Ultrastructural observations on the germ line of Xenopus laevis

Summary The development of the male germ line in Xenopus laevis has been examined by electron microscopy. Findings have been compared to the parallel process in the female. Three structures unique to the germ line were found in both male and female cells: a fibrillar nuclear region free of DNA; largely proteinaceous masses of nuage material; and a chromatoid body. Germ plasm bodies of the egg and early embryo appear to represent a form of nuage material. The finding of a structure which can be identified as a chromatoid body in the female germ line is unique, as is its presence in sexually undifferentiated primordial germ cells. The chromatoid body in Xenopus, unlike that in mammals, does not persist in the spermatozoon. Instead, it dissociates into a series of coated vesicles during spermatogenesis. The chromosomal ultrastructure of meiotic prophase stages in Xenopus is similar in both sexes until diplotene, when male bivalents condense and enter meiotic metaphase instead of entering the extended lampbrush stage characteristic of the female. The multiple nucleoli present in gonia are lost at the onset of meiotic prophase, but no obvious mechanism for this process was observed.The author would like to thank Drs. Joseph Gall and Bernard Tandler for their helpful suggestions during the course of this investigation. The author is a postdoctoral fellow of the National Institutes of Health, U.S.A. This research was supported by N.I.H. Grants 51823 and 12427.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary 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.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda)

Summary A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37–39° C under anaerobic conditions (95% N₂, 5% CO₂). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5–15 min, is totally inhibited at 4° C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22–9° C. Activation also requires favorable pH (6.8–8.7) and continual exposure to sufficiently high sodium concentrations (134–154mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 g/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.Adult Ascaris suum were provided through the generosity of Wilson and Company, Cedar Rapids, Iowa, U.SA. This study was supported by grant number 5T01 HD00152 and postdoctoral fellowship 1F 32AI05646 from the National Institute of Health, U.S.A.     

LTCI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression

A novel chymotrypsin inhibitor of the potato I protease inhibitor family from the earthworm Lumbricus terrestris was purified. The inhibitor, named LTCI, was isolated by methanol extraction, affinity chromatography on immobilized methylchymotrypsin, and ion exchange chromatography followed by RP–HPLC. The 7076 Da inhibitor consists of a single polypeptide chain of 64-amino-acid residues without disulfide bridges. LTCI is the first of the potato I protease inhibitors with Tyr in position P1 of the reactive site. cDNA analysis revealed that LTCI is produced as a 86-amino-acid precursor with a 22-amino-acid secretory signal peptide. RT–PCR analysis demonstrates that LTCI mRNA is expressed in body wall, intestine, and coelomocytes. The recombinant LTCI was produced in Escherichia coli as a fusion protein with intein and chitin binding domain using IMPACT™–CN system.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

A Cl channel in Ascaris suum selectivity conducts dicarboxylic anion products of glucose fermentation and suggests a role in removal of waste organic anions

The permeability of organic anions (produced from anaerobic fermentation of glucose) through a nonselective membrane Cl channel was examined. Single channel recording techniques were used to study the permeabilities of the anions: oxalate, succinate, oxaloacetate, malate, lactate and pyruvate in Ascaris muscle cell membranes. All of the anions, except malate, were found to be conducted through the channel. The relative permeability of most anions could be predicted from the component structure of the anions. The failure of the channel to conduct malate prevents an energy drain on the cell. These studies further the hypothesis that a Cl channel functions to transport waste organic anions across the cell membrane. This mechanism does not require specific exchange carriers for the anions. Channels with properties like the nonselective anion channels of Ascaris, are suitable for transport of carboxylic anions through cell membranes, down a concentration or potential gradient.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.).     

Mutagenic profiles of carbazole in the male germ cells of Swiss albino mice

Mutagenic effect of carbazole was evaluated by employing dominant lethal mutation and sperm head abnormality assays in male Swiss albino mice. For the dominant lethal mutation assay, adult male mice were treated for five consecutive days either with 30 or 60 mg/kg body weight (b.w.) of carbazole by single intraperitoneal (i.p.) injection. For the sperm head abnormality assay mice were treated with 50, 100, 150, 200 and 300 mg/kg b.w as a single i.p. injection. Treatment of adult male mice with carbazole resulted in induction of dominant lethal mutation and abnormal sperm heads. The results show that carbazole is mutagenic in male germ cells of mice.     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

Effects of Disinfectants on Larval Development of Ascaris suum Eggs

The objective of this study was to evaluate the effects of several different commercial disinfectants on the embryogenic development of Ascaris suum eggs. A 1-ml aliquot of each disinfectant was mixed with approximately 40,000 decorticated or intact A. suum eggs in sterile tubes. After each treatment time (at 0.5, 1, 5, 10, 30, and 60 min), disinfectants were washed away, and egg suspensions were incubated at 25˚C in distilled water for development of larvae inside. At 3 weeks of incubation after exposure, ethanol, methanol, and chlorohexidin treatments did not affect the larval development of A. suum eggs, regardless of their concentration and treatment time. Among disinfectants tested in this study, 3% cresol, 0.2% sodium hypochlorite and 0.02% sodium hypochlorite delayed but not inactivated the embryonation of decorticated eggs at 3 weeks of incubation, because at 6 weeks of incubation, undeveloped eggs completed embryonation regardless of exposure time, except for 10% povidone iodine. When the albumin layer of A. suum eggs remained intact, however, even the 10% povidone iodine solution took at least 5 min to reasonably inactivate most eggs, but never completely kill them with even 60 min of exposure. This study demonstrated that the treatment of A. suum eggs with many commercially available disinfectants does not affect the embryonation. Although some disinfectants may delay or stop the embryonation of A. suum eggs, they can hardly kill them completely.     

The Ca-activated chloride channel of Ascaris suum conducts volatile fatty acids produced by anaerobic respiration: A patch-clamp study

Plasma membrane vesicles prepared from the bag region of the somatic muscle cell of the parasite Ascaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of carboxylic acids, a number of which are products of anaerobic respiration, was investigated using the patch-clamp technique and isolated inside-out patches of muscle membrane. The channel has a conductance of 140 pS in symmetrical 140 mm chloride. Replacement of internal chloride with various carboxylic acids (140 mm) caused large hyperpolarizing shifts in the reversal potential. Permeability ratios, relative to chloride, were calculated for each acid. The relationship between permeability ratio and ionic size is inverse and linear predicting a pore diameter of 6.55 Å. This simple relationship was not observed between ionic size and conductance. Calculation of the transition state energy required to transfer a single methyl group from aqueous phase to the binding site afforded a value that was low but favorable, indicating a cationic binding site of low field strength. As the channel is able to open fully at the resting membrane potential of Ascaris and is permeable to fatty acids produced by anaerobic respiration, the possible role of this channel in the removal of metabolic products across the muscle membrane is discussed.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.). M. Valkanov was sponsored by The British Council.     

Effects of Some Pesticides on Development of Ascaris suum Eggs

To evaluate the effects of pesticides to parasite eggs, Ascaris suum eggs were incubated with 5 different pesticides (1:1,500-1:2,000 dilutions of 2% emamectin benzoate, 5% spinetoram, 5% indoxacarb, 1% deltamethrin, and 5% flufenoxuron; all v/v) at 20℃ for 6 weeks, and microscopically evaluated the egg survival and development on a weekly basis. The survival rate of A. suum eggs incubated in normal saline (control eggs) was 90±3% at 6 weeks. However, the survival rates of eggs treated with pesticides were 75-85% at this time, thus significantly lower than the control value. Larval development in control eggs commenced at 3 weeks, and 73±3% of eggs had internal larvae at 6 weeks. Larvae were evident in pesticide-treated eggs at 3-4 weeks, and the proportions of eggs carrying larvae at 6 weeks (36±3%-54±3%) were significantly lower than that of the control group. Thus, pesticides tested at levels similar to those used in agricultural practices exhibited low-level ovicidal activity and delayed embryogenesis of A. suum eggs, although some differences were evident among the tested pesticides.     

Effects of kimchi extract and temperature on embryostasis of Ascaris suum eggs

To determine the effects of kimchi extracts at different temperatures on larval development, Ascaris suum eggs were mixed with soluble part of 7 different brands of commercially available kimchi and preserved at either 5℃ or 25℃ for up to 60 days. A. suum eggs incubated at 25℃ showed marked differences in larval development between kimchi extract and control group. While all eggs in the control group completed embryonation by day 21, only 30% of the eggs in the kimchi extract group became embryonated by day 36 and about 25% never became larvated even at day 60. At 5℃, however, none of the eggs showed larval development regardless of the incubation period or type of mixture group. To determine the survival rate of A. suum eggs that showed no embryonation after being preserved at 5℃, eggs preserved in kimchi extracts for 14, 28, and 60 at 5℃ were re-incubated at 25℃ for 3 weeks in distilled water. While all eggs in the control group became larvated, eggs in the kimchi extract group showed differences in their embryonation rates by the incubation period; 87.4 % and 41.7% of the eggs became embryonated after being refrigerated for 14 days and 28 days, respectively. When refrigerated for 60 days, however, no eggs mixed in kimchi extract showed larval development. Our results indicate that embryogenesis of A. suum eggs in kimchi extract was affected by duration of refrigeration, and that all eggs stopped larval development completely in kimchi kept at 5℃ for up to 60 days.     

Ascaris suum: immunoglobulin responses in mice

The immune response in mice to infection with Ascaris suum was characterized by determining (1) changes in serum immunoglobulin levels and (2) changes in the relative proportions of immunoglobulin-containing cells in major lymphoid tissues and sites of possible local immunoglobulin production.     

Maintenance of Dispersed Reproductive Cells from Male and Female Ascaris suum

In vitro cultivation of tissues and cells provides an experimental methodology to define and manipulate physiological mechanisms that are not possible with in vivo techniques. Tissues from the germinative-growth zones of adult Ascaris suum gonads were excised and minced, and then enzymatically dispersed and transferred to an artificial, perienteric fluid-fetal calf-serum-medium complex. Cells were maintained in a viable state for 8 days, with medium replacement every 48 hours. During this period, morphological changes in the gonadal cells included decreased size, dedifferentiation, and degeneration. Two indices of metabolism, evolution of ¹⁴CO₂ from radiolabelled glucose and reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), decreased by approximately 50% and 60%, respectively. The in vitro procedures developed provide the first opportunity to examine specific cellular functions of nematode reproductive tissues over an extended period of time.     

Intermolecular interactions of homologs of germ plasm components in mammalian germ cells

In some species such as flies, worms, frogs and fish, the key to forming and maintaining early germ cell populations is the assembly of germ plasm, microscopically distinct egg cytoplasm that is rich in RNAs, RNA-binding proteins and ribosomes. Cells which inherit germ plasm are destined for the germ cell lineage. In contrast, in mammals, germ cells are formed and maintained later in development as a result of inductive signaling from one embryonic cell type to another. Research advances, using complementary approaches, including identification of key signaling factors that act during the initial stages of germ cell development, differentiation of germ cells in vitro from mouse and human embryonic stem cells and the demonstration that homologs of germ plasm components are conserved in mammals, have shed light on key elements in the early development of mammalian germ cells. Here, we use FRET (Fluorescence Resonance Energy Transfer) to demonstrate that living mammalian germ cells possess specific RNA/protein complexes that contain germ plasm homologs, beginning in the earliest stages of development examined. Moreover, we demonstrate that, although both human and mouse germ cells and embryonic stem cells express the same proteins, germ cell-specific protein/protein interactions distinguish germ cells from precursor embryonic stem cells in vitro; interactions also determine sub-cellular localization of complex components. Finally, we suggest that assembly of similar protein complexes may be central to differentiation of diverse cell lineages and provide useful diagnostic tools for isolation of specific cell types from the assorted types differentiated from embryonic stem cells.     

Vaccination with recombinant Ascaris suum 24-kilodalton antigen induces a Th1/Th2-mixed type immune response and confers high levels of protection against challenged Ascaris suum lung-stage infection in BALB/c mice

Previous studies have shown that antigens from various life-cycle stages of Ascaris suum can induce host-protective immunity against challenge infections with infective eggs of A. suum. This study evaluated whether Escherichia coli-expressed recombinant 24-kDa antigen from A. suum (rAs24) was a suitable vaccine candidate for the control of Ascaris infections by examining its performance in a mouse model. Immunization of BALB/c mice in three consecutive doses with rAs24 in Freund's Complete Adjuvant (FCA) results in protection against challenge infections as manifested by a 58% reduction (P<0.001) in recovery and stunted development of A. suum lung-stage larvae at day 7 post-challenge. Sera obtained from immune protected mice had a significantly increased level of immunoglobulin G (IgG) (P<0.0001) but had no IgE response. Analysis of IgG-subclass profiles revealed that IgG1 (P<0.0001) showed the greatest increase followed by IgG2b (P<0.005), IgG2a (P<0.006) and IgG3 (P<0.04). Splenic T cells from rAs24-FCA immunized mice secreted significantly high levels of both Th1 cytokine gamma-interferon (P<0.005) and Th2 cytokine interleukin-10 (P<0.001) after stimulation with rAs24 in vitro. Interestingly, affinity purified anti-rAs24 IgG was shown to inhibit moulting of A. suum lung-stage L3 to L4 in vitro by 26%, indicating an in vivo function of the endogenous As24 in the moulting processes. An intense expression of endogenous As24 in the hypodermis and gut epithelium of A. suum lung-stage L3 by immunofluorescence supports a function for endogenous As24. These findings may contribute to the understanding of rAs24-induced Th1/Th2-mediated effector mechanisms required for the protection of A. suum lung-stage larval infection.     

Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.     

Cytogenetic study of <Emphasis Type="Italic">Ascaris</Emphasis> trypsin inhibitor in cultured human lymphocytes with metabolic activation

The trypsin inhibitor (ATI) isolated from gastrointestinal nematode Ascaris suum was tested in vitro for induction of chromosome aberrations and sister chromatid exchanges (SCE). Genotoxicity assessment of purified ATI was carried out on metaphase plates received from peripheral blood lymphocyte macroculture (48 h test of structural chromosome aberrations and 72 h test of SCE) with exogenous metabolic activation. ATI was tested in dose of 25, 50 and 100 μg per ml of culture. Kinetics of cell divisions were determined by the replication index (RI). The mitotic index (MI) was expressed as a number of metaphases per 1000 nuclei analysed. Analysis of chromosome aberrations showed that higher doses of ATI (50 and 100 μg/ml) significantly increased the frequency of chromosome aberrations (mainly of chromatid gaps and breaks) compared to the negative control. All concentrations of ATI caused a statistically significant reduction in the MI and RI. In comparison with the negative control, a significant increase in the SCE frequency was observed in all applied doses of ATI. Thus, in the presence of S9 activation, the Ascaris trypsin inhibitor showed potential clastogenic activity and inhibition of the dynamics of lymphocyte divisions.