Drug effects on the 5-HT response of Schistosoma mansoni

Several vertebrate 5-HT antagonists at concentrations around 0.1 mM reduced 5-HT-induced increases in the motor activity of the parasitic blood fluke Schistosoma mansoni. The order of potency for 5-HT response antagonism was haloperidol greater than cyproheptadine greater than mianserin greater than trazodone greater than spiperone greater than methysergide. Nisoxetine, a 5-HT uptake inhibitor in vertebrate preparations, was also a potent antagonist of the 5-HT response in schistosomes. The potent antischistosomal praziquantel reduced the 5-HT response similarly to the other antiserotonergic drugs, but at much lower concentrations, beginning around 0.1 microM. The 5-HT agonist quipazine stimulated worm activity at 1-0.1 mM when applied alone, but reduced the 1 mM 5-HT response when quipazine and 5-HT were administered concurrently. Dopamine (DA) alone had no effect on the overall activity of S. mansoni. Although no drug was found to have absolute species specificity, quantitative differences were observed between the relative activity of drugs in schistosomes and vertebrates.     

Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.     

Insertion of bacteriorhodopsin into polymerized diacetylenic phosphatidylcholine bilayers

We have developed a method to incorporate the membrane protein bacteriorhodopsin into polymerized bilayers composed of a diacetylenic phosphatidylcholine, 1,2-bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) and a non-polymerizable phospholipid, dinonanoylphosphatidylcholine (DNPC). The extent of DC8,9PC polymerization in the bilayer was significantly improved when 2:1 mole ratio DNPC-DC8,9PC was used. Octyl glucopyranoside-solubilized bacteriorhodopsin was inserted into the polymerized DNPC-DC8,9PC bilayers by overnight incubation at 4 degrees C followed by dialysis to remove the detergent. The protein was inserted into the membranes after photo-polymerization to avoid inactivation of the protein due to the UV irradiation. The insertion of bacteriorhodopsin into the polymerized DNPC-DC8,9PC membranes was confirmed by density gradient centrifugation, UV/visible spectroscopy, and freeze fracture electron microscopy. The polymerized DNPC-DC8,9PC membranes containing bacteriorhodopsin were about 10% protein by weight. These results suggest that mixed lipid systems such as the DNPC-DC8,9PC can be used to improve both the extent of polymerization and the efficiency of membrane protein incorporation in the polymerized bilayer.     

Insights into the dynamics of DMSO in phosphatidylcholine bilayers.

The solvation effects of dimethyl sulfoxide (DMSO) on the phase stability of dimyristoylphosphatidylcholine (DMPC) have been fully characterized using differential scanning calorimetry (DSC) and fluorescence spectroscopy with 1,6-diphenyl-1,3,5-hexatriene (DPH). The temperatures of the sub-, pre-, and main transitions of DMPC were found to increase linearly with increasing mole fraction of DMSO up to mole fraction X=0.13 DMSO/H(2)O. Beyond X=0.13, the pre-transition peak started to merge with the peak representing the main transition. Simultaneously, the subtransition peak began to disappear as its transition temperature also decreased. At X=0.18, with both the subtransition and pre-transition absent, the main transition between the planar gel and the liquid-crystalline phase was observed at 30.3 degrees C. Transition enthalpy values indicated that the subgel, planar gel and rippled gel phases are most stable at X=0.11, 0.16 and 0.20 DMSO/H(2)O, respectively. This demonstrates that DMSO exerts distinct effects on each respective phase and corresponding transition. Temperature-dependent fluorescence emission scans show an increase in hydration as the system proceeds from the subgel phase all the way to the liquid-crystalline phase and correlated well with the effects of DMSO on the transition temperatures of DMPC observed in our calorimetry data. Initial observations for the sub- and main transition are further confirmed by fluorescence anisotropy using DPH as a probe. The results illustrate the differences in the microviscosity of each phase and how DMSO affects the phase transitions. Ultimately, our results suggest the most likely mechanism governing the biological actions of DMSO may involve the regulation of the solvation effects of water on the phospholipid bilayer.     

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.     

Schistosoma mansoni: incorporation and metabolism of protein amino acids in vitro

1. The amino acid composition of total proteins in six stages of the life cycle of Schistosoma mansoni was determined by routine autoanalysis of acid hydrolysates. Aspartate, glutamate and glycine were consistently the most abundant protein amino acids in all stages. 2. Incorporation of each of the protein amino acids into adult and egg proteins was determined using 72 hr cultures in complex media. Incorporation rates varied widely and there was no correlation between abundance in protein and the rate of incorporation. 3. Only five amino acids were interconverted to other amino acids which were themselves incorporated into worm and egg proteins (ala, arg, asp, gly, ser); of these only two (glu from ala and pro from arg) appeared to be of quantitative significance. Exogenous glucose yielded only three protein amino acids (ala, asp, glu). 4. The data are considered in the light of differences in egg and adult protein synthesis and with particular regard to potential chemotherapy at this level.     

Partitioning of perfluorooctanoate into phosphatidylcholine bilayers is chain length-independent

The chain length dependence of the interaction of PFOA, a persistent environmental contaminant, with dimyristoyl- (DMPC), dipalmitoyl- (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated using steady-state fluorescence anisotropy spectroscopy, differential scanning calorimetry (DSC) and dynamic light scattering (DLS). PFOA caused a linear depression of the main phase transition temperature T_m while increasing the width of the phase transition of all three phosphatidylcholines. Although PFOA's effect on T_m and the transition width decreased in the order DMPC > DPPC > DSPC, its relative effect on the phase behavior was largely independent of the phosphatidylcholine. PFOA caused swelling of DMPC but not DPPC and DSPC liposomes at 37 °C in the DLS experiments, which suggests that PFOA partitions more readily into bilayers in the fluid phase. These findings suggest that PFOA's effect on the phase behavior of phosphatidylcholines depends on the cooperativity and state (i.e., gel versus liquid phase) of the membrane. DLS experiments are also consistent with partial liposome solubilization at PFOA/lipid molar ratios > 1, which suggests the formation of mixed PFOA–lipid micelles.     

The effect of maintenance in vitro on glucose uptake and the incorporation of glucose into glycogen by adult Schistosoma mansoni

The effect of maintenance in vitro on glucose uptake and the incorporation of glucose into glycogen by adult Schistosoma mansoni. International Journal for Parasitology16: 253–261. Adult male Schistosoma mansoni rapidly depleted their glycogen reserves in vitro. Both sexes also exhibited a gradual reduction in glycogen content during prolonged maintenance. Paired and separated worms were incubated in [³H] glucose and rates of glucose uptake and incorporation into glycogen were determined following periods of maintenance in vitro. The glucose uptake rate declined during long-term maintenance and was higher for separated males and females than for equivalent paired worms. Increasing the medium glucose concentration also increased the rate of uptake. Glucose continued to be incorporated into glycogen throughout 10 days in vitro, with evidence from paired schistosomes suggesting that the rapid depletion of male glycogen could be due to a decrease in incorporation rate in vitro. The incubation of separated worms and the use of higher glucose concentrations in media both effected an increase in incorporation rate. These results are discussed in the light of observations of the depletion of schistosome glycogen in vitro.     

Schistosoma mansoni: effects of in vitro serotonin (5-HT) on aerobic and anaerobic carbohydrate metabolism

The effect of Serotonin on carbohydrate metabolism, excreted end products, and adenine nucleotide pools in Schistosoma mansoni was determined following 60 min in vitro incubations under air (= 21% O2) and anaerobic (95% N2:5% CO2) conditions. In the presence of 0.25 mM Serotonin, glucose uptake increased by 82-84% and lactate excretion increased by 77-78%; levels of excreted lactate were significantly higher under aerobic than under anaerobic conditions. The tissue pools of glucose, hexosephosphates, fructose 1,6-bisphosphate, pyruvate, and lactate were significantly increased under anaerobic conditions compared to air incubation; the presence of Serotonin decreased tissue glucose pools and increased the size of the pyruvate and lactate tissue pools. The glycolytic carbon pool was significantly greater under anaerobic than under aerobic conditions, irrespective of Serotonin. Serotonin increased adenosine 5'-diphosphate and adenosine 5'-monophosphate levels under aerobic conditions; neither Serotonin nor gas phase significantly affected total adenine nucleotide levels or the adenylate energy charge. Serotonin increased energy requirements by S. mansoni due to increased muscle contractions; demand was met by enhanced rates of carbohydrate metabolism. Irrespective of gas phase, 74-78% of available carbohydrate was converted to lactate. In the presence of Serotonin, conversion of glucose to lactate was reduced to 63-67%. In view of the requirements by S. mansoni for an abundant supply of glycoprotein and glycolipid precursors for surface membrane renewal, it is suggested that carbohydrate (glucose and glycogen) that was not converted to lactate may have been incorporated into biosynthetic processes leading to membrane synthesis.     

A freeze fracture study of the developing tegumental outer membrane of Schistosoma mansoni

The freeze fracture technique has been used to quantify changes in the integral components of the double outer membrane of Schistosoma mansoni during the 6-week period of development within the mouse. The intramembraneous particle (IMP) density on the P1 face begins to rise within 6 h of host penetration, reaches a maximum at day 4 and then falls rapidly after day 9, so that it is at a low level between 3 and 6 weeks. The E1 face IMP density follows the same course as that of the P1 face except that maximum particle density is recorded on day 1 and the counts begin to fall on day 5. The IMP density on the P2 face remains at a consistently low level throughout development. The E2 face IMP density rises gradually to a peak at day 4, when the parasites have migrated to the lungs, and remains thereafter at a similar level, so that by 6 weeks the E2 face has a higher IMP density than the other three fracture faces. The E2 face IMP show a marked increase in size on day 4. Morphological studies indicate that a different type of inclusion body makes a transient appearance in the tegument of the lung worms, and immunocytochemical techniques show the lung worms to be nonimmunogenic. It is suggested, therefore, that the E2 face IMP may represent complexes of parasite antigens and acquired host antigens. The tegumental membranes of cultured specimens have also been examined by freeze fracturing and the IMP densities compared with those obtained from in vivo parasites; the cultured schistosomula have a lower E2 face particle density than the in vivo specimens.     

Quantitation of cholesterol incorporation into extruded lipid bilayers

Cholesterol incorporation into lipid bilayers, in the form of multilamellar vesicles or extruded large unilamellar vesicles, has been quantitated. To this aim, the cholesterol contents of bilayers prepared from phospholipid:cholesterol mixtures 33-75 mol% cholesterol have been measured and compared with the original mixture before lipid hydration. There is a great diversity of cases, but under most conditions the actual cholesterol proportion present in the extruded bilayers is much lower than predicted. A quantitative analysis of the vesicles is thus required before any experimental study is undertaken.     

Specific incorporation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid into phosphatidylcholine in human endothelial cells

The incorporation of hydroxyeicosatetraenoic acids (HETEs) into cellular lipids was studied in cultures of human umbilical vein endothelial cells. 5-[3H]HETE was incorporated into the phospholipids (8%) and neutral lipids (15.5%). The uptake was at half maximum after 15 min and reached a plateau after 1 h. The incorporation occurred mainly into phosphatidylcholine (6.3%) with minimal uptake into phosphatidylserine and phosphatidylinositol (0.6%) or phosphatidylethanolamine (1.2%). There was no uptake of 12-[3H]HETE, 15-[3H]HETE or [3H]leukotriene B4 into phospholipids. Treatment of the phosphatidylcholine fraction with phospholipase A2 released 64% of the 5-[3H]HETE with 26% remaining in the lysophosphatidylcholine fraction. This indicates that the majority of the 5-HETE was in the sn-2 position. Unlabeled 5-HETE and arachidonic acid inhibited the uptake of 5-[3H]HETE into phosphatidylcholine with an ID50 of 2.5 and 1.25 microM, respectively. Stearic acid and 15-HETE were not effective inhibitors. Histamine, which activates phospholipases, increased the uptake of 5-[3H]HETE into phosphatidylcholine by 3-fold. Both 5-[3H]HETE and 12-[3H]HETE were incorporated into the neutral lipids of the cells. Analysis of the neutral lipid fraction revealed that 5-[3H]HETE was incorporated into mono-, di- and triacylglycerols but not cholesterol esters. Incorporation of 5-HETE into cellular lipids reduced histamine- and arachidonic acid-stimulated synthesis of 6-ketoprostaglandin F1 alpha and prostaglandin E2 in a concentration-related manner. Angiotensin I converting enzyme activity was not changed. Thus, 5-HETE is incorporated specifically into phosphatidylcholine and glycerol esters of human endothelial cells and this incorporation inhibits prostaglandin synthesis in these cells.     

Hormonal and pharmacologic agents affecting choline uptake and incorporation in Schistosoma mansoni adults

Initial uptake of choline by Schistosoma mansoni (2-min uptake) revealed no differences between male, female or paired worms for any of the control or experimental groups. After a 30-min uptake period, however, males showed significantly higher uptake of choline in the presence of mitomycin C, cytochalasin B and calcium ionophore A23187, while paired worms showed significantly reduced uptake in the presence of actinomycin D, puromycin, mitomycin C, cytochalasin B, colchicine, insulin, thyroxine and lysine. Choline uptake by females was elevated, in the presence of cytochalasin B at 30 min, although not significantly. Significantly increased incorporation of choline into phosphatidylcholine was observed following a 30-min incubation with 5-hydroxytryptamine (males), puromycin and thyroxine (females) and calcium ionophore A23187 (males, females and pairs). These effects on phosphatidylcholine synthesis are discussed in relation to the uptake data and to previous work concerning the outer membrane complex of the parasite as an important facet of parasite resistance to the host immune response.     

Schistosoma mansoni: stimulus and transformation of cercariae into schistosomules

Schistosoma mansoni schistosomules prepared from cercariae by seven in vitro techniques had not all reached the same state of development at the end of the incubation period as scored by seven parameters: water tolerance; Cercarienhüllen Reaktion; presence of the glycocalyx; condition of the surface membrane; nuclear state; granule migration; and cryopreservability. At the end of the specific incubation period for each technique, the level of development was judged with respect to schistosomules which had developed in situ for 1 hr after penetration of the ear skin of mice. In descending order of their correspondence to in vivo schistosomules, those derived in vitro (by the procedures listed) ranked as follows: first, penetration of dried rat skin; second, centrifuging and vortexing, or incubation in serum-supplemented medium; and third, syringe passage, omnimixing, centrifuging, and incubating, or incubating alone. The only treatment common to all techniques was incubation in 37 C culture medium for 2 hr or more. This is suggested as the stimulus for the cercaria-to-schistosomule transformation.     

Serotonin-induced muscular activity in Schistosoma mansoni larval stages: importance of 5-HT transport and role in daughter sporocyst production

Mother sporocysts of Schistosoma mansoni transport exogenously supplied serotonin (5-hydroxytrypamine; 5-HT), and respond to it with increases in motility. In the present study, we investigated the importance of 5-HT transporter activity in the manifestation of these 5-HT-induced motility changes, and further examined the role of 5-HT in the development of daughter sporocysts in vitro. Serotonin-induced motility of in vitro-derived sporocysts is not inhibited by antidepressant compounds, e.g., fluoxetine, that block 5-HT transport, suggesting that the receptors responsible for motility responses to 5-HT are surface exposed. Using a sporocyst in vitro culture system, we show that depletion of larval stores of 5-HT reduces production of daughter sporocysts, the second intramolluscan larval stage. Moreover, we demonstrate a strong correlation between endogenous 5-HT levels and basal mother sporocyst muscle activity. Overall, these data suggest that larval stages of S. mansoni can detect exogenous 5-HT via surface-exposed receptors, and they are consistent with the hypothesis that endogenous stores of 5-HT are important for the proper regulation of muscular contractions in mother sporocysts, and for the successful emergence of daughter sporocysts.     

Schistosoma mansoni: levels of antioxidants and resistance to oxidants increase during development

The effects of cell-free generated oxidants on migrating and developing stages of Schistosoma mansoni were investigated and the levels of antioxidant enzymes and of glutathione were determined for each stage. Schistosomula and 2-week-old parasites recovered from the livers of infected mice showed similar susceptibility to killing by added hydrogen peroxide and t-butylhydroperoxide. However, when glucose (0.5 mM)-glucose oxidase (2.5 mU ml-1) and xanthine (0.5 mM) or hypoxanthine (0.5 mM)-xanthine oxidase (5.0 mU ml-1) systems were used to generate hydrogen peroxide and oxygen free-radicals, schistosomula were more susceptible to oxidative killing than the 2-week-old parasites. The 4- and 8-week-old worms were more resistant to oxidants than all of the younger stages. High levels of superoxide dismutase (16.2-24.8 U mg-1 protein) were present in all stages. Catalase was not detected. Glutathione peroxidase activity with cumene hydroperoxide as substrate was not detectable in the schistosomula but the activity was present in the 2-week-old parasites. However, hydrogen peroxide-sensitive glutathione peroxidase activity was present in all the stages with a threefold difference in activity between schistosomula and the adult stages. Glutathione-s-transferase activity was significantly lower in the schistosomula, lung stages, and the 2-week-old parasites than in the older stages. Progressive increases in the levels of glutathione reductase and glutathione were also observed with development. The differences in the levels of antioxidants between different stages of development may partly explain the increase in resistance to oxidant-mediated damage as the parasite develops.     

Permeability of phosphatidylcholine and phosphatidylethanolamine bilayers

Sodium and glucose effluxes were measured in liposomes formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Vesicles composed of a saturated PC display a local permeability maximum in the region of the lipid transition temperature. The height of this maximum is predominantly a function of the thickness of the hydrocarbon chain region. Liposomes formed from a saturated PE do not display such a permeability maximum and in these vesicles the permeability process appears to be controlled by the head group region. It is postulated that the control exerted by the ethanolamine group is due to the reorganization of water structure it induces at the bilayer surface.