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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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward.

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., (Copyright (c) 2006 Wiley-Liss, Inc.)

We rediscover and review the brilliant work of Theodore Boveri, over a 100 years ago, on the centrosome of the round worm Ascaris and show how it impacts on our understanding of human fertilization and embryogenesis. Boveri was able to make fundamental predictions on the mechanics of fertilization and the dominant role of the sperm centrosome (Boveri's rule), which is now applicable to most animals. Using advanced digital imaging by light and electron microscopy, we explore centrosomal dynamics during Ascaris fertilization and the first cell cycle during cleavage. Twenty figures are presented in this visual publication. Humans follow Boveri's rule, as do most mammals excluding some rodents, and there is a remarkable similarity of the events of fertilization and cleavage in Ascaris and humans, the latter of which has been documented since 1991. The role of the sperm centrosome (centriole) in egg activation, polarity, embryogenesis, infertility and cancer is discussed. An attempt is made to portray the images Boveri may have visualized, in his painstaking drawings presented in his thesis in 1888. We now know the origins of the centrosome in human somatic cells--predominantly from the sperm cell. The impact of Boveri's work on human development is highlighted in this age of assisted reproductive technology.

Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated alpha- and beta-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that alpha-MSP is 142 Da larger and 0.6 pH unit more basic than beta-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but alpha-MSP formed filaments at lower solvent concentration than beta-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.

A fluorescent derivative of the anthelmintic ivermectin (4''-5,7-dimethyl bodipy proprionylivermectin, referred to hereafter as bodipy ivermectin) was synthesized for an investigation of the distribution of avermectins. Injected into adult Ascaris suum at doses greater than 0.1 micron per worm, it produced a dose-dependent immobilization. Fluorescent microscopy of frozen sections revealed the distribution of the probe in the whole nematode. Staining of collagenase-isolated muscle cells was studied using bath application of bodipy ivermectin. The trypan-blue quenching technique showed that the ivermectin probe was located in the outer monolayer of the muscle membrane. The cytoplasm was not stained. The interpretation of these observations is discussed in view of the known lipophilic nature of avermectins. Staining of the muscle membrane and nerve cord is consistent with the view that avermectins act at these sites. The significance of the hypodermal and lateral line staining is also discussed.

Morphology of the female reproductive system of Ascaris lumbricoides L. was studied in the region starting with the junction between the oviduct and the uterus (O-U) up to the junction of both uterine branches into the vagina with regard to the process of fertilization and formation of egg-shells. In the O-U junction morphology differed in two following sections: a continuous simple squamous up to simple cuboidal epithelium, and simple cuboidal up to columnar epithelium with broad intercellular spaces leading into the lumen of the tubular reproductive organ filled with sperm. The area in the O-U junction zone was found where the wall of the organ was formed by elongated club-shaped cells attached to the common basal lamina by a narrow pedicle. Intercellular spaces thus formed "crypts" which was covered with dilated parts of cells towards the tubular lumen. Crypts were found to be filled with sperm. This area resembles the structure known as the receptaculum seminis where the stored sperm survive. Epithelial cells of the uterus are of cuboidal up to columnar shape with signs of merocrine secretion. In the distal part of the uterus the secretory active cells probably produce viscous secreta allowing the transfer of the eggs towards the vagina. The cells of the uterus wall are elongated and because of their longer axis, they are orientated longitudinally. In centripetal parts, the cell walls do not have contact with each other and form elongated, deep furrows ("canyons") through which the sperm can run against the flow of uterus content up to the junction of the O-U, where they are stored in the spermatheca-like structure. At any time they are disposal for fertilization.

During the process of chromatin diminution, which takes place in all presomatic cells of the early Ascaris embryo, the heterochromatic termini of the chromosomes are lost. Here we show that the newly formed ends of the reduced somatic chromosomes carry tandem repeats of the telomeric sequence TTAGGC. Comparison of a cloned somatic telomere with the corresponding germline chromosomal region revealed that these telomeric repeats are not present at or near the chromosomal breakage site. They are most likely added by a telomerase-mediated event. Chromosomal breakage, which precedes the telomere addition process, takes place within a short, specific chromosomal region (CBR); however, it does not occur at a single locus, but rather at many different sites. Altogether, our data show that chromatin diminution in Ascaris is a complex molecular process that includes site-specific chromosomal breakage, new telomere formation, and DNA degradation.

The process of colonization of Ascaris lumbricoides eggs by the fungus Verticillium chlamydosporium was studied by scanning electron microscopy. The preparations were made by fractionation of egg suspension exposed to the fungus for four days and frozen in liquid nitrogen according to Stĕrba and Milácek (1986). Ovicidal fungus forms an abundant ramifying mycelial network in the area between the eggs. However, egg-shells are penetrated only by some hyphae without any penetration organs produced (simple hyphal penetration). In a liquid medium, after penetration, hyphae inside the eggs rapidly grow among inner structures of egg-shells and on the surface of developing larvae. In the next phase, hyphae colonize the developing larva. The eggs attacked by this fungus remain morphologically unchanged for a long time except the sites of penetration. Verticillium chlamydosporium is a fungus with unique ovicidal properties. It colonizes the eggs of Ascaris lumbricoides at all stages of embryo development and also attacks larvae inside the eggs.

The insoluble cuticlin from the cortical zone of the cuticle of adult Ascaris suum was purified and used to raise antibodies in C57/bl mice. The specificity of the antibodies for the external cortical layer was shown in the indirect immunofluorescence antibody test and by immunoelectronmicroscopy. A very high specificity for the external cortical layer was found. Some cross-reactions with cuticular collagens occurred, and increased after booster immunizations. The anti-cuticlin antibody cross-reacted with the electron-dense layers of the cortical zones of adult Acanthocheilonema viteae and Brugia pahangi. A very weak reaction was found in the cortical zone of adult Onchocerca volvulus. In the cuticles of third stage larvae of all three species mainly epitopes in the cortical zones were labelled. In no case did the anti-cuticlin antibody interact with the outermost surface of the cuticle.

To facilitate biochemical, pharmacological, and biophysical studies on the membrane of the body muscle of Ascaris suum, a method for preparing intact vesicles was developed. Vesicles were prepared by incubating a muscle flap preparation with 1 mg/ml collagenase in a saline solution and then washing in saline without enzyme. The vesicles then formed gradually over the next hour as outgrowths of the original surface membrane from the bag region of the muscle. The vesicles were harvested readily by suction using a Pasteur pipette. The structure of the vesicles was examined with the transmission electron microscope. The whole-cell patch-clamp technique showed that the vesicles had a high input resistance and that the membrane was complete. The vesicle membrane was shown to contain Ca-activated Cl channels and gamma-aminobutyric acid-activated Cl channels. The vesicles also were shown to be suitable for fluorescence recovery after photobleaching studies designed to examine lateral and vertical movement of a lipid probe (5-N [octadecanoyl]-aminofluorescein) in the membrane. This probe had a mean lateral diffusion coefficient (DL) of 8.1 x 10(-9) cm2/sec, but only a proportion (68.4%) of the probe was mobile. The latter observation illustrated the nonuniform nature of the membrane. Ivermectin (10(-7) M) had no effect on DL or percent recovery. Trypan blue quenching experiments showed that the lipid probe remained in the outer monolayer of the membrane. These observations illustrate the experimental value of the vesicles; they are potentially useful in discerning anthelmintic mode of action and in drug screening.

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.

Six subcellular fractions were isolated by differential centrifugation of the homogenate of spermatozoa of Ascaris suum. The cellular constituents of pelleted fractions, as identified by electron microscopy, were membranes and membranous organelles (fraction A1), microsomal (A2), cytoplasmic (A3), large refringent granules (B1), small refringent granules (B2) and a detergent-soluble fraction (B3). Polypeptide analysis of SDS-PAGE showed that the 18,400-dalton band, one of the major spermatozoan proteins, is detectable in all of the fractions. However, the cytoplasmic (A1) and refringent-granule (B1) fractions contained the highest level. The isolated refringent granules consisted of 2-6% lipid while the nonlipid fraction formed an insoluble matrix with a fibrillar network morphology. This fibrillar matrix contained three polypeptides of small molecular weight (7,000-14,000) in addition to the 18,400-dalton polypeptide. These small polypeptides (7,000--14,000 MW) are detectable only in fractions of the refringent granules and are therefore called the refringent-granule proteins (RGP). These RGP are sensitive to tryptic hydrolysis and have solubility properties similar to the protein, ascaridine.

The course of penetration of the ovicidal fungus Verticillium chlamydosporium Goddard through the Ascaris lumbricoides egg-shells was studied in electron miorographs. The contact area between the egg and the fungus is smooth at the stage of the contact of the ovicidal fungus with the egg surface, as well as at the stage of adhesion. No special attaching organs are formed which indicates that no mechanical penetration of the fungal hypha through the egg-shells is involved. Traces of enzymatical activity of the fungus penetrating upright through the egg-shells can be observed during the penetration stage proper. At the consumption stage, the branching of the ovicidal fungus becomes more dense and some hyphae grow through the egg envelopes parallelly under the egg surface. The fungus grows through all parts of the infected egg, even through its contents.

Investigations were carried out into the properties and localization of chitin synthase (EC 2.4.1.16) in connection with the formation of Ascaris suum egg-shells. The chitin synthase of eggs exhibited properties very similar to those found in other chitin-synthesizing organisms. It was stimulated by the presence of ATP, GlcNAc and Mg2+ with maximal activity at pH 7.0. In the phase of active chitin synthesis, during formation of the primary shell layers, chitin synthase was bound to the structures of the eggs. On completion of chitin synthesis in the eggs, the amount of the enzyme increased in the soluble fraction. Two forms of chitin synthase have been found in the eggs, namely an inactive (zymogenic) and an active form. Transformation of these two enzyme forms represents a key mechanism in the regulation of chitin synthesis in A. suum eggs.

Complex II of Ascaris suum mitochondria, which functions as fumarate reductase in physiological conditions, contains three types of iron-sulfur clusters. These correspond to clusters S-1, S-2 and S-3 and are distinguishable by low-temperature ESR studies. Cluster S-1 is reduced by succinate, giving ESR signals with gz, gy and gx values at 2.033, 1.939 and 1.920. The existence of cluster S-2 is suggested by an enhancement of the S-1 spin relaxation induced upon reduction of S-2 by dithionite. Cluster S-3 is ESR detectable under air-oxidized conditions and gives a strong signal at g = 2.025. Cluster S-3 was only partially reduced even with an excess amount of sodium succinate, which is a common characteristic of fumarate reductase but this is not seen in the mitochondrial complex II.

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment. Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO2. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen. In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O2/H2O2 couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e- transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.

The rate of 14CO2 evolution from 1-[14C]pyruvate by intact Ascaris mitochondria was very slow, but increased with increasing concentrations of pyruvate. At all concentrations of pyruvate, in an aerobic environment, pyruvate decarboxylation was stimulated greatly by the addition of fumarate, malate, or succinate. However, under anaerobic conditions, only malate and fumarate stimulated pyruvate decarboxylation; succinate had no effect. This implies that the aerobic metabolism of succinate, presumably to other dicarboxylic acids, may be required for the stimulation. Incubation of sonicated mitochondria with pyruvate plus fumarate, under rate-limiting concentrations of NAD+, resulted in approximately equal quantities of pyruvate utilized and succinate formed, suggesting that pyruvate oxidation and fumarate reduction may be linked. Branched-chain, volatile fatty acids were not formed during incubations with either malate or succinate, or succinate plus acetate. However, incubations of intact Ascaris mitochondria with pyruvate plus succinate yielded 2-methylbutyrate and 2-methylvalerate, whereas incubations with pyruvate plus propionate yielded almost exclusively 2-methylvalerate. Oxygen dramatically inhibited the synthesis of the branched-chain acids from succinate plus pyruvate, attesting to the apparent anaerobic nature of Ascaris mitochondrial metabolism. Significantly, the addition of glucose plus ADP stimulated the formation of all volatile fatty acids. Therefore, the synthesis of branched-chain acids may be related directly to increased energy generation. Alternatively, they may function in the regulatory role of maintaining the mitochondrial redox balance.

Results of various cytochemical tests demonstrate large deposits of glycogen within the intestinal absorptive cells of Ascaris suum. Carbohydrate material is also associated with the microvilli surface and basal lamella. Staining produced by the periodate-thiocarbohydrazide-osmium procedure was abolished by analine or m-aminophenol. Diastase digestion did not alter the staining on the microvilli surface. Similar results were seen using the silver methenamine procedure. A positive reaction was noted on the microvilli surface, vesicles in both the apical and basal cytoplasm, Golgi apparatus, and basal lamella. Lanthanum nitrate stained the microvilli surface and intercellular spaces between absorptive cells. Alcian blue or cetylpyridinium chloride in combination with lanthanum enhanced the staining produced by lanthanum alone. These results suggest the presence of acidic glycans on both the microvilli surface and basal lamella.

The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane. A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.

Under the light microscope the chitin-protein layer of egg-shells in ascarids appears to be a regular, hyaline and nonstructural layer of 1.5 to 2.00 microns in thickness. The outer uterine layer is usually removed during the preparation. The lipid (ascaroside) layer covers the inner surface of the chitinous layer and seems to be irregularly undulated and regularly thick over the whole surface, with the thickness up to 1 micron. In electron micrographs the fibrous structure of the lipid layer is not evident as a rule. This is probably due to washing the lipids away from this layer during the dehydration of deeper layers of egg-shells that are imperfectly fixed with glutaraldehyde. A very low permeability of the egg-shells is typical of geohelminth eggs. The layer lipid shows a distinct lamellate structure only after a prolonged fixation with osmium at higher temperature. This is supported by the studies using the method of freeze-fracturing.

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.

Disrupted Ascaris mitochondria formed 2-methylbutyrate (2-MB) and 2-methylvalerate (2-MV) when incubated anaerobically with acetyl CoA, propionyl CoA and NADH. However, when mitochondrial membranes were removed by high speed centrifugation and the mitochondrial soluble fraction was incubated with the same substrates, 2-methylcrotonate (tiglate) and a compound tentatively identified as 2-methyl-2- pentenoate accumulated rather than 2-MB or 2-MV. These data suggest that the terminal reduction of the unsaturated intermediates to the saturated 2-MB and 2-MV was catalyzed by an enzyme system at least partially bound to membranes. This supposition was further supported by the findings that disrupted Ascaris mitochondria also formed 2-MB and lesser amounts of 2-MV when incubated with tiglyl CoA plus NADH, and both soluble and membrane-bound components appear to be involved in this reduction. The possibility that electron transport associated ATP synthesis may be coupled to these reductions remains to be examined.

On microphotographs the morphological peculiarities of the eggs of Ascaris lumbricoides are demonstrated. Diagnostic problems caused by these anomalies are discussed. Study of the variations of this parasite can contribute to a better understanding of the morphology, ecology and biology of intestinal parasites.

Twelve synaptonemal complexes are present in both oocyte and spermatocyte pachytene nuclei of Ascaris lumbricoides var. suum, as determined by 3-D reconstruction of the nuclear contents from electron microscropy of serial sections and therefore, n=12 in the strain of Ascaris described here. In the female the heterochromatic end of each synaptonemal complex is attached to the nuclear envelope and the other end is free in the nucleoplasm. In the male neither end of the synaptonemal complex is attached, but there is a heterochromatic knob at one end of each complex. Five additional large heterochromatic masses are present in the spermatocyte nucleus and these may be the sex chromosomes described by earlier workers.

The fixation of mercuric ions on the uterin layer of the Ascaris lumbricoides egg wall is proved by light microscopic histochemical technics and electron microscopic microanalysis. These observations support the hypothesis of a possible reaction between the egg surface components and those of the dilution reagents. This reaction is used in the methods of concentration in coprology. In the case of mercuric salts, the free thiol components which are missing in all the Ascaris egg wall layers are not the factors of the reaction.

The body muscle cells of the nematode Ascaris lumbricoides are characterized by massive amounts of intermediate filaments (IF). These occur in all three regions of this giant cell type. They traverse the cytoplasm of the balloon-like belly, which houses the nucleus, and occur as bundles in the arm-like extensions to the nerve. The organization of IF in the third region, the contractile fiber, was analyzed further by serial sections and three-dimensional reconstruction. IF bundles traverse the glycogen-rich lumina of the fiber and reach as baskets around the sarcomeres. Together with numerous dense bodies they form the Z-band-like arrangements. IF bundles reach the plasma membrane at hemidesmosome-like specializations often situated at deep membrane invaginations filled with a fibrillar component of the extracellular matrix. The ultrastructural appearance of IF bundles is connected to the contractional state of the sarcomeres. They appear straight in extended muscle but coil up upon contraction. In the pharynx massive IF bundles are oriented longitudinally. A second type of IF bundles follows the radially oriented sarcomeres. These reveal pronounced Z-band type structures with massive disks. IF surround the sarcomeres and seem to terminate at these disks. We discuss possible functions of the complex IF organization in body muscle and pharynx.

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.

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.

Ascariasis is considered to be one of the rare infectious diseases in Japan, but recently it has been slightly increasing. This paper reports three ascariasis cases who seemed to be infected recently in the Kitakyushu area, Japan. Case 1: A 59-year-old woman in Kitakyushu City passed a round worm after continuous abdominal pain. The patient was discharged from the hospital because of no further abnormal intestinal symptoms and findings. Case 2: An 85-year-old woman in Nakama City, who suffered from cerebral infarction, vomited a round worm before hospitalization. Many ascarid eggs were detected after admission, and after treatment with pyrantel pamoate (Combantrin) two round worms were passed and egg detection became negative. Case 3: A 77-year-old man in Saikawa Town vomited 3 round worms after gastrectomy due to early gastric cancer. Many unfertilized eggs were also detected from the stool together with hook worm eggs, but no eggs were found after administration of pyrantel pamoate. Morphological examination was made by a scanning electron microscope on the denticles on the dentigerous lip ridges of the worms to differentiate from possible infection with a pig parasite, Ascaris suum. The three cases were diagnosed as ascariasis due to human Ascaris lumbricoides based on the following evidences that the expelled worms had 1) less pointed tips of the denticles and shallower or wider interdenticle notches, and 2) far more denticles of smaller size along the dentigerous ridges, compared with Ascaris suum. The necessity of differentiating pig- from human-ascarids, when considering human infection with Ascaris suum, is discussed.

The cuticle of the parasitic roundworm Ascaris lumbricoides suum has been found to contain apparent pores, using scanning and transmission electron microscopy. However, X-ray spectral analysis and dot mapping analysis of diffusing divalent cations in the cuticle found these ions to be randomly distributed on the pseudocoelomic surface of the cuticle. This indicates that the pores seen with the electron microscope were not true structural pores and that the cuticle is homogeneously permeable to ions.

Accessory nuclei were observed in early meiotic prophase (leptotene) in female Ascaris suum but such structures were absent in meiosis in males. Accessory nuclei also were observed during the prediminution embryonic divisions. All embryos did not have accessory nuclei, thus, those embryos with these structures may be female since the accessory nuclei are maternal-limited. Accessory nuclei were not observed after the fourth embryonic division, at which stage the process of chromatin diminution begins. The eliminated chromatin may be composed in part of DNA from accessory nuclei.

Cells comprising the oviduct-uterine junction of the female reproductive tract in Ascaris suum were shown to have a distinctive ultrastructure. The free luminal surface of each epithelial cell was bordered by an electron-dense layer that measured 90 to 120 nm in thickness and could be removed by treatment with pronase. Numerous membrane-bound, filament-containing bodies were present in the cytoplasm near the apical margin. These filaments, measuring 6.5 nm in diameter, coalesce (or condense) to form a larger coiled, or homogeneous, electron-dense substance. These membrane-bound, filament-containing bodies often fused with the plasma membrane and released their contents into the lumen. In other instances, the filamentous arrangements were not membrane bound and remained within the cytoplasm where they formed electron-dense masses adjacent to the plasma membrane. Basal infoldings suggestive of water or ion transport occurred in each cell. Externally, the cells were bordered by a basement lamina and muscle cells.

1. Enzymes hydrolysing acetylsalicylic acid were found in the cytosol of the cestode, Moniezia expansa, and in the cytosol of the intestinal epithelial cells and cytosol of the reproductive tract of the nematode, Ascaris lumbricoides var suum. 2. Enzymes hydrolysing 2-naphthyl acetate and 4-methylumbelliferyl acetate were found throughout the proglottid of the cestode and in the reproductive tract, intestine, mesenchyme fluid and cuticle of the nematode. These enzymes had mol. wt. of 30 000-300 000 whereas those hydrolysing acetylsalicylic acid in both species had mol. wt. of about 87 000. 3. The acetylsalicylic acid hydrolases from both helminths showed pH optima of about 7.0, and activity was enhanced by Ca2+ and low-mol. wt. thiols. Cu2+, Cd2+, Hg2+, Zn2+, La3+, F- and EDTA at 1 mM inhibited activity. N-Ethylmaleimide, p-chloromercuribenzoate, haloxon and paraoxon also inhibited hydrolase activity.

The ultrastructural changes in the intestines of Ascaris suum and Syngamus trachea, induced by in vivo treatment of the hosts with the anthelmintic mebendazole, are reported. The primary site of drug action seemed to be the organelles involved in the secretory mechanism of the intestinal cells. The block in the transport of secretory granules and in the movement of other subcellular organelles coincided clearly with the disappearance of cytoplasmic microtubules. On the other hand, the microtubular system of the host cells was unaffected by the treatment. Degenerative changes in the intestinal cells of the parasites observed afterwards were correlated with the primary deteriorative effect of the drug on cytoplasmic microtubules.

Triglyceride utilization is correlated with glycogen resynthesis in developing Ascaris suum larvae. Glycogen content, determined by amyloglucosidase hydrolysis at 2-day intervals, decreases sharply during embryonation from 15% (per mg dry weight) at day 4 to 2% at day 12. Thereafter glycogen increases 3-fold through day 20, marking the resynthesis period. Triglyceride lipid droplets are confirmed primarily to the posterior half of the larvae and mark the site of the interconversion. Although they serve as precursors to glycogen, only a small diminution was observed ultrastructurally. Glycogen accumulation, on the other hand, correlates well with increases determined biochemically. alpha-glycogen builds up among lipid droplets, while beta-glycogen concentrates in the cytoplasm of somatic muscle cells paralleling myofibril development. Dense granules, restricted to the lipid body region, are considered as the possible subcellular site for the enzymatic conversion.