Schistosoma mansoni: a complement-dependent receptor on adult male parasites

The dorsal surface of adult male Schistosoma mansoni exhibits an affinity for Salmonella typhi when these bacteria are preincubated in normal serum from mice, guinea pigs, or humans. The complement (C) system was shown to be responsible for the bacterial binding. Bacteria not preincubated with normal serum or preincubated in normal serum which had been treated with ethylenediamine tetraacetic acid (EDTA), or cobra venom factor (CoF), or heated at 56 C for 60 min did not bind to the parasite's surface. Further experiments utilizing ethylene glycol tetraacetic acid (EGTA) plus Mg²⁺, heat inactivation at 50 C for 30 min, and zymosan treatment of the serum indicated the C fixation and deposition on the bacteria occurs via the alternative C pathway. These observations indicate the presence of a complement-dependent receptor on the dorsal tegumental surface of the adult male parasite.     

Spirocamallanus cricotus (Nematoda): isoelectric focusing and spectrophotometric characterization of its hemoglobin and that of its piscine host, Micropogonias undulatus

The hemoglobins of Spirocamallanus cricotus, a reddish-colored, camallanid nematode, and its Atlantic croacker fish host, Micropogonias undulatus, were characterized with spectrophotometry and isoelectric focusing. Hemoglobin from female parasites' perienteric fluid and homogenized male parasites gave Spectrophotometric peaks at 412, 539, and 575 nm, whereas female worms drained of perienteric fluid and homogenized differed by having a Soret peak of 408 nm. Changing the ionic strength of the buffer from 0.1 to 0.01 M shifted the Soret peak to 406 nm for the female parasites' perienteric fluid and ground male parasites and 404 nm for homogenized female parasites. In all cases, the β band had a higher absorption than the α band suggesting a high O₂ affinity for the parasite hemoglobin. Host hemoglobin had peaks of 406, 437, and 577 nm. Isoelectric focusing not only confirmed the Spectrophotometric evidence that host and parasite hemoglobins differed, but also showed that the parasite's analyzed hemoglobin fractions differed from one another by having different isoelectric points.     

Insights into the mode of action of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89) against adult Schistosoma mansoni worms

Control of morbidity associated with schistosomiasis via chemotherapy largely relies on the drug praziquantel. Repeated therapy with praziquantel has created concerns about the possible selection of resistant worms and necessitated the search for novel drugs to treat schistosomiasis. Here, a murine model was infected with Schistosoma mansoni and treated with oral 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89), which caused a significant reduction in fecundity and egg burden and reduced morbidity when administered at 5-weeks post-infection.The analysis showed that the mode of action occurred through the ingestion of activated N-89 by the worms, and that there was no direct external effect on the S. mansoni worms. Ultrastructural analysis of the treated worms showed disruptions in the gut lumen and the presence of large volumes of material, suggestive of undigested blood meals or red blood cells. In addition, there were reduced vitelline cells in female worms and damage to sub-tegmental musculature in male worms. Eggs recovered from the treated mice showed both damage to the eggs and the production of immature eggs. Expression of mRNA responsible for gut and digestive function and egg production was also significantly affected by N-89 treatment, whereas control genes for musculature showed no significant changes.Thus, N-89 drastically affected the total digestive function and egg production of S. mansoni worms. Physiological processes requiring heme uptake such as egg production and eggshell formation were subsequently affected, suggesting that the compound could be a possible therapeutic drug candidate for schistosomiasis control.     

Hydroxycinnamoyl acid amides and aromatic amines in the inflorescences of some araceae species

Hydroxycinnamoyl acid amides (HCA's) were found to be important components in the inflorescences of different Araceae species. HCA's occurred in large amount in spathes and in the male and female flowers, and were totally absent from the sterile flowers, commonly found on Araceae spadices. Differences in the distribution of HCA's were noted between male and female flowers. Thus the amount of neutral HCA's was always greater in the male than in the female flowers and the female flowers generally contained more basic HCA'S. In the inflorescences of some Araceae species in the Monsteroideae and Philodendroideae (genera Monstera, Raphidophora and Philodendron), the aromatic amines tyramine and dopamine were very abundant, with concentrations ranging from 1 to 4 mg of each amine per g fr. wt.     

THE POTASSIUM EQUILIBRIUM IN MUSCLE

1. Analyses were made of the K and HCO₃ content, the irritability, and weight change of isolated frog sartorius muscles after immersion for 5 hours in Ringer''s solutions modified as to pH and potassium content. 2. At each pH a concentration of potassium in the solution was found which was in diffusion equilibrium with the potassium in the muscle. In greater concentrations potassium moved into the muscle against the concentration gradient and vice versa. 3. The greater the alkalinity of the solution the smaller the concentration of the potassium at equilibrium so that the product of the concentrations of OH and K in the solution at equilibrium tends to remain approximately constant. 4. The pH inside the muscle is approximately equal to that outside when first dissected but it tends to change during immersion so as to follow the changes in the pH of the solution. This finding is in direct conflict with the theory according to which the high potassium concentration inside should be accompanied by an equally high hydrogen ion concentration in relation to that outside. 5. The diffusion of potassium into the muscle makes its contents more alkaline but the increase in alkalinity is not always, nor usually, equivalent to the amount of potassium which has diffused and conversely, the pH inside can change in either direction according to the pH outside without there being any diffusion of potassium. Hence potassium is not the only penetrating ion. 6. The irritability of the muscles is at a maximum in concentrations of potassium which are greater than that in normal Ringer''s solution, or about 20 mg. per cent potassium. This optimum does not seem to be a function of pH and is therefore not dependent upon the direction of movement of the potassium but probably on the ratio of potassium outside to that inside. 7. Swelling of the muscles occurs in solutions which injure the muscle so as to permit both cations and anions to enter without permitting the organic protein anions to escape. Anion impermeability is necessary to prevent this same osmotic swelling under normal conditions. 8. An increase in the CO₂ tension in muscle and solution causes a greater increase in acidity in the solution than in the muscle and leads to a loss of potassium. One expects therefore a potassium shift from tissues to blood comparable to the chlorine shift from plasma to corpuscles.     

Schistosoma mansoni: Calcium efflux and effects of calcium-free media on responses of the adult male musculature to praziquantel and other agents inducing contraction

When male Schistosoma mansoni were incubated in a Ca²⁺-free medium their responsiveness to the contracture inducing agents, praziquantel (PZ), dinitrophenol (DNP), 60 mM K⁺ (high K⁺), ouabain, and low temperature, was rapidly attenuated. After 5 min in a zero Ca²⁺ medium the responsiveness to PZ was reduced by 60% but a residual response of 20% remained even after 40 min in a calcium-free medium. The contracture induced by ouabain or low temperature was totally lost within 1 min exposure to a zero Ca²⁺ medium. The efflux of ⁴⁵Ca²⁺ from parasites incubated in a medium containing no Ca²⁺ closely parallels the drop in responsiveness of their musculature to high K⁺, DNP, and PZ. The total amount of Ca²⁺ in the parasite was reduced by only 30% after 60 min in zero Ca²⁺ medium. A relatively rapid exponential decline in muscle tension was noted when parasites, previously treated with PZ, high K⁺, or DNP, were transferred to a medium containing these agents but with no Ca²⁺. Parasites that had been contracted with ouabain or low temperature showed no significant relaxation 16 min after transfer to a zero Ca²⁺ medium. The ⁴⁵Ca²⁺ efflux from worms bathed in zero Ca²⁺ medium was not significantly altered by the presence of ouabain. These results suggest the presence of active Ca²⁺ transport at the level of the parasites' muscle membranes.     

Salts and the Infectivity of Romanomermis culicivorax

The effects of common inorganic ions found in freshwater on the infectivity of Romanomermis culicivorax and the survival of its host, Culex pipiens, were tested. In general, the median lethal concentrations found for R. culicivorax were greater than the reported median concentrations of these ions in freshwater but less than the reported maximum natural concentrations. The ion toxicity for R. culicivorax (on a molar basis) increased in the following order: sodium < potassium < calcium; aml chloride < carbonate = sulfate < nitrate < nitrite < phosphate. The larvae of C. pipiens were generally 20 to 75 times as tolerant of higher ion concentrations as were the preparasitic stages of R. culicivorax.     

Studies on resistance in snails: Interference by nonirradiated echinostome larvae with natural resistance to Schistosoma mansoni in Biomphalaria glabrata

Most of the genetically selected juvenile Biomphalaria glabrata snails, normally strongly resistant to Schistosoma mansoni, lost their juvenile resistance to this parasite when other trematodes were concurrently present in the snail. Three echinostome species all were able to reduce this genetically controlled juvenile resistance: Echinostoma lindoense, E. paraensei, and e. liei. Subsequently, adult resistance to S. mansoni, clearly present in control snails of the same age and strain that were not doubly infected, failed to develop in most of the snails that also harbored echinostomes. Other snails, selected for resistance as adults to S. mansoni, also usually became susceptible to this parasite following infection with E. paraensei. The capacity of E. paraensei to interfere with the snails' resistance to S. mansoni was greater than that of E. lindoense. Destruction by predation of primary sporocysts of S. mansoni by echinostome rediae prevented completion of development of the S. mansoni infections. In a number of snails all primary S. mansoni sporocysts were consumed before secondary sporocysts could be formed. In most experimental snails, however, some of the schistosomes survived, often as a small number of degenerated secondary S. mansoni sporocysts. The capability of flukes to interfere with the natural defense of snails may be an important phenomenon whereby trematode species survive in their snail hosts.     

Schistosoma mansoni: Astiban-induced damage to tegument and the male reproductive system

Astiban produced structural damage in male Schistosoma mansoni (Puerto Rican strain) in mice. The degree of disorganization was directly related to the dosage administered, although initial changes in structure for the first three doses (3 × 30–40 mg/kg) varied between individual worms of the same infection. More consistent damage to the tegument, parenchyma, and reproductive organs occurred after 6 × 40 mg/kg of Astiban injections. Exposure of the subtegumentary musculature preceded appearance of an increased number of noncytoplasmic spaces in various tissues, probably a result of osmotic stress. Testicular disorganization was prominent initially in spermatozoa and spermatids, but became more generalized with drug accumulation. The sustentacular cells showed increased phagocytic activity with testicular damage. Continuous administration of drug resulted in a general distortion of the worm's morphology. However, partial recovery occurred within 22 days following cessation of drug administration.     

The evolutionary transition to sideways-walking gaits in brachyurans was accompanied by a reduction in the number of motor neurons innervating proximal leg musculature

The forwards-walking portly crab, Libinia emarginata is an ancient brachyuran. Its phylogenetic position and behavioral repertoire make it an excellent candidate to reveal the adaptations, which were required for brachyuran crabs to complete their transition to sideways-walking from their forwards-walking ancestors. Previously we showed that in common with other forwards-walking (but distantly related) crustaceans, L. emarginata relies more heavily on its more numerous proximal musculature to propel itself forward than its sideways-walking closer relatives. We investigated if the proximal musculature of L. emarginata is innervated by a greater number of motor neurons than that of sideways-walking brachyurans. We found the distal musculature of spider crabs is innervated by a highly conserved number of motor neurons. However, innervation of its proximal musculature is more numerous than in closely-related (sideways-walking) species, resembling in number and morphology those described for forwards-walking crustaceans. We propose that transition from forward- to sideways-walking in crustaceans involved a decreased role for the proximal leg in favor of the more distal merus–carpus joint.     

Plasmodium iophurae: cationized ferritin staining, an electron microscope cytochemical method for differentiating malarial parasite and host cell membranes

The surface membranes of erythrocyte-free Plasmodium lophurae and its host cell, the duckling erythrocyte, stain differentially when exposed to cationized ferritin (CF). At low CF concentrations (0.18 mg/ml) only the outer surface of the red cell stains, whereas at the standard concentration (0.7 mg/ml) both the red cell and the parasitophorous vacuolar membranes (PVM) were stained on their outer faces. By using a high CF concentration (3.7 mg/ml), the parasite's plasma membrane (PM) could be distinguished from that of the PVM: The former did not bind CF, whereas the latter was stained on its outer surface. At this level of CF the red cell membrane stained on both faces if these surfaces were exposed to stain. Although the PVM is formed by red cell endocytosis of the parasite, it can be distinguished from the membrane of the erythrocyte as well as that of the PM.     

Schistosomiasis mansoni, haematobia, and japonica in hamsters: liver granuloma measurements

Differences in the granulomatous response of hamsters infected with Schistosoma mansoni, Schistosoma haematobium, or Schistosoma japonicum were studied by measuring granulomas formed around eggs in the livers at 1, 3, 5, 11, and 19 week intervals after patency of the infections. For each species, the mean diameters of both granulomas containing only 1 egg and granulomas selected at random were determined. The mean volume of single egg granulomas in S. mansoni-infected hamsters was greater than in those with other species at all time intervals studied. The decrease in single egg granuloma size with time occurred more gradually in S. mansoni infections. The mean volume of granulomas measured at random was greatest in S. japoracum-infected animals at 1, 3, and 11 weeks after patency; was greatest in S. haematobium-iufected. hamsters at 5 and 19 weeks; and was least in S. mansoni-infected hamsters at all time periods. During the 19 week period following patency, the mean volume of randomly measured granulomas increased with time in S. haematobium infections, decreased gradually in S. mansoni infections, and decreased markedly in S. japonicum infections. Multi-egg granulomas represented 2–6% of all granulomas measured in S. mansoni infections, 38–68% in S. haematobium infections, and 20–53% in S. japonicum infections. An estimated proportion of the liver occupied by granulomas, i.e., a lesion-to-tissue ratio, was computed. There was no consistent difference in this ratio among the 3 species when the data were grouped in comparable intervals of mean number of eggs per g of liver. The lesion-to-tissue ratio increased with increasing numbers of eggs per g of liver. The host response of hamsters to these 3 species of schistosomes differed markedly even at comparable levels of eggs per g of liver. This study provides further evidence of an intrinsic qualitative difference in the eggs of the 3 species. The species of eggs present in tissues apparently has greater influence on the host response than does the quantity of eggs present.     

Eosinophil-mediated destruction of S. mansoni eggs IV. Effects of several inhibitory substances on eosinophil function

The destruction of isolated Schistosoma mansoni eggs in vitro by eosinophil-rich peritoneal exudative cells obtained from S. mansoni-infected mice is known to involve both specific anti-egg antibody and non-specific activation of the cells by lymphokine(s). The current study reports the effects of various metabolic inhibitors and other reagents known to alter cell function. The addition of inhibitors of glycolysis (Iodoacetate, 2 deoxy-d glucose) ablated the egg-destructive activity of eosinophils, but also resulted in considerable cell mortality during the 24 hr incubation period. Antimycin A, an inhibitor of aerobic respiration, also inhibited egg destruction. The presence of divalent cations, Ca²⁺ in particular, was found to be essential for eosinophil-mediated egg destruction to occur. Cytochalasin B also inhibited this eosinophil-dependent activity. Tosyl-lysine-chloromethyl ketone decreased the extent of egg destruction, but when used at non-cytotoxic concentrations this effect was marginal. The metabolic requirements for eosinophils, and other cell types, in this and other systems are compared.     

Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology.     

Effects of extracellular potassium concentration on meiotic and cytoplasmic maturation in the porcine oocyte

Effects of extracellular potassium (K⁺) concentration in maturation media on the meiotic and cytoplasmic maturation of porcine oocytes were examined. Oocyte-cumulus cell complexes or cumulus cell denuded oocytes were cultured in Whitten's medium containing 0, 3, 6, 12 or 16 mM potassium. Absence of K⁺ in the media did not inhibit germinal vesicle breakdown (GVBD) in cumulus intact oocytes, but significantly decreased the frequency of meiotic maturation. In cumulus cell denuded oocytes, both GVBD and meiotic maturation were inhibited in K⁺-free medium. Millimole concentrations of K⁺ channel blockers, 4-aminopyridine or tetraethyl ammonium chloride inhibited GVBD and almost completely suppressed progression of meiotic maturation. The effect of varying the concentration of K⁺ on cytoplasmic maturation of pig oocytes was evaluated by the ability to form a male pronucleus after in vitro fertilisation. The percentage of sperm penetration or monospermic penetration was not different among treatments (P > 0.1). However, male pronuclear formation in oocytes in medium with 6 mM K⁺ was higher than in media with 12 and 16 mM K⁺. These results suggest that extracellular K⁺ is required for GVBD and meiotic maturation, and high concentrations (12 or 16 mM) of K⁺ in maturation media impair cytoplasmic maturation.     

The Time Course of the Loss and Recovery of Contracture Ability in Frog Striated Muscle Following Exposure to Ca-Free Solutions

Using area under the contracture curve to quantitate contractures, the diffusion coefficient of calcium ions within the frog toe muscle during washout in a calcium-free solution and subsequent recovery after reintroduction of calcium to the bathing solution was calculated to be about 2 x 10^-6 cm²/sec. The diffusion coefficient measured during washout was found to be independent of temperature or initial calcium ion concentration. During recovery it was found to decrease if the temperature was lowered. This was likely due to the repolarization occurring after the depolarizing effect of the calcium-free solution. The relation between contracture area and [Ca]_o was found to be useful over a wider range than that between maximum tension and [Ca]_o. The normalized contracture areas were larger at lower calcium concentrations if the contractures were produced with cold potassium solutions or if NO₃ replaced Cl in the bathing solutions. Decreasing the potassium concentration of the contracture solution to 50 mM from 115 mM did not change the relation between [Ca]_o and the normalized area. If the K concentration of the bathing solution was increased, the areas were decreased at lower concentrations of Ca.     

Schistosoma mansoni: Tyrosine,a putative in vivo substrate of phenol oxidase

l-Tyrosine, l-[3,4]dihydroxyphenylalanine (l-DOPA), and dopamine are known to be in vitro substrates for Schistosoma mansoni phenol oxidase. Since all three compounds are present in the female schistosome, it is not clear which one serves as the substrate for phenol oxidase in intact S. mansoni. However, the concentration of l-tyrosine in the female schistosome (252 ng/mg worm) is 4-fold higher than the K_m of phenol oxidase for this amino acid while the concentrations of l-DOPA and dopamine (0.954 and 0.790 ng/mg worm, respectively) are 100- and 500-fold lower than the K_m of these substrates. Tri-l-tyrosine methyl ester is oxidized at less than 3% of the rate of l-tyrosine methyl ester. A tyrosine:lysine peptide and chymotrypsinogen are not oxidized. Female S. mansoni do not incorporate l-tyrosine into proteins to a significantly greater extent than l-leucine. The results suggest that free l-tyrosine is the substrate for S. mansoni phenol oxidase in vivo.     

Schistosoma mansoni: Uptake in vitro and incorporation of adenine by adults

The rates of adenine uptake and incorporation into nucleic acids by adult male and female Schistosoma mansoni were determined during periods of up to 10 days in vitro, and comparisons were made between paired and separated worms. Adenine uptake by separated males and females exceeded that exhibited by equivalent paired worms. The rate of incorporation of adenine into nucleic acids was higher in separated females than in paired females. In contrast, the state of pairing had little effect on adenine incorporation by male S. mansoni. There was no correlation between rates of adenine uptake and incorporation and the reproductive activity of S. mansoni adults in vitro. Uptake and incorporation rates appeared to reflect the changing somatic requirements of both male and female worms.