements of the cuticle. The soluble proteins from the L2 differed from the adult and other larval stages in both the number and molecular weight of protein bands and their lack of collagenase sensitivity. Antibodies made against the soluble cuticular proteins reacted with the medial and basal layers of the cuticle but not the external cortical or epicuticular regions. A significant amount of the cuticle was not solubilized by 2ME and was not digested by bacterial collagenase. These insoluble cuticular proteins were probably derived from the epicuticular and external cortical regions of the cuticle. Different developmental stages of A. suum were biotinylated and examined by electron microscopy. An organic soluble biotin reagent labeled all stages in a transcuticular pattern, while an aqueous soluble biotin labeled only the external cortical and epicuticular regions of the L4 and adult cuticle. These data indicate the presence of a hydrophobic barrier in the cuticle of later stages of the parasite.

Eosinophilia and mast cell hyperplasia induced by parasite phospholipid.

Phospholipid preparations from the nematode Ascaris suum or cysts of Echinococcus granulosus (hydatid cysts) induced an eosinophilia when injected into the peritoneal cavity of rats. Peritoneal eosinophilia persisted throughout 21 days of daily injections of Ascaris lipid and was accompanied by blood eosinophilia, mast cell granule lysis and mast cell hyperplasia. The active material consisted of lecithin and lecithin plasmalogen, and in aqueous suspension had a membrane-like appearance. Electron microscopy revealed that the phospholipid was ingested by all types of cells in the peritoneal cavity, including mast cells, and was rapidly broken down by eosinophils. Phagocytosis was found to be complement dependent. The lipid combined with properdin in human serum and stimulated complement breakdown via the alternative complement pathway.

Attenuation of rat conjunctival response by repeated hapten applications.

Attenuation of the rat conjunctival response by repeated topical challenge with dinitrophenyl (DNP) hapten was demonstrated in our study. Adult rats were immunized by intraperitoneal injections of dinitrophenylated Ascaris suum extract (DNP-Asc) and alum. Serum levels of anti-DNP homocytotropic antibody were determined by passive cutaneous anaphylaxis in rats prepared with antibody 48 hours earlier. In other animals, topical challenge was performed by applying N,N'-di-2,4-DNP-L-lysine (di-DNP-lysine) in phosphate-buffered saline (PBS) to one eye; PBS alone was applied to the fellow eye. The degree of conjunctival reaction was assessed clinically, and ocular tissues were processed for histological evaluation. The intensity of the conjunctival reaction and extent of mast cell degranulation were significantly greater after one challenge with di-DNP-lysine than after multiple challenges. In the multiple-challenge group, the contralateral eye remained responsive to a single challenge with di-DNP-lysine. These results may have implications for therapeutic interventions in ocular anaphylaxis.

The bacterial flora of the intestine of Ascaris suum and 5-hydroxytryptamine production.

Representative facultative anaerobes of the bacterial flora from the intestine of female Ascaris suum were isolated and identified. The number of bacteria in the intestine was approximately 4 X 10(9) per g wet weight of intestine. Seventeen of 19 of the isolated colonies were found to secrete 5-hydroxytryptamine in culture. Holding A. suum in an antibiotic-containing medium did not affect the levels of 5-hydroxytryptamine in the worm, which were 231 +/- 14 ng/g in antibiotic-media as compared to 250 +/- 16 ng/g in control media. This implied that the bacteria may not be contributing to the level of 5-hydroxytryptamine in the tissues of A. suum.

Structural analysis of the calcium-binding protein calmodulin from Ascaris suum obliquely striated muscle.

Calmodulin was purified from the obliquely striated skeletal muscle of Ascaris suum. The calmodulin had a molecular weight of 16,400 and the amino acid composition indicated it is highly similar to other purified calmodulins, showing insignificant variation in 12 of 17 residues. In the residues that showed variation, a trend towards conservative substitution was observed. Spectrophotometric absorption maxima of 276 nm and 283 nm were observed. A molar absorption coefficient of 7,800 was calculated. Calcium-dependent binding to phenothiazine Affi-Gel confirmed that calcium binding induces conformation changes characteristic of calmodulin. Double reciprocal analysis of phosphodiesterase activation by A. suum calmodulin gave a Kapp of 40 nM.

Inhibition of human blood clotting by extracts of Ascaris suum.

The effects of soluble extracts of Ascaris suum on human blood coagulation were investigated. Whole worm supernatant solution prolonged the whole blood clotting time and the kaolin-activated, partial thromboplastin time but it did not alter the prothrombin time. These data indicate impairment of the intrinsic pathway of blood coagulation. Whole worm supernate inhibited platelet aggregation induced by ADP and ristocetin. This supernate did not affect fibrinolysis. The maximal concentration of anticoagulant activity was found in the pseudocoelomic fluid of the worm. Activity was also noted in the cuticle and secretory/excretory products. Perhaps inhibition of blood clotting by helminths may facilitate their passage through the blood stream.

Ultrastructural changes in Ascaris suum intestine after mebendazole treatment in vivo.

The effect of in vivo treatment with mebendazole on the ultrastructural morphology of Ascaris suum intestine was investigated. Pigs, infected with A. suum, were fed ad libitum a medicated food containing mebendazole at a concentration of 30 ppm. Control and treated animals were killed 6, 9, 15, and 24 hr after the onset of feeding. The parasites were quickly collected from the pig intestinal tract and prepared for ultrastructural and cytochemical examination. Absence of secretory granules in the terminal web, accumulation of secretory granules in the Golgi region, formation of autophagic vacuoles in the apical cell part, and loss of glycogen were the characteristic changes observed after 6 and 9 hr of treatment. Degenerative changes were very pronounced after 15 and 24 hr and involved almost the entire cytoplasm. Microvilli were decreased in number and appeared swollen in the majority of absorptive cells. Some more severely altered cells were completely devoid of microvilli. Cytochemistry revealed that the accumulated secretory granules in the Golgi area contained glycoproteins or polysaccharides. Microvilli, lysosomes, and Golgi apparatus were reactive for acid phosphatase in the control intestinal cells. After treatment, the enzyme activity was localized in numerous autophagic vacuoles, whereas the secretory granules remained unstained. The acid phosphatase activity in the microvilli decreased or was completely absent. The possible significance of these modifications in view of mebendazole's anthelmintic activity is discussed.

Studies on species specificity of proteins in Ascaris lumbricoides and Ascaris suum.

The separate identity of A. lumbricoides and A. suum was confirmed by means of the disc electrophoresis method in that differences were established in the protein profile of somatic and ribosomal proteins. In addition differences were found in the representation of lipoproteins, glycoproteins, and in the activity of LDH, SDH, peroxidase, esterase and alkalic phosphatase.