A Ca2+ influx associated with exocytosis is specifically abolished in a Paramecium exocytotic mutant.

A Paramecium possesses secretory organelles called trichocysts which are docked beneath the plasma membrane awaiting an external stimulus that triggers their exocytosis. Membrane fusion is the sole event provoked by the stimulation and can therefore be studied per se. Using 3 microM aminoethyl dextran (AED; Plattner, H., H. Matt, H.Kersken, B. Haake, and R. Stürz, 1984. Exp. Cell Res. 151:6-13) as a vital secretagogue, we analyzed the movements of calcium (Ca2+) during the discharge of trichocysts. We showed that (a) external Ca2+, at least at 3 X 10(-7) M, is necessary for AED to induce exocytosis; (b) a dramatic and transient influx of Ca2+ as measured from 45Ca uptake is induced by AED; (c) this influx is independent of the well-characterized voltage-operated Ca2+ channels of the ciliary membranes since it persists in a mutant devoid of these channels; and (d) this influx is specifically abolished in one of the mutants unable to undergo exocytosis, nd12. We propose that the Ca2+ influx induced by AED reflects an increase in membrane permeability through the opening of novel Ca2+ channel or the activation of other Ca2+ transport mechanism in the plasma membrane. The resulting rise in cytosolic Ca2+ concentration would in turn induce membrane fusion. The mutation nd12 would affect a gene product involved in the control of plasma membrane permeability to Ca2+, specifically related to membrane fusion.

Flavonoids and related compounds in parasitic disease control.

Flavonoids are natural plant compounds increasingly used in therapeutic applications. Their large spectrum of activities depends on their structures and cellular targets. Most recent research shows they are promising drugs for controlling human and animal parasitic diseases. Their multiple effects make it difficult to understand their modes of action, but some of them have been elucidated. This review also deals with their toxicity in mammals.

Response of goat sperm to hypoosmotic steps modelled probit analysis.

Hypoosmotic swelling test (HOS) has been proposed by many authors to evaluate the functional integrity of the sperm membrane. Our approach in this experiment has consisted in exposing spermatozoa to a wide range of osmotic pressures then evaluating the reacted sperm cells by flow cytometry and finally modelling the sperm cell responses. Semen samples were diluted in skim milk or NPPC (native phosphocaseinate) extenders, and stored at 4 degrees C for 3 days. At D0 and D3 aliquots from each ejaculate (n=12) were submitted to seven hypoosmotic solutions varying from 230 to 10mOsm/kg. Sperm samples were analyzed using flow cytometry to determine two populations of spermatozoa identified by propidium iodide (PI): PI+ (including PI, red fluorescence) and PI- (excluding PI, no fluorescence). Spermatozoa PI+ were considered as spermatozoa with membrane damages. PI+ exhibited a high variation from 230 to 10mOsm/kg which was considered as a dose-response curve. Data were modelled using Mixed procedure and probit analysis to a sigmoid curve. Each model curve characterized the profile of response of the variable PI+ to the range of osmotic pressure from 230 to 10mOsm/kg. The estimated parameters modelling the sigmoid curves are discussed in order to evaluate the effect of extender (skim milk versus NPPC) and duration of preservation (D0 versus D3). Such modelling could help to differentiate storage method ejaculates within males or between male, contributing therefore to improve semen technology.

The ERK-dependent signalling is stage-specifically modulated by FSH, during primary Sertoli cell maturation.

Primary cultures of Sertoli cells provide an interesting model to study how signalling pathways induced by a single hormone in a single cell type evolve, depending on the developmental stage. In vivo, follicle-stimulating hormone (FSH) induces proliferation of Sertoli cells in neonate and controls the subsequent differentiation of the entire population. Molecular mechanisms underlying Sertoli cell pleiotropic responses to FSH have long been investigated. But to date, only cAMP-dependent kinase (PKA) activation has been reported to account for most FSH biological activities in male. Here, we demonstrate that FSH activates the ERK MAP kinase pathway following dual coupling of the FSH-R both to Gs and to Gi heterotrimeric proteins, in a PKA- and also Src-dependent manner. This activation is required for FSH-induced proliferation of Sertoli cells isolated 5 days after birth. Consistently, we show that the ERK-mediated FSH mitogenic effect triggers upregulation of cyclin D1. In sharp contrast, at 19 days after birth, as cells proceed through their differentiation program, the ERK pathway is dramatically inhibited by FSH treatment. Taken together, these results show that FSH can exert opposite effects on the ERK signalling cascade during the maturation process of Sertoli cells. Thus, signalling modules triggered by the FSH-R evolve dynamically throughout development of FSH natural target cells.

A rapid calcium influx during exocytosis in Paramecium cells is followed by a rise in cyclic GMP within 1 s.

The synchrony of trichocyst exocytosis in Paramecium allows temporal correlation of associated events. Using quenched flow we observed a Ca2+ influx concurrent with exocytosis within 80 ms after stimulation with the secretagogue aminoethyldextran. Cyclic AMP did not change in depency of stimulation. Cyclic GMP transiently increased after 500 ms, culminating at 2 s, and thus considerably lags behind exocytosis induction and influx of Ca2+. Both Ca2+ influx and rise in cGMP are known to be induceable also by Ba2+ or veratridine, allegedly via the opening of ciliary Ca2+ channels. However, only veratridine stimulated exocytosis. We conclude that both aminoethyldextran and veratridine induce an exocytosis-associated Ca2+ influx, which is responsible for the rise in cGMP, through an as yet unknown pathway.

Annexin A5 D226K structure and dynamics: identification of a molecular switch for the large-scale conformational change of domain III.

The domain III of annexin 5 undergoes a Ca(2+)- and a pH-dependent conformational transition of large amplitude. Modeling of the transition pathway by computer simulations suggested that the interactions between D226 and T229 in the IIID-IIIE loop on the one hand and the H-bond interactions between W187 and T224 on the other hand, are important in this process [Sopkova et al. (2000) Biochemistry 39, 14065-14074]. In agreement with the modeling, we demonstrate in this work that the D226K mutation behaves as a molecular switch of the pH- and Ca(2+)-mediated conformational transition. In contrast, the hydrogen bonds between W187 and T224 seem marginal.

The xenotransplantation of goat and human hematopoietic cells to sheep fetuses.

Hematopoietic xenografts were carried out in three experiments using goat fetal liver (44-48 days, experiments I and II) or purified human CD 34+ cells (experiment III) as the donor cells. Recipients were sheep fetuses at 41-47 days of gestation. Goat fetal liver cells were either injected without any pretreatment or stimulated by preincubation in a culturing in goat phytohemagglutinin-stimulated lymphocyte supernatant. Human CD 34+ myeloid progenitor cells were purified from bone marrow by minimacs immunomagnetic purification and cultured in medium supplemented with stem cell factor, IL3, and IL6. Goat-sheep chimerism was assessed by flow cytometry analysis (FCA) of peripheral blood and bone marrow cells using a mouse anti-goat CD 45 monoclonal antibody and by karyotype analysis of peripheral blood from goat/sheep chimeras. Human cell engraftment was assessed by polymerase chain reaction amplification of the human DAX1 gene in blood and bone marrow DNA from sheep which had received human cells. In the three experiments, a mean of 76% (26 of 34) of injected fetuses were born alive without any clinical evidence of graft-versus-host disease. Three lambs were found to be goat/sheep chimeric after flow cytometry analysis (peripheral blood and bone marrow) and karyotype (peripheral blood) analysis. Both tissues continued to express goat cells at 6 or 12 months (last assessment) depending on the experiment. No human chimerism was detected using polymerase chain reaction amplification in peripheral blood and bone marrow of any of the six sheep grafted with human cells. These data and those also obtained on other species (human, pig/sheep) show that it is possible to carry out hematopoietic xenografts using the sheep fetus as recipient provided both donor and recipient fetal cells are processed during the period of tolerance to foreign antigens.

Worm population characteristics and pathological changes in lambs after a single or trickle infection with Teladorsagia circumcincta.

The regulation of the worm population and of its pathological effects was studied after a single or trickle infection with T. circumcincta. One hundred and twenty lambs, 60 Romanov and 60 Mérinos d'Arles, 3 months old, were distributed in four balanced groups: non-infected (G0), infected with 7000 L3 per animal once and slaughtered after 4 weeks (G14) or 8 weeks (G18), and infected 8 times and slaughtered after 8 weeks (G88). Parasitological, histological, haematological parameters and weight gains were recorded on each animal. Female and artificially nursed lambs had lower worm burdens and egg counts (epg) than males and naturally suckled lambs. No difference in parasitological parameters was seen between the two breeds, but Mérinos lambs infected once, had a higher increase in pepsinogen concentrations than Romanov lambs. In the infected animals, a significant proliferation of mast and eosinophil cells was observed in the abomasum wall. Serum pepsinogen concentrations were significantly higher 3 weeks p.i. and the weight gain was depressed during the first month p.i. The worm population was more numerous and younger in group G14 compared with G18 in which 24% of the worms had been expelled during the second month p.i. The female worms in G18 were longer and had more eggs in utero and higher egg output. After the trickle infection (G88) the take was reduced, female worms were longer with more eggs in vagina (pars ejectrix) and there was a higher variability in the number of eggs (compared with G18 data). The pepsinogen rise was smaller but no specific effect was seen on histological and haematological parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization and migration of benzimidazole resistant and susceptible adult Cooperia curticei in the small intestine of sheep after treatment with thiabendazole.

Four groups of three lambs per group were experimentally infected with Cooperia curticei susceptible (two groups) or resistant (two groups) to benzimidazoles, and distributions of adult worms in the small intestine were studied. For each Cooperia isolate, one group was treated with thiabendazole (TBZ) (5 or 50mg/kg bodyweight) 28 days after infection. In the two untreated groups, the population of C. curticei were present from the second to the tenth meter of intestine from the pylorus with a maximum in the sixth meter for both isolates. After treatment with TBZ, the size of the resistant worm population did not significantly decrease but a large number of worms were found towards the proximal sections of the intestine. In contrast, the susceptible population was reduced by about 40% but the surviving worms remained at this same site of predilection after treatment. Measurements of the concentration of TBZ and 5OH-thiabendazole (5OH-TBZ) in the intestinal segments do not indicate a clear relationship between the localization of worms and TBZ or 5OH-TBZ concentrations at least 12h after the anthelmintic treatment. The hypothesis of an enhanced expression of the mechanisms of resistance in the first few meters of small intestine is suggested.

Use of free living stages to study the effects of thiabendazole, levamisole, pyrantel and ivermectin on the fine structure of Haemonchus contortus and Heligmosomoides polygyrus.

Ultrastructural changes induced in vitro by thiabendazole, levamisole, pyrantel and ivermectin in the free living larval stages of two trichostrongyles (Heligmosomoides polygyrus and Haemonchus contortus) were analysed. The observed damage for each anthelmintic is related to the known mode of action and compared to the damage commonly described in adults. The advantage of using larvae to study the effects of anthelmintics on the fine structure of nematodes rather than adults is described. Thiabendazole induced alteration of the cellular organization especially epithelial cells of the digestive tract. Changes in mitochondria were also seen. Levamisole caused contraction of muscle fibres whereas no specific lesions were observed with pyrantel. Ivermectin caused an hypertrophy of muscular groups. The interest of such a technique in research on the modes of action of anthelmintics is emphasized.

Response of cattle treated with a fenbendazole slow release bolus to challenge from nematodes the following season.

Nematode infection of cattle treated in their first year at pasture with the fenbendazole slow release bolus ('Bolus group') was compared during the second year with that of untreated cattle. Ostertagia was the most prevalent parasite associated with Cooperia. Except for the Dictyocaulus spp. which caused clinical signs of bronchitis in the 'Bolus' group, the infection during the second year resulted in a moderate response of the cattle whatever the group. Rises in both pepsinogen and gastrin levels were correlated with the number of Ostertagia L3 on herbage. Damages in the abomasal mucosa were more frequent and severe in the 'Bolus' group where more inflammatory signs were observed in spite of a smaller number of worms. Nevertheless, the differences in total weight gains were not significant thanks to a compensatory effect during the second part of the grazing season in the 'Bolus' group. Hypotheses related to a minimum threshold of infection during the first year necessary to develop high enough protection during the second year are discussed. The pathological effects of gastrointestinal nematodes seem to vary more according to the inflammatory response than to the number of worms.

Persistent efficacy of topical moxidectin against Dictyocaulus viviparus and Ostertagia ostertagi.

The persistent activity of moxidectin topically administered at the dose rate of 0.5 mg kg-1 bodyweight was evaluated against experimental nematode infection in 30 calves randomly allocated to six groups. Five groups were treated on days -42, -35, -28, -21 and -14. The 6th group remained untreated as a control. On Day 0, the calves were infected experimentally with 1000 Dictyocaulus viviparus and 50,000 Ostertagia ostertagi larvae and killed 3 weeks later. The formulation of moxidectin showed excellent activity against both parasites for up to 5 weeks (> 99%). Six weeks after treatment the reduction in the number of D. viviparus was still high (> 90%). No adverse reactions to moxidectin were observed in any of the animals.

The control of parasitic gastroenteritis of grazing cattle in Normandy, France using the morantel sustained release bolus.

The efficacy of the morantel sustained release bolus in controlling parasitic gastroenteritis in 153 first-season grazing cattle was assessed in three separate field trials conducted in Normandy, France. In each trial, comparisons were made on weight gain performance and parasitology data (faecal worm egg counts, herbage larval counts and- in two of the trials- worm counts from principal animals sacrificed at the end of the grazing season) when bolus treatment was given either at spring turnout or in mid-season in order to determine the optimum time for bolus administration. Cattle were allocated into three groups, each group maintained on a separate but equivalent paddock constructed from the division of a larger pasture. A morantel sustained release bolus was administered to one group of animals at the time of turnout and to a second group of animals in midsummer. The third group of animals in each trial remained nontreated. The effect of the treatment on the contamination of pasture, and parasite levels and weight gain of the principal trial animals was assessed. Similar results were observed in all three trials. Faecal worm egg counts were reduced during the first part of the grazing season in animals receiving the bolus at turnout compared with mid-season treated animals where egg counts followed a pattern similar to the controls until bolus treatment at which time counts abruptly dropped to a low level. Likewise, levels of infective larvae on pastures grazed by control and mid-season treated animals followed similar patterns, increasing to a high level in late summer, while larval levels on pastures grazed by early-season treated animals remained at low levels throughout most of the season. Serum pepsinogen levels, worm counts and weight gain reflected the results from faecal worm egg and herbage larval counts indicating that early-season treatment with the bolus provided the most efficient treatment time for controlling parasitic gastroenteritis throughout the grazing season. The overall mean weight gain advantage of the early-season bolus-treated animals over the controls was 37.2 kg (P less than 0.01) while the advantage of the mid-season treated animals over controls was 13.7 kg.

New staining methods for sperm evaluation estimated by microscopy and flow cytometry.

New staining methods and automated instruments are now available to evaluate the sperm cell in vitro. Individual compartments of the sperm cell, such as the nucleus and the plasma and acrosomal membranes, may be investigated, as well as the cell function as shown by mitochondria activity and capacitation. Various probes are used and they can be analyzed by direct light or fluorescent microscopy or by flow cytometry. The automated instruments allow objective and accurate analysis and quantification as well as the ability to evaluate large population of cells in a shorter time, thus providing accurate evaluation of sperm quality. However, before these test can be recommended for routine clinical and investigational use, in the stallion, they need to be confirmed on a larger number of stallions and their correlation with traditional semen parameters and with stallion fertility has to be demonstrated.

Population dynamics of Trichostrongylus colubriformis in sheep following anthelmintic treatment.

Three groups of five, three-and-a-half to four-month-old lambs were put to graze on three plots contaminated by Trichostrongylus colubriformis. One group (control) was free of parasites, while the other two were infected just before turning out with 3 X 700 third stage larvae. Depending on whether or not the lambs were treated with 50 mg kg-1 thiabendazole nine days after the first infection, the worm burden and the egg output increased or decreased during the grazing period when compared with the control group. In tracer animals put to graze on the same plots afterwards, it was found that the smaller the number of eggs excreted by the first lambs, the higher were the worm burdens of the tracer lambs.

Winter survival of trichostrongyle larvae: a study using tracer lambs.

Six groups of tracer lambs grazed from April to August on a pasture ungrazed since the previous November. A gradual decrease was observed in the worm burdens until July when a marked increase both in the number of worms and in egg counts was recorded. Haemonchus species were solely responsible for this sudden increase and it seems most likely that infection originated from larvae of the previous year rather than from overwintering eggs. Survival of Haemonchus species larvae in soil is suggested.

Winter survival of third stage Trichostrongylus colubriformis larvae: effects on infectivity and fecundity of adult worms.

Two groups of six lambs were fed with grass cut from two plots which had been contaminated by spreading Trichostrongylus colubriformis larvae. One plot received larvae in December, the other in April. The post mortem examination results showed that the infectivity of overwintering larvae was significantly higher than that of fresh larvae. An inverse relationship was demonstrated between infectivity of larvae and egg production of adult worms developed from these larvae. The results are compared with those obtained under laboratory conditions.

A high efficiency technique for the long-term preservation of infective nematode larvae.

Improvements are suggested for the existing long term techniques for the preservation of nematode larvae. Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis and Cooperia curticei larvae exsheathed in sodium hypochlorite and then suspended in phosphate buffered saline (PBS pH 7.2) are cooled in the gas over liquid nitrogen at a cooling rate of -1 degree C min-1 down to -50 degrees C. Larvae are then stored in liquid nitrogen at -196 degrees C. After warming at 30 degrees C and reactivation at 20 degrees C for at least 12 h, their percent motility is maintained (approximately 85%) providing that no more than 3000 to 5000 larvae are suspended in 1.8 mL of PBS in cryotubes. Infectivity does not significantly decrease: 46% of larvae cooled for 2 or 6 mo develop to adult stages compared to 52% for larvae stored at 4 degrees C for 2 mo.

Efficacy of a pour-on formulation of eprinomectin (MK-397) against nematode parasites of cattle, with emphasis on inhibited early fourth-stage larvae of Ostertagia spp.

OBJECTIVE: To evaluate efficacy of topically applied eprinomectin against inhibited early fourth-stage larvae (IL4) of Ostertagia spp in calves. ANIMALS: 4 groups (n = 6 [replicates]) for dose titration; 2 groups (n = 8 calves [replicates]) for dose confirmation. PROCEDURE: 2 dose titration studies-0, 125, 250, and 500 micrograms of eprinomectin/kg of body weight-Louisiana and Georgia- and 2 dose confirmation studies of selected therapeutic dosage (500 micrograms/kg) in Scotland and France. Monitor calves were used to determine inhibition percentage of Ostertagia IL4. Test calves were ranked by weight in replicates of 4 (titration trials) or 2 (confirmation trials) animals each, and within replicates, were randomly allocated to treatment groups. Drug treatments were done on day 0, and animals were euthanatized by replicate, with holding time between treatment and euthanasia varying among trials from 14 to 27 days. RESULTS: Observations indicated high efficacy (> 99%) of 500 micrograms of eprinomectin/kg in removal of Ostertagia IL4. Ostertagia and Cooperia were only genera common across sites, with efficacy of aforementioned dosage against adult and larval stages of both genera consistently high (> 99%). Results of 1 or both titration studies (500 micrograms/kg) indicated > 99 to 100% efficacy against adult Haemonchus placei, Trichostrongylus axei, T colubriformis, Bunostomum phlebotomum, Dictyocaulus viviparus, and Oesophagostomum radiatum. Lower efficacy values were observed at minimal (125 micrograms/kg) dosage. In France, 500 micrograms/kg was 85% effective against Trichostrongylus spp adults; however, numbers of control calves infected with Trichostrongylus spp and degree of infection were low. Adverse reactions were not evident. CONCLUSION: Eprinomectin given topically (500 micrograms) was highly effective against Ostertagia IL4 and other common nematodes of cattle.

A microlarval development assay for the detection of anthelmintic resistance in sheep nematodes.

A microlarval development test for the detection of anthelmintic resistance in nematodes is described. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis eggs were cultured to third stage larvae in the presence of Earle's balanced salt solution, yeast extract and bacteria in a total volume of 150 microliters. Good dose-response data were obtained with thiabendazole, levamisole, pyrantel tartrate and ivermectin allowing the determination of the 50 per cent lethal concentration and of resistance factors when resistant strains were available. The test was found to be accurate, sensitive, easy to carry out and applicable to the routine detection of resistance.